Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

Biomarker of food intake for assessing the consumption of dairy and egg products

Dairy and egg products constitute an important part of Western diets as they represent an excellent source of high-quality proteins, vitamins, minerals and fats. Dairy and egg products are highly diverse and their associations with a range of nutritional and health outcomes are therefore heterogeneous. Such associations are also often weak or debated due to the difficulty in establishing correct assessments of dietary intake. Therefore, in order to better characterize associations between the consumption of these foods and health outcomes, it is important to identify reliable biomarkers of their intake. Biomarkers of food intake (BFIs) provide an accurate measure of intake, which is independent of the memory and sincerity of the subjects as well as of their knowledge about the consumed foods. We have, therefore, conducted a systematic search of the scientific literature to evaluate the current status of potential BFIs for dairy products and BFIs for egg products commonly consumed in Europe. Strikingly, only a limited number of compounds have been reported as markers for the intake of these products and none of them have been sufficiently validated. A series of challenges hinders the identification and validation of BFI for dairy and egg products, in particular, the heterogeneous composition of these foods and the lack of specificity of the markers identified so far. Further studies are, therefore, necessary to validate these compounds and to discover new candidate BFIs. Untargeted metabolomic strategies may allow the identification of novel biomarkers, which, when taken separately or in combination, could be used to assess the intake of dairy and egg products.     

From insulin receptor signalling to Glut 4 translocation abnormalities in obesity and insulin resistance

Insulin resistance is commonly associated with obesity in rodents. Using mice made obese with goldthioglucose (GTG-obese mice), we have shown that insulin resistance results from defects at the level of the receptor and from intracellular alterations in insulin signalling pathway, without major alteration in the number of the Glut 4 glucose transporter. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundly affected in response to insulin. This defect appears very early in the development of obesity, together with a marked decrease in IRS 1 tyrosine phosphorylation. In order to better understand the abnormalities in glucose transport in insulin resistance, we have studied the pathway leading from the insulin receptor kinase stimulation to the translocation of the Glut 4 containing vesicles. This stimulation involves the activation of PI 3-kinase, which in turns activates protein kinase B. We have then focussed at the mechanism of vesicle exocytosis, and more specifically at the role of the small GTPase Rab4 in this process. We have shown that Rab4 participates, first in the intracellular retention of the Glut 4 containing vesicles, second in the insulin signalling pathway leading to glucose transporter translocation.     

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.     

Phylogeny, traits, environment, and space in cerrado plant communities at Emas National Park (Brazil)

Soil, drought, and fire are abiotic factors that may act as environmental filters in the cerrado, the Brazilian savanna. We used a framework to analyze environmental filtering in geographic and phylogenetic context, sampling woody species in one of the largest cerrado reserves. In 100 quadrats, we measured 10 functional traits on each woody individual. We also measured several soil variables, altitude and slope as a rough surrogate of water availability, interval between fires, and time since last fire. Almost all environmental variables were spatially auto-correlated. We found an overall trait clustering, but not an overall phylogenetic clustering. Nevertheless, we found a phylogenetic signal for some traits. Linking phylogeny, traits, environment, and space, we were able to detect a major dichotomy between two geomorphological units. The flat tableland was positively related with altitude, fire frequency, and nutrient-richer soil. Environmental filtering caused by water availability and fire lead to trait clustering, with smaller shrubs and trees that presented thicker barks, denser woods, sclerophyllous leaves, highlighted by the prevalance of Myrtaceae. The other geomorphological unit, hilly terrain, was positively related with slope, low fire frequency, and nutrient-poorer soil. Environmental filtering was caused especially by nutrient-poor soil that lead to trait clustering, assembling taller trees, with thinner barks, lighter woods, and compound, large, tender, nutrient-richer leaves, distributed across many lineages, including Fabaceae. Hence, the high environmental variability in space with different environmental filters assembled different combination of plant traits and lineages, increasing the overall diversity in cerrado.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Synthesis and antimalarial and antituberculosis activities of a series of natural and unnatural 4-methoxy-6-styryl-pyran-2-ones, dihydro analogues and photo-dimers

Previous studies have identified the 3,6-dialkyl-4-hydroxy-pyran-2-one marine microbial metabolites pseudopyronines A and B to be modest growth inhibitors of Mycobacterium tuberculosis and a range of tropical diseases including Plasmodium falciparum and Leishmania donovani. In an effort to expand the structure-activity relationship of this compound class towards infectious diseases, a library of natural product and natural product-like 4-methoxy-6-styryl-pyran-2-ones and a subset of catalytically reduced examples were synthesized. In addition, the photochemical reactivity of several of the 4-methoxy-6-styryl-pyran-2-ones were investigated yielding head-to-head and head-to-tail cyclobutane dimers as well as examples of asymmetric aniba-dimer A-type dimers. All compounds were evaluated for cytotoxicity and activity against M. tuberculosis, P. falciparum, L. donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi. Of the styryl-pyranones, natural product 3 and non-natural styrene and naphthalene substituted examples 13, 18, 21, 22 and 23 exhibited antimalarial activity (IC(50) <10 μM) with selectivity indices (SI) >10. Δ(7) Dihydro analogues were typically less active or lacked selectivity. Head-to-head and head-to-tail photodimers 5 and 34 exhibited moderate IC(50)s of 2.3 to 17 μM towards several of the parasitic organisms, while the aniba-dimer-type asymmetric dimers 31 and 33 were identified as being moderately active towards P. falciparum (IC(50) 1.5 and 1.7 μM) with good selectivity (SI ~80). The 4-tert-butyl aniba-dimer A analogue 33 also exhibited activity towards L. donovani (IC(50) 4.5 μM), suggesting further elaboration of this latter scaffold could lead to the identification of new leads for the dual treatment of malaria and leishmaniasis.     

Diabetic β-Cells Can Achieve Self-Protection against Oxidative Stress through an Adaptive Up-Regulation of Their Antioxidant Defenses

Background

Oxidative stress (OS), through excessive and/or chronic reactive oxygen species (ROS), is a mediator of diabetes-related damages in various tissues including pancreatic β-cells. Here, we have evaluated islet OS status and β-cell response to ROS using the GK/Par rat as a model of type 2 diabetes.

Methodology/Principal Findings

Localization of OS markers was performed on whole pancreases. Using islets isolated from 7-day-old or 2.5-month-old male GK/Par and Wistar control rats, 1) gene expression was analyzed by qRT-PCR; 2) insulin secretion rate was measured; 3) ROS accumulation and mitochondrial polarization were assessed by fluorescence methods; 4) antioxidant contents were quantified by HPLC. After diabetes onset, OS markers targeted mostly peri-islet vascular and inflammatory areas, and not islet cells. GK/Par islets revealed in fact protected against OS, because they maintained basal ROS accumulation similar or even lower than Wistar islets. Remarkably, GK/Par insulin secretion also exhibited strong resistance to the toxic effect of exogenous H₂O₂ or endogenous ROS exposure. Such adaptation was associated to both high glutathione content and overexpression (mRNA and/or protein levels) of a large set of genes encoding antioxidant proteins as well as UCP2. Finally, we showed that such a phenotype was not innate but spontaneously acquired after diabetes onset, as the result of an adaptive response to the diabetic environment.

Conclusions

The GK/Par model illustrates the effectiveness of adaptive response to OS by β-cells to achieve self-tolerance. It remains to be determined to what extend such islet antioxidant defenses upregulation might contribute to GK/Par β-cell secretory dysfunction.