The inflammatory response in the MPTP model of Parkinson's disease is mediated by brain angiotensin: relevance to progression of the disease

The neurotoxin MPTP reproduces most of the biochemical and pathological hallmarks of Parkinson's disease. In addition to reactive oxygen species (ROS) generated as a consequence of mitochondrial complex I inhibition, microglial NADPH-derived ROS play major roles in the toxicity of MPTP. However, the exact mechanism regulating this microglial response remains to be clarified. The peptide angiotensin II (AII), via type 1 receptors (AT1), is one of the most important inflammation and oxidative stress inducers, and produces ROS by activation of the NADPH-oxidase complex. Brain possesses a local angiotensin system, which modulates striatal dopamine (DA) release. However, it is not known if AII plays a major role in microglia-derived oxidative stress and DA degeneration. The present study indicates that in primary mesencephalic cultures, DA degeneration induced by the neurotoxin MPTP/MPP⁺ is amplified by AII and inhibited by AT1 receptor antagonists, and that protein kinase C, NADPH-complex activation and microglial activation are involved in this effect. In mice, AT1 receptor antagonists inhibited both DA degeneration and early microglial and NADPH activation. The brain angiotensin system may play a key role in the self-propelling mechanism of Parkinson's disease and constitutes an unexplored target for neuroprotection, as previously reported for vascular diseases.     

Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

Background

Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.

Methodology

Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.

Conclusions

The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.     

Gene Dosage Effects of the Imprinted Delta-Like Homologue 1 (Dlk1/Pref1) in Development: Implications for the Evolution of Imprinting

Genomic imprinting is a normal process that causes genes to be expressed according to parental origin. The selective advantage conferred by imprinting is not understood but is hypothesised to act on dosage-critical genes. Here, we report a unique model in which the consequences of a single, double, and triple dosage of the imprinted Dlk1/Pref1, normally repressed on the maternally inherited chromosome, can be assessed in the growing embryo. BAC-transgenic mice were generated that over-express Dlk1 from endogenous regulators at all sites of embryonic activity. Triple dosage causes lethality associated with major organ abnormalities. Embryos expressing a double dose of Dlk1, recapitulating loss of imprinting, are growth enhanced but fail to thrive in early life, despite the early growth advantage. Thus, any benefit conferred by increased embryonic size is offset by postnatal lethality. We propose a negative correlation between gene dosage and survival that fixes an upper limit on growth promotion by Dlk1, and we hypothesize that trade-off between growth and lethality might have driven imprinting at this locus.     

Effects of caloric restriction and gender on rat serum paraoxonase 1 activity

Paraoxonase 1 (PON1) associates to specific high-density lipoproteins (HDLs)--those containing apolipoprotein A-I (apoA-I) and apolipoprotein J (apoJ)--and is largely responsible for their antiatherogenic properties. Caloric restriction (CR) has been shown to reduce major atherosclerotic risk factors. The aims of this work were to study PON1 activity response to CR (40% over 14 weeks) and to elucidate whether there are adaptive differences related to gender. Serum and liver paraoxonase and arylesterase activities, serum triglyceride, total and HDL cholesterol concentrations, serum PON1, apoA-I and apoJ contents and liver PON1 mRNA levels were measured. No effects of CR or gender were observed in triglyceride, total cholesterol concentration and PON1 mRNA levels. HDL cholesterol was higher in female rats than in male rats and increased with CR only in the latter animals. Serum PON1 activities tended to be higher in female rats and dropped with CR, with females showing the biggest decrease. Serum PON1 content was higher in female rats and decreased in both genders with CR, whereas apoA-I and apoJ contents, which were higher in female rats too, decreased only in the former animals, accounting for the high PON1 activity decrease observed in these animals. In conclusion, the short-term CR-associated reduction of serum PON1 activity and PON1, apoA-I and apoJ levels points toward a reduced stability of HDL-PON1 complexes and/or HDL particle levels responsible for PON1 transport and function in the blood. Moreover, the variations in PON1 activity and apolipoprotein levels show gender-related differences that are indicative of a different adaptive strategy of male and female rats when faced with a period of food restriction.     

Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

Background

Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells.

Methodology/Principal Findings

Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras^−/−,N-ras^−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras^−/−) embrionary fibroblasts by mating of K-ras^+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras^−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras^−/− fibroblasts.

Conclusions/Significance

We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations.     

Mouse Bone Marrow-Derived Mesenchymal Stromal Cells Turn Activated Macrophages into a Regulatory-Like Profile

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-α, IL-6, IL-12p70 and interferon-γ while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE₂ we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-α and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia^b and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.     

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.     

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.