Ecological risk assessment of pesticides in sediments of Pampean streams, Argentina.

After their application in agricultural areas, pesticides are dispersed throughout the environment, causing contamination problems. In Argentina, the main promoter of transgenic biotechnology in the region, the total consumption of agrochemicals has increased significantly in recent years. Most chemicals dumped near surface waters eventually end up in bottom sediments and can be toxic to the organisms that live there. However, published data on the mixing of pesticides in this compartment is still scarce. The objective of this work was to detect and quantify pesticide residues in the sediment of rural streams in the Pampas region and to carry out acute and chronic risk assessment in these aquatic ecosystems. The study area comprises the mountainous system of Tandilia, located in one of the most productive agricultural areas in the country. The concentration of atrazine, acetochlor, chlorpyrifos, cypermethrin, and 2,4-D in the sediment of four rural streams was determined in three different seasons, and the toxic units (TU) and the risk ratios (RQ) were calculated. All the compounds analyzed were detected in most of the sampling seasons and study sites, at concentrations higher than those established in the national and international quality guidelines for the protection of aquatic biota in surface waters and for human consumption. Chlorpyrifos, cypermethrin, and acetochlor were the main pesticides contributing to the TU and RQ values, representing a medium or high ecological risk in most of the sites. Therefore, the evaluation of these pesticides in the bottom sediments could be a decisive factor in assessing the risk to the aquatic environment.

Endovascular repair of postoperative vascular graft related complications after aorto-iliac surgery.

AIM: Para-anastomotic aneurysms, leakage due to anastomotic failure, aorto- and arterioenteric fistulas are some of the serious complications after aorto-iliac surgical reconstructions. Treatment of these complications is challenging and is either done by open surgery or by endovascular therapy. The mortality and morbidity is higher compared to the initial treatment. We present twelve patients with these complications which were treated by an endovascular approach. METHODS: From January 2008 through January 2013 our radiological records were searched for cases with post surgical vascular complications treated with endovascular intervention. These comprised of anastomotic pseudoaneurysm, suture leakage and arterial enteric fistulas. Patients with limb occlusions were not included in this study. RESULTS: Twelve patients with graft related complications treated with endovascular intervention were recorded. There were four women and eight men with a mean age 75,3 years (range 48-80). At the time of diagnosis, 9 patients (75%) had symptoms and three (25%) was incidentally discovered. Six patients had leakage due to suture failure. All infective parameters were within normal limits. Four patients presented with anastomotic pseudoaneurysms without leak, of which three had proximal anastomotic pseudoaneurysms and one had distal iliac anastomotic pseudoaneurysm. Implanted stent graft were Endurant (Medtronic) bifurcated endoprostheses in three patients and Excluder (Gore) prosthesis in a two cases. Tubular Medtronic endoprosthesis was implanted in one case and in two cases aortic cuff was used. Fluency periphery stent grafts were used in four cases. There was a 100% technical success. Intervention related early mortality was 8%. One patient with pseudoaneurysm died 28 months after endovascular treatment because of cardiac infarct and one patient with previously infected arterio-enteric fistula and advanced malignancy died 7 months after second endovascular treatment. Overall the mortality was 25%. There was no procedure related morbidity or complications during hospitalization and follow-up of mean 12, 3 months (range 1-36 months) in the other 9 patients. There were no complications like endoleaks or limb occlusions. CONCLUSION: Endovascular treatment of vascular graft related postsurgical complications is a valuable therapeutic option followed by lower mortality and morbidity rates compared with re-operation. Short and midterm follow-up is without severe complications and if it occurs most of them can be treated by endovascular means again.

Autophagy in neurons: a review.

Macroautophagy is a process of regulated turnover of cellular constituents that occurs during development and under conditions of stress such as starvation. Defects in autophagy have serious consequences, as they have been linked to neurodegenerative disease, cancer, and cardiomyopathy. This process, which exists in all eukaryotic cells, is tightly controlled, but in extreme cases results in the death of the cell. While major insights into the molecular and biochemical pathways involved have come from genetic studies in yeast, little is known about autophagic pathways in mammalian cells, particularly in neurons. Recently, research in neuronal culture models has begun to identify some characteristics of neuronal macroautophagy. The results suggest that macroautophagy in neurons may provide a neuroprotective mechanism. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurons.

Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death.

Alpha-synuclein mutations have been identified in certain families with Parkinson's disease (PD), and alpha-synuclein is a major component of Lewy bodies. Other genetic data indicate that the ubiquitin-dependent proteolytic system is involved in PD pathogenesis. We have generated stable PC12 cell lines expressing wild-type or A53T mutant human alpha-synuclein. Lines expressing mutant but not wild-type alpha-synuclein show: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins; (2) enhanced baseline nonapoptotic death; (3) marked accumulation of autophagic-vesicular structures; (4) impairment of lysosomal hydrolysis and proteasomal function; and (5) loss of catecholamine-secreting dense core granules and an absence of depolarization-induced dopamine release. Such findings raise the possibility that the primary abnormality in these cells may involve one or more deficits in the lysosomal and/or proteasomal degradation pathways, which in turn lead to loss of dopaminergic capacity and, ultimately, to death. These cells may serve as a model to study the effects of aberrant alpha-synuclein on dopaminergic cell function and survival.

Brain-derived neurotrophic factor inhibits apoptosis and dopamine-induced free radical production in striatal neurons but does not prevent cell death.

In hereditary Huntington's disease, a triplet repeat disease, there is extensive loss of striatal neurons. It has been shown that brain-derived neurotrophic factor (BDNF) protects striatal neurons against a variety of insults. We confirmed that BDNF enhances survival and DARPP-32 expression in primary striatal cultures derived from postnatal mice. Furthermore, BDNF inhibited intracellular oxyradical stress triggered by dopamine, and partially blocked basal and dopamine-induced apoptosis. Nevertheless, BDNF failed to rescue striatal neurons from dopamine-induced cell death. Therefore, BDNF inhibits free radical and apoptotic pathways in medium spiny neurons, but does so downstream from the point of commitment to cell death.

Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells.

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration.

Huntington's disease (HD) is caused by an expanded CAG repeat in exon 1 of the gene coding for the huntingtin protein. The cellular pathway by which this mutation induces HD remains unknown, although alterations in protein degradation are involved. To study intrinsic cellular mechanisms linked to the mutation, we examined dissociated postnatally derived cultures of striatal neurons from transgenic mice expressing exon 1 of the human HD gene carrying a CAG repeat expansion. While there was no difference in cell death between wild-type and mutant littermate-derived cultures, the mutant striatal neurons exhibited elevated cell death following a single exposure to a neurotoxic concentration of dopamine. The mutant neurons exposed to dopamine also exhibited lysosome-associated responses including induction of autophagic granules and electron-dense lysosomes. The autophagic/lysosomal compartments co-localized with high levels of oxygen radicals in living neurons, and ubiquitin. The results suggest that the combination of mutant huntingtin and a source of oxyradical stress (provided in this case by dopamine) induces autophagy and may underlie the selective cell death characteristic of HD.

Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury.

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.

Increased MAP1B expression without increased phosphorylation in manganese-treated PC12Mn cells.

Regulation of MAP1B expression and phosphorylation is thought to play an important role in neuronal development, particularly with regard to axon growth. The present work utilizes a novel PC12 cell variant [26] which exhibits many of the early morphological features of neurite outgrowth when stimulated with manganese chloride. Expression of MAP1B was determined by immunoblots and phosphorylation was assessed by metabolic radiolabeling with [32P]orthophosphate or with a phospho-specific antibody. The results indicate that MAP1B protein levels rise within 12 to 24 h, but there is no significant change in the phosphorylation of MAP1B. The latter conclusion is based on (i) experiments utilizing SMI 31, a monoclonal antibody that specifically reacts with phospho-MAP1B and (ii) assessments of both MAP1B phosphorylation and MAP1B protein within that same isloated protein band on Western blots. Thus, manganese increases MAP1B expression without affecting its relative phosphorylation. Although manganese does not cause neurite formation in the parental PC12 cell line, manganese is capable of inducing transient neurite regeneration from NGF-primed cells. These studies provide further evidence that the onset of neurite outgrowth may proceed without increased phosphorylation of MAP1B. During sustained neurite regeneration, however, NGF increases phosphate incorporation into MAP1B. Based on all of these findings, we conclude that early phases of neurite outgrowth (cell spreading and formation of short tapered extensions) do not necessarily require elevated phosphorylation of MAP1B.

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture.

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1-12-3 recognized only one protein of identical mobility on the two-dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.

Pediatric renal transplantation without steroids.

Pediatric renal transplant patients present a number of challenges and problems, especially the inhibited post-transplant growth seen in children receiving standard immunosuppressive triple therapy that includes steroids. We report the successful use of steroid-free immunosuppression since 1990 in 14 pediatric renal allograft recipients who received a 10-day initial course of anti-lymphocyte globulin and surface area-adjusted doses of cyclosporine, 7 of whom also received mycophenolate mofetil (MMF) as maintenance immunosuppression. Only 1 patient died (3 months after transplantation as a result of a primary Epstein-Barr virus infection-induced lymphoproliferative disorder), 1 patient's graft never functioned, and another patient lost his graft after 3 years because of chronic rejection. Three patients experienced early acute cellular rejection, which resolved in 2 cases with OKT3, and in the 3rd with MMF. There were no late acute rejections. All patients evidenced growth and a growth spurt under this regimen. We conclude that all the pediatric patients benefited from our steroid-free protocol and that this protocol is superior to conventional triple therapies, which entail the eventual reduction and discontinuation of steroids, a procedure that not only inhibits growth but also carries an additional risk of acute rejection due to a steroid-adapted immune response.

Percutaneous transluminal treatment of an iliac pseudoaneurysm with endoprosthesis implantation. A case report.

PURPOSE: Evaluation of a covered stent in the treatment of an iliac pseudoaneurysm. MATERIAL AND METHODS: During PTCA, a 61-year-old man with angina pectoris was shown to have an asymptomatic pseudoaneurysm, 5 x 4 x 4 cm, of the left common iliac artery (CIA). The patient was not a candidate for surgery. The pseudoaneurysm was treated by insertion of a covered stent (8 mm/6 cm). RESULTS: By 30 s after stent placement, the pseudoaneurysm was angiographically excluded. Twelve and 17 months after stent implantation, the patient had no symptoms from the lower extremities and the left CIA was open with normal flow in the stent. CONCLUSION: Our case illustrates the ability of a covered stent to exclude a pseudoaneurysm. Percutaneous intravascular stent placement in the management of iliac pseudoaneurysms might be the treatment of choice in patients with increased risk of major anesthesia and surgery.

Activity and activation of the complement system in patients being operated on for cancer of the colon.

OBJECTIVE: To find out if there was any local activation of complement in the vicinity of a colonic cancer, and any fluctuation in the function of the complement system during operation. DESIGN: Prospective study. SETTING: One university and two district hospitals in Denmark. SUBJECTS: 29 selected patients undergoing emergency and elective operations for colonic cancer. INTERVENTIONS: Measurements of systemic and local complement fixation capacity and complement activation in samples of serum or plasma taken before, during, and after operation. MAIN OUTCOME MEASURES: Changes in complement fixation capacity and complement activation during operation. RESULTS: Haemodilution during operation caused a significant reduction in the complement fixation capacity of serum and in the activation of the complement system as measured by generation of C3c. We were unable to confirm the presence of complement inhibitors during operation. Haemodilution caused a 30% reduction in fixation capacity of C3b (12/29 samples of serum had values more than 2SD below the mean of the reference range compared with 4/29 before operation). The activity of C4 was reduced by 25% during operation and the capacity of the complement system to fix C3b and C4b was restored to baseline nine days postoperatively. Concentration of C3d was significantly higher in serum from tumour venous blood compared with that from peripheral blood during operation. CONCLUSION: The presence of complement activation products in the general circulation reflects local activation of the complement system in the vicinity of the tumour, but this may have been influenced by tissue necrosis or subclinical infection. Haemodilution causes a significant reduction in the capacity of the complement system during operation, whereas inhibitory factors associated with the cancer or operation and anaesthesia could not be demonstrated. We found no correlation between complement activity and clinical data.