Exogenous pulmonary surfactant prevents the development of intra‐abdominal adhesions in rats

Intra‐abdominal adhesions are major post‐operative complications for which no effective means of prevention is available. We aimed to evaluate the efficacy of exogenous pulmonary surfactant administration in the prevention of post‐operative abdominal adhesions. Rats were randomly assigned to undergo laparotomy (L) or gastroenterostomy (GE) and then treated with surfactant (groups L‐S and GE‐S, respectively). Intra‐abdominal adhesions, collagen fibre content, metalloproteinase (MMP)‐9, expression of growth factors (TGF‐β, KGF and VEGF), type III procollagen (PCIII) and pro‐caspase 3, as well as isolectin B4 and ED1‐positive cells expressing MMP‐9, were evaluated. Groups treated with surfactant (GE‐S and L‐S) exhibited fewer adhesions. A significant reduction in collagen fibre content was observed in GE‐S compared to GE animals (P < 0.001). In situ and gelatin zymography analysis showed higher MMP‐9 expression and activity in the GE‐S group compared to the GE group (P < 0.05). ED1‐positive cell counts were significantly higher in the GE‐S group (P < 0.001) than in the GE group. Virtually all cells positive for ED1 were MMP‐9+. Double‐labelling of MMP‐9 with IB4 showed no significant differences between GE‐S and GE groups. TGF‐β, KGF, PCIII and pro‐caspase‐3 mRNA expression decreased significantly in GE‐S compared to GE animals (P < 0.05). Surfactant administration also reduced apoptosis in the GE‐S group. These findings suggest that surfactant reduces the intra‐abdominal adhesions triggered by laparotomy and gastrointestinal anastomosis, thus preventing fibrosis formation at the peritoneal surfaces. This preclinical study suggests an innovative treatment strategy for intra‐abdominal adhesions with surfactant and to endorse its putative mechanism of action.     

Simple agrometeorological models for estimating Guineagrass yield in Southeast Brazil

The objective of this work was to develop and evaluate agrometeorological models to simulate the production of Guineagrass. For this purpose, we used forage yield from 54 growing periods between December 2004–January 2007 and April 2010–March 2012 in irrigated and non-irrigated pastures in São Carlos, São Paulo state, Brazil (latitude 21°57′42″ S, longitude 47°50′28″ W and altitude 860 m). Initially we performed linear regressions between the agrometeorological variables and the average dry matter accumulation rate for irrigated conditions. Then we determined the effect of soil water availability on the relative forage yield considering irrigated and non-irrigated pastures, by means of segmented linear regression among water balance and relative production variables (dry matter accumulation rates with and without irrigation). The models generated were evaluated with independent data related to 21 growing periods without irrigation in the same location, from eight growing periods in 2000 and 13 growing periods between December 2004–January 2007 and April 2010–March 2012. The results obtained show the satisfactory predictive capacity of the agrometeorological models under irrigated conditions based on univariate regression (mean temperature, minimum temperature and potential evapotranspiration or degreedays) or multivariate regression. The response of irrigation on production was well correlated with the climatological water balance variables (ratio between actual and potential evapotranspiration or between actual and maximum soil water storage). The models that performed best for estimating Guineagrass yield without irrigation were based on minimum temperature corrected by relative soil water storage, determined by the ratio between the actual soil water storage and the soil water holding capacity.irrigation in the same location, in 2000, 2010 and 2011. The results obtained show the satisfactory predictive capacity of the agrometeorological models under irrigated conditions based on univariate regression (mean temperature, potential evapotranspiration or degree-days) or multivariate regression. The response of irrigation on production was well correlated with the climatological water balance variables (ratio between actual and potential evapotranspiration or between actual and maximum soil water storage). The models that performed best for estimating Guineagrass yield without irrigation were based on degree-days corrected by the water deficit factor.     

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe₃S₄ cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII_int) with four ¹H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII_ox and FdII_int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same ¹H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdII_ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII_int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm⁻¹ and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII_int. This study, together with the spin delocalization mechanism proposed here for DgFdII_int, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII_ox and DgFdII_int.     

Morphological and growth alterations in Vero cells transformed by cisplatin

Cisplatin is an antineoplastic agent used to treat solid tumours, such as ovarian, testicular and bladder tumours. However, studies in vitro and in vivo have shown that cisplatin is mutagenic, genotoxic and tumorigenic in other tissues and organs. In this work, we examined the effect of cisplatin on Vero cells, a fibroblast-like cell line. The morphological characteristics were investigated using phase contrast microscopy, scanning electron microscopy and the actin cytoskeleton was labelled with fluorescein isothiocyanate-phalloidin. Cell proliferation was assessed based on the growth curve. Cultured Vero cells treated with cisplatin showed behavioural and morphological alterations associated with cellular transformation. The transformed cells grew in multilayers and formed cellular aggregates. The proliferation and morphological characteristics of the transformed cells were very different from those of control ones. Since transformed Vero cells showed several characteristics related to neoplastic growth, these cells could be a useful model for studying tumour cells in vitro.     

Increased SOD1 association with chromatin,DNA damage,p53 activation,and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly⁹³ → Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.     

Optimized methodology for extraction of (1 → 3)(1 → 6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative

The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment.