32 research outputs found
How melatonin interacts with lipid bilayers: a study by fluorescence and ESR spectroscopies
AbstractESR spectra of spin labels placed at the membrane surface and at different depths of the bilayer core, and melatonin fluorescence in the presence of lipid vesicles, suggest an average shallow position for the hormone in the membrane. However, according to the melatonin ability to cross lipid bilayers, nitroxides placed deep in the bilayer were able to quench the melatonin fluorescence. Melatonin membrane partition coefficients were calculated for bilayers in different packing states, and similar and rather high values were found. The data presented here may be quite important to the understanding of melatonin physiological actions at the membrane level
Pioglitazone Treatment Increases Survival and Prevents Body Weight Loss in Tumor-Bearing Animals: Possible Anti-Cachectic Effect
Cachexia is a multifactorial syndrome characterized by profound involuntary weight loss, fat depletion, skeletal muscle wasting, and asthenia; all symptoms are not entirely attributable to inadequate nutritional intake. Adipose tissue and skeletal muscle loss during cancer cachexia development has been described systematically. the former was proposed to precede and be more rapid than the latter, which presents a means for the early detection of cachexia in cancer patients. Recently, pioglitazone (PGZ) was proposed to exhibit anticancer properties, including a reduction in insulin resistance and adipose tissue loss; nevertheless, few studies have evaluated its effect on survival. for greater insight into a potential anti-cachectic effect due to PGZ, 8-week-old male Wistar rats were subcutaneously inoculated with 1 mL (2x10(7)) of Walker 256 tumor cells. the animals were randomly assigned to two experimental groups: TC (tumor + saline-control) and TP5 (tumor + PGZ/5 mg). Body weight, food ingestion and tumor growth were measured at baseline and after removal of tumor on days 7, 14 and 26. Samples from different visceral adipose tissue (AT) depots were collected on days 7 and 14 and stored at -80oC (5 to 7 animals per day/group). the PGZ treatment showed an increase in the survival average of 27.3%(P<0.01) when compared to TC. It was also associated with enhanced body mass preservation (40.7 and 56.3%, p<0.01) on day 14 and 26 compared with the TC group. the treatment also reduced the final tumor mass (53.4%, p<0.05) and anorexia compared with the TC group during late-stage cachexia. the retroperitoneal AT (RPAT) mass was preserved on day 7 compared with the TC group during the same experimental period. Such effect also demonstrates inverse relationship with tumor growth, on day 14. Gene expression of PPAR-gamma, adiponectin, LPL and C/EBP-alpha from cachectic rats was upregulated after PGZ. Glucose uptake from adipocyte cells (RPAT) was entirely re-established due to PGZ treatment. Taken together, the results demonstrate beneficial effects of PGZ treatment at both the early and final stages of cachexia.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Mogi das Cruzes, Integrated Grp Biotechnol, Lab Adipose Tissue Biol, Mogi Das Cruzes, BrazilUniv São Paulo, Inst Biomed Sci, Canc Metab Res Grp, São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Physiol Lab, São Paulo, BrazilUniv Estadual Maringa, Dept Physiol Sci, Maringa, Parana, BrazilUniversidade Federal de São Paulo, Dept Biomed Engn, Sao Jose Dos Campos, BrazilBoston Sch Med, Dept Biochem, Boston, MA USAUniversidade Federal de São Paulo, Dept Biomed Engn, Sao Jose Dos Campos, BrazilFAPESP: 2010/51078-1FAPESP: 2008/54091-9FAPESP: 2012/51094-1Web of Scienc
Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase
Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs
Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples
Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts
How melatonin interacts with lipid bilayers: a study by fluorescence and ESR spectroscopies
The effect of heparin on steady-state FRET-peptide hydrolysis by HNE.
<p>The influence of heparin concentration upon steady-state HNE kinetics parameters <i>k</i><sub>cat</sub> [A] and <i>K</i><sub>M</sub> [B] was determined spectrofluometricallyas described under “Experimental Procedures”. The fluorescence increase of FRET-peptide hydrolysis (0.2 - 10 µM) by 12.6 nM HNE were performed in 10 mM Hepes, pH 7.4, containing 140 mM NaCl and 0,05% Triton X-100 at 37°C.</p
Steady-state kinetic parameters for hydrolysis of fluorogenic substrates by human neutrophil elastase in the presence of heparin.
a<p>Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021525#pone.0021525-Spencer1" target="_blank">[4]</a>.</p