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41.
Manuela Trabi Marianne D. Keller Nicholas N. Jonsson 《Metabolomics : Official journal of the Metabolomic Society》2013,9(5):1041-1047
Freezers in research institutions often contain a plethora of samples left over from studies performed years or even decades ago. Along with samples stored in biobanks, these could prove to be treasure troves for metabonomic research. Although the influence of sample handling and short to medium term storage on conventionally determined blood parameters has been reported, little is known about the effects of long term storage (years to decades) on plasma samples. The aim of this study was to investigate the influence of long term storage on the metabolite profile and to assess the value of archived samples for metabonomic studies. Heparinised plasma of 22 heifers that had been stored at ?20 °C for between 2 and 15 years was analysed using NMR spectroscopy and statistical analysis techniques. Lactate (principal component 1) explained 79.6 % of variance between all spectra, but was not correlated with storage time. The highest correlation with storage time (R 2 = 0.474) was found for betaine, with other metabolites (acetoacetate, histidines, glycerol, lipids and glucose) also showing moderate correlation (R 2 values between 0.217 and 0.437). Our results indicate that samples stored for extended periods of time can potentially be used in metabonomics studies, if precautions are taken during data analysis. 相似文献
42.
FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy
Christine L. Kragh Gwena?lle Fillon Amanda Gysbers Hanne D. Hansen Manuela Neumann Christiane Richter-Landsberg Christian Haass Bernard Zalc Catherine Lubetzki Wei-Ping Gai Glenda M. Halliday Philipp J. Kahle Poul H. Jensen 《PloS one》2013,8(1)
Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention. 相似文献
43.
Manuela Polimeni Elena Valente Daniela Ulliers Ghislain Opdenakker Philippe E. Van den Steen Giuliana Giribaldi Mauro Prato 《PloS one》2013,8(8)
Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis. 相似文献
44.
Brian Ponnaiya Sally A. Amundson Shanaz A. Ghandhi Lubomir B. Smilenov Charles R. Geard Manuela Buonanno David J. Brenner 《Radiation and environmental biophysics》2013,52(4):523-530
While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses. 相似文献
45.
46.
Cláudia Marques Diana Teixeira Ana Cunha Manuela Meireles Diogo Pestana Elisa Keating Conceição Calhau Rosário Monteiro Ana Faria 《Cell biology and toxicology》2013,29(4):293-302
Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and 3H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 μM). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. 3H-Deoxyglucose (3H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and 3H-thymidine incorporation (P?<?0.05). MTX (0.1 μM) treatment increased lipid accumulation and basal 3H-DG uptake by adipocytes (P?<?0.05). In 0.1 μM MTX-treated adipocytes, insulin stimulation did not result in an increase of 3H-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX. 相似文献
47.
48.
Manuela Gellert Simone Venz Jessica Mitl?hner Catherine Cott Eva-Maria Hanschmann Christopher Horst Lillig 《The Journal of biological chemistry》2013,288(49):35117-35125
Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development. 相似文献
49.
Ellen Preuss Manuela Hugle Romy Reimann Marcel Schlecht Simone Fulda 《The Journal of biological chemistry》2013,288(49):35287-35296
The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS. 相似文献
50.
Cleoni Alves Mendes de Lima Harrison Magdinier Gomes Maraníbia Aparecida Cardoso Oelemann Jesus Pais Ramos Paulo Cezar Caldas Carlos Eduardo Dias Campos Márcia Aparecida da Silva Pereira Fátima Fandinho Onofre Montes Maria do Socorro Calixto de Oliveira Philip Noel Suffys Maria Manuela da Fonseca Moura 《Memórias do Instituto Oswaldo Cruz》2013,108(4):457-462
The main cause of pulmonary tuberculosis (TB) is infection with
Mycobacterium tuberculosis (MTB). We aimed to evaluate the
contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in
patients from the state of Rondônia using respiratory samples and
epidemiological data from TB cases. Mycobacterium isolates were identified using
a combination of conventional tests, polymerase chain reaction-based restriction
enzyme analysis of hsp65 gene and hsp65 gene
sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded
bacterial cultures, including 369 cases positive for MTB and 75 cases positive
for NTM. Within the latter group, 14 species were identified as
Mycobacterium abscessus, Mycobacterium
avium, Mycobacterium fortuitum,
Mycobacterium intracellulare, Mycobacterium
gilvum, Mycobacterium gordonae,
Mycobacterium asiaticum, Mycobacterium
tusciae, Mycobacterium porcinum,
Mycobacterium novocastrense, Mycobacterium
simiae, Mycobacterium szulgai,
Mycobacterium phlei and Mycobacterium
holsaticum and 13 isolates could not be identified at the species
level. The majority of NTM cases were observed in Porto Velho and the relative
frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately
half of the TB subjects with NTM, a second sample containing NTM was obtained,
confirming this as the disease-causing agent. The most frequently observed NTM
species were M. abscessus and M. avium and
because the former species is resistant to many antibiotics and displays
unsatisfactory cure rates, the implementation of rapid identification of
mycobacterium species is of considerable importance. 相似文献