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51.
Mercedes Rodriguez-Teja Claudia Breit Mitchell Clarke Kamil Talar Kai Wang Mohammad A. Mohammad Sage Pickwell Guillermina Etchandy Graeme J. Stasiuk Justin Sturge 《Journal of visualized experiments : JoVE》2016,(115)
Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed. 相似文献
52.
Grier JD Xiong S Elizondo-Fraire AC Parant JM Lozano G 《Molecular and cellular biology》2006,26(1):192-198
The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development. 相似文献
53.
An ER‐resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse
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Guillermina Hernando Christian Schultheis Diego Rayes Jie Pan Thorsten Schedletzky Harald Hutter Cecilia Bouzat Alexander Gottschalk 《The EMBO journal》2009,28(17):2636-2649
Nicotinic acetylcholine receptors (nAChRs) are homo‐ or heteropentameric ligand‐gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell‐surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA‐2 or NRA‐4 (n icotinic r eceptor a ssociated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype‐specific agonists of these nAChRs is altered differently, as demonstrated by whole‐cell voltage‐clamp of dissected adult muscle, when applying exogenous agonists or after photo‐evoked, channelrhodopsin‐2 (ChR2) mediated acetylcholine (ACh) release, as well as in single‐channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra‐2 mutants. Cell‐surface expression of different subunits of the ‘levamisole‐sensitive’ nAChR (L‐AChR) is differentially affected in the absence of NRA‐2 or NRA‐4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. 相似文献
54.
Bortolotti A Pérez-Dorado I Goñi G Medina M Hermoso JA Carrillo N Cortez N 《Biochimica et biophysica acta》2009,1794(2):199-210
Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR. 相似文献
55.
56.
Daniel Larocque Gabriela Fragoso Jinghan Huang Walter E. Mushynski Martin Loignon Stéphane Richard Guillermina Almazan 《PloS one》2009,4(6)
Background
The quaking viable (qkv) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qkv mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.Methodology/Principal Findings
To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27KIP1 and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Conclusions/Significance
Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination. 相似文献57.
Occurrence, fate, and biodegradation of estrogens in sewage and manure 总被引:10,自引:0,他引:10
Sarah Combalbert Guillermina Hernandez-Raquet 《Applied microbiology and biotechnology》2010,86(6):1671-1692
The estrogens estrone (E1), 17α-estradiol (E2α), 17β-estradiol (E2β), and estriol (E3) are natural sex hormones produced by
humans and animals. In addition, there are some synthetic estrogens, such as 17α-ethinylestradiol (EE2), used for contraception
purposes. These compounds are able to produce endocrine disruption in living organisms at nanogram-per-liter levels. In both
humans and animals, estrogens are excreted in urine and feces, reaching the natural environment through discharge from sewage
treatment plants (STP) and manure disposal units. In STPs, hormone removal depends on the type of treatment process and on
different parameters such as the hydraulic and sludge retention times. Thus, hormone elimination rates vary from 0% to 90%
in different STPs. Animals are also an important source of estrogens in the environment. Indeed, animals produce high concentrations
of hormones which will end up in manure which is typically spread on land. Hence, waste-borne animal hormones may transfer
these pollutants to the soil. The purpose of this review is to highlight the significance for both health and the environment
of pollution by estrogens and critically review the existing knowledge on their fate and removal in different treatment processes.
Relevant information on the microbial degradation of hormones and metabolic pathways is also included. 相似文献
58.
Evgueni Doukhanine Christina Gavino Jeffery D. Haines Guillermina Almazan Stéphane Richard 《Molecular biology of the cell》2010,21(17):3029-3040
The quaking viable (qkv) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qkv mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3′-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qkv/qkv mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1. 相似文献
59.
Katherine Standish Guillermina Kuan William Avilés Angel Balmaseda Eva Harris 《PLoS neglected tropical diseases》2010,4(3)
Dengue is a major public health problem in tropical and subtropical regions; however, under-reporting of cases to national surveillance systems hinders accurate knowledge of disease burden and costs. Laboratory-confirmed dengue cases identified through the Nicaraguan Pediatric Dengue Cohort Study (PDCS) were compared to those reported from other health facilities in Managua to the National Epidemiologic Surveillance (NES) program of the Nicaraguan Ministry of Health. Compared to reporting among similar pediatric populations in Managua, the PDCS identified 14 to 28 (average 21.3) times more dengue cases each year per 100,000 persons than were reported to the NES. Applying these annual expansion factors to national-level data, we estimate that the incidence of confirmed pediatric dengue throughout Nicaragua ranged from 300 to 1000 cases per 100,000 persons. We have estimated a much higher incidence of dengue than reported by the Ministry of Health. A country-specific expansion factor for dengue that allows for a more accurate estimate of incidence may aid governments and other institutions calculating disease burden, costs, resource needs for prevention and treatment, and the economic benefits of drug and vaccine development. 相似文献
60.