|Immunogen||Highly purified whole mouse IgG.|
|Specificity||Recognizes mouse IgG, both heavy and light chains.|
|Antibody Type||Polyclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20ºC from date of receipt.|
|Material Size||500 µg|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 48 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries. |
Tung, CP; Chen, IC; Yu, CM; Peng, HP; Jian, JW; Ma, SH; Lee, YC; Jan, JT; Yang, AS
Scientific reports 5 15053 2015
Broadly neutralizing antibodies developed from the IGHV1-69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.
|E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3. |
Chesarino, NM; McMichael, TM; Yount, JS
PLoS pathogens 11 e1005095 2015
Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.
|Mesenchymal stem cells in rabbit meniscus and bone marrow exhibit a similar feature but a heterogeneous multi-differentiation potential: superiority of meniscus as a cell source for meniscus repair. |
Ding, Z; Huang, H
BMC musculoskeletal disorders 16 65 2015
The restoration of damaged meniscus has always been a challenge due to its limited healing capacity. Recently, bone marrow-derived mesenchymal stem cells (BMSCs) provide a promising alternative to repair meniscal defects. However, BMSCs are not ideal chondroprogenitor cells for meniscus repair because they have a high propensity for cartilage hypertrophy and bone formation. Our hypothesis is that mesenchymal stem cells (MSCs) reside in meniscus maintain specific traits distinct from others which may be more conducive to meniscus regeneration.MSCs were isolated from bone marrow and menisci of the rabbits. The similarities and differences between BMSCs and MMSCs were investigated in vitro by a cell culture model, ex vivo by a rabbit meniscus defect model and in vivo by a nude rat implantation model using histochemistry, immunocytochemistry, qRT-PCR and western blotting.Our data showed that two types of MSCs have universal stem cell characteristics including clonogenicity, multi-potency and self-renewal capacity. They both express stem cell markers including SSEA-4, Nanog, nucleostemin, strol-1, CD44 and CD90. However, MMSCs differed from BMSCs. MMSC colonies were much smaller and grew more slowly than BMSC colonies. Moreover, fewer MMSCs expressed CD34 than BMSCs. Finally, MMSCs always appeared a pronounced tendency to chondrogenic differentiation while BMSCs exhibited significantly greater osteogenic potential, whatever in vitro and in vivo.This study shows the similarities and differences between MMSCs and BMSCs for the first time. MMSCs are a promising source of mesenchymal stem cells in repairing meniscus defect.
|Mutations in MAB21L2 result in ocular Coloboma, microcornea and cataracts. |
Deml, B; Kariminejad, A; Borujerdi, RH; Muheisen, S; Reis, LM; Semina, EV
PLoS genetics 11 e1005002 2015
Ocular coloboma results from abnormal embryonic development and is often associated with additional ocular and systemic features. Coloboma is a highly heterogeneous disorder with many cases remaining unexplained. Whole exome sequencing from two cousins affected with dominant coloboma with microcornea, cataracts, and skeletal dysplasia identified a novel heterozygous allele in MAB21L2, c.151 Cgreater than G, p.(Arg51Gly); the mutation was present in all five family members with the disease and appeared de novo in the first affected generation of the three-generational pedigree. MAB21L2 encodes a protein similar to C. elegans mab-21 cell fate-determining factor; the molecular function of MAB21L2 is largely unknown. To further evaluate the role of MAB21L2, zebrafish mutants carrying a p.(Gln48Serfs*5) frameshift truncation (mab21l2Q48Sfs*5) and a p.(Arg51_Phe52del) in-frame deletion (mab21l2R51_F52del) were developed with TALEN technology. Homozygous zebrafish embryos from both lines developed variable lens and coloboma phenotypes: mab21l2Q48Sfs*5 embryos demonstrated severe lens and retinal defects with complete lethality while mab21l2R51_F52del mutants displayed a milder lens phenotype and severe coloboma with a small number of fish surviving to adulthood. Protein studies showed decreased stability for the human p.(Arg51Gly) and zebrafish p.(Arg51_Phe52del) mutant proteins and predicted a complete loss-of-function for the zebrafish p.(Gln48Serfs*5) frameshift truncation. Additionally, in contrast to wild-type human MAB21L2 transcript, mutant p.(Arg51Gly) mRNA failed to efficiently rescue the ocular phenotype when injected into mab21l2Q48Sfs*5 embryos, suggesting this allele is functionally deficient. Histology, immunohistochemistry, and in situ hybridization experiments identified retinal invagination defects, an increase in cell death, abnormal proliferation patterns, and altered expression of several ocular markers in the mab21l2 mutants. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma and highlight the power of genome editing manipulation in model organisms for analysis of the effects of whole exome variation in humans.
|Restraint of the G2/M transition by the SR/RRM family mRNA shuttling binding protein SNXAHRB1 in Aspergillus nidulans. |
James, SW; Banta, T; Barra, J; Ciraku, L; Coile, C; Cuda, Z; Day, R; Dixit, C; Eastlack, S; Giang, A; Goode, J; Guice, A; Huff, Y; Humbert, S; Kelliher, C; Kobie, J; Kohlbrenner, E; Mwambutsa, F; Orzechowski, A; Shingler, K; Spell, C; Anglin, SL
Genetics 198 617-33 2014
Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2(cdc2) that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2(cdc) (2), nimE6(cyclinB), and nimT23(cdc) (25), but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10(cyclinB) mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA(Hrb) (1) is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA(HRB1) is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2(CDC2)/NIME(CYCLINB) kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIME(CYCLINB) at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway.
|Novel autoimmune response in a tauopathy mouse model. |
Nogueras-Ortiz, CJ; De Jesús-Cortes, HJ; Vaquer-Alicea, J; Vega, IE
Frontiers in neuroscience 7 277 2014
Molecular diagnostic tools with non-invasive properties that allow detection of pathological events in Alzheimer's disease (AD) and other neurodegenerative tauopathies are essential for the development of therapeutics. Several diagnostic strategies based on the identification of biomarkers have been proposed. However, its specificity among neurodegenerative disorders is disputable as the association with pathological events remains elusive. Recently, we showed that Amphiphysin-1 (AMPH1) protein's abundance is reduced in the central nervous system (CNS) of the tauopathy mouse model JNPL3 and AD brains. AMPH1 is a synaptic protein that plays an important role in clathrin-mediated endocytosis and associates with BIN1, one of the most important risk loci for AD. Also, it has been associated with a rare neurological disease known as Stiff-Person Syndrome (SPS). Auto-antibodies against AMPH1 are used as diagnostic biomarkers for a paraneoplastic variant of SPS. Therefore, we set up to evaluate the presence and abundance of auto-AMPH1 antibodies in tau-mediated neurodegeneration. Immunoblots and enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of auto-AMPH1 antibodies in sera from euthanized mice that developed neurodegeneration (JNPL3) and healthy control mice (NTg). Results showed increased levels of auto-AMPH1 antibodies in JNPL3 sera compared to NTg controls. The abundance of auto-AMPH1 antibodies correlated with motor impairment and AMPH1 protein level decrease in the CNS. The results suggest that auto-AMPH1 antibodies could serve as a biomarker for the progression of tau-mediated neurodegeneration in JNPL3 mice.
|An ITAM in a nonenveloped virus regulates activation of NF-κB, induction of beta interferon, and viral spread. |
Stebbing, RE; Irvin, SC; Rivera-Serrano, EE; Boehme, KW; Ikizler, M; Yoder, JA; Dermody, TS; Sherry, B
Journal of virology 88 2572-83 2014
Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: μ2, σ2, and λ2. We demonstrate for the first time that μ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, μ2 and μNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the μ2 ITAM. Moreover, both the μ2 ITAM and Syk are required for maximal μ2 activation of NF-κB. A mutant virus lacking the μ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-β) than does wild-type virus despite similar replication. Notably, the consequences of these μ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the μ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the μ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-β. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.
|High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells. |
Chiu, HC; Hannemann, H; Heesom, KJ; Matthews, DA; Davidson, AD
PloS one 9 e93305 2014
Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.
|Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations. |
Turner, J; Quek, LE; Titmarsh, D; Krömer, JO; Kao, LP; Nielsen, L; Wolvetang, E; Cooper-White, J
PloS one 9 e112757 2014
As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion.
|Abelson interactor 1 (ABI1) and its interaction with Wiskott-Aldrich syndrome protein (wasp) are critical for proper eye formation in Xenopus embryos. |
Singh, A; Winterbottom, EF; Ji, YJ; Hwang, YS; Daar, IO
The Journal of biological chemistry 288 14135-46 2013
Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.
|The dynamics of connexin expression, degradation and localisation are regulated by gonadotropins during the early stages of in vitro maturation of swine oocytes. |
Santiquet, N; Robert, C; Richard, FJ
PloS one 8 e68456 2013
Gap junctional communication (GJC) plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx), proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.
|Rev3, the catalytic subunit of Polζ, is required for maintaining fragile site stability in human cells. |
Bhat, A; Andersen, PL; Qin, Z; Xiao, W
Nucleic acids research 41 2328-39 2013
It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G(2)/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G(2)/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.
|Aerobic exercise training induces metabolic benefits in rats with metabolic syndrome independent of dietary changes. |
Caponi, PW; Lehnen, AM; Pinto, GH; Borges, J; Markoski, M; Machado, UF; Schaan, BD
Clinics (São Paulo, Brazil) 68 1010-7 2013
We evaluated the effects of aerobic exercise training without dietary changes on cardiovascular and metabolic variables and on the expression of glucose transporter Type 4 in rats with metabolic syndrome.Twenty male spontaneously hypertensive rats received monosodium glutamate during the neonatal period. The animals were allocated to the following groups: MS (sedentary metabolic syndrome), MS-T (trained on a treadmill for 1 hour/day, 5 days/week for 10 weeks), H (sedentary spontaneously hypertensive rats) and H-T (trained spontaneously hypertensive rats). The Lee index, blood pressure (tail-cuff system), insulin sensitivity (insulin tolerance test) and functional capacity were evaluated before and after 10 weeks of training. Glucose transporter Type 4 expression was analyzed using Western blotting. The data were compared using analysis of variance (ANOVA) (pless than 0.05).At baseline, the MS rats exhibited lower insulin sensitivity and increased Lee index compared with the H rats. Training decreased the body weight and Lee index of the MS rats (MS-T vs. MS), but not of the H rats (H-T vs. H). There were no differences in food intake between the groups. At the end of the experiments, the systolic blood pressure was lower in the two trained groups than in their sedentary controls. Whole-body insulin sensitivity increased in the trained groups. Glucose transporter Type 4 content increased in the heart, white adipose tissue and gastrocnemius muscle of the trained groups relative to their respective untrained groups.In conclusion, the present study shows that an isolated aerobic exercise training intervention is an efficient means of improving several components of metabolic syndrome, that is, training reduces obesity and hypertension and increases insulin sensitivity.
|A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats. |
Xiao, Y; Sharma, N; Arias, EB; Castorena, CM; Cartee, GD
Age (Dordrecht, Netherlands) 35 573-82 2013
Exercise has been demonstrated to enhance subsequent insulin-stimulated glucose uptake (GU) by predominantly type II (fast-twitch) muscle of old rats, but previous research has not evaluated exercise effects on GU by type I (slow-twitch) muscle from old rats. Accordingly, we studied male Fischer 344/Brown Norway rats (24 months old) and determined GU (0, 100, 200, and 5,000 μU/ml insulin) of isolated soleus (predominantly type I) and epitrochlearis (predominantly type II) muscles after one exercise session. Epitrochlearis (100, 200, and 5,000 μU/ml insulin) and soleus (100 and 200 μU/ml insulin) GU were greater at 3-h postexercise vs. age-matched sedentary controls. Insulin receptor tyrosine phosphorylation (Tyr1162/1163) was unaltered by exercise in either muscle. Akt phosphorylation (pAkt) was greater for exercised vs. sedentary rats in the epitrochlearis (Ser473 and Thr308 with 100 and 200 μU/ml, respectively) and soleus (Ser473 with 200 μU/ml). AS160 phosphorylation (pAS160) was greater for exercised vs. sedentary rats in the epitrochlearis (Thr642 with 100 μU/ml), but not the soleus. Exercised vs. sedentary rats did not differ for total protein abundance of insulin receptor, Akt, AS160, or GLUT4 in either muscle. These results demonstrate that both predominantly type I and type II muscles from old rats are susceptible to exercise-induced improvement in insulin-mediated GU by mechanisms that are independent of enhanced insulin receptor tyrosine phosphorylation or altered abundance of important signaling proteins or GLUT4. Exercise-induced elevation in pAkt, and possibly pAS160, may contribute to this effect in the epitrochlearis of old rats, but other mechanisms are likely important for the soleus.
|The SIRT1 modulators AROS and DBC1 regulate HSF1 activity and the heat shock response. |
Raynes, R; Pombier, KM; Nguyen, K; Brunquell, J; Mendez, JE; Westerheide, SD
PloS one 8 e54364 2013
The heat shock response, the cellular response to protein damaging stress, is critical in maintaining proteostasis. The heat shock response is regulated by the transcription factor HSF1, which is activated upon heat shock and other stresses to induce the expression of molecular chaperones. SIRT1 has previously been shown to activate HSF1 by deacetylating it, leading to increased DNA binding ability. We have investigated how the heat shock response may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD(+)/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators AROS and DBC1 have an impact on hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. Therefore, AROS and DBC1 are now two new targets available for therapeutic regulation of the heat shock response.
|Gpr125 modulates Dishevelled distribution and planar cell polarity signaling. |
Li, X; Roszko, I; Sepich, DS; Ni, M; Hamm, HE; Marlow, FL; Solnica-Krezel, L
Development (Cambridge, England) 140 3028-39 2013
During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.
|An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain. |
Kopanic, JL; Al-Mugotir, M; Zach, S; Das, S; Grosely, R; Sorgen, PL
Frontiers in pharmacology 4 106 2013
A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.
|Increased copper levels in in vitro and in vivo models of Niemann-Pick C disease. |
Mary Carmen Vázquez,Pablo Martínez,Alejandra R Alvarez,Mauricio González,Silvana Zanlungo
Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 25 2012
Niemann-Pick type C disease (NPC) is a hereditary neurovisceral atypical lipid storage disorder produced by mutations in the NPC1 and NPC2 genes. The disease is characterized by unesterified cholesterol accumulation in late endosomal/lysosomal compartments and oxidative stress. The most affected tissues are the cerebellum and the liver. The lysotropic drug U18666A (U18) has been widely used as a pharmacological model to induce the NPC phenotype in several cell culture lines. It has already been reported that there is an increase in copper content in hepatoma Hu7 cells treated with U18. We confirmed this result with another human hepatoma cell line, HepG2, treated with U18 and supplemented with copper in the media. However, in mouse hippocampal primary cultures treated under similar conditions, we did not find alterations in copper content. We previously reported increased copper content in the liver of Npc1 (-/-) mice compared to control animals. Here, we extended the analysis to the copper content in the cerebella, the plasma and the bile of NPC1 deficient mice. We did not observe a significant change in copper content in the cerebella, whereas we found increased copper content in the plasma and decreased copper levels in the bile of Npc1(-/-) mice. Finally, we also evaluated the plasma content of ceruloplasmin, and we found an increase in this primary copper-binding protein in Npc1 (-/-) mice. These results indicate cell-type dependence of copper accumulation in NPC disease and suggest that copper transport imbalance may be relevant to the liver pathology observed in NPC disease.
|p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease. |
Li, KL; Chen, J; Li, ZH; Zhao, L; He, YN
Cardiovascular journal of Africa 23 e1-9 2012
To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53-/- mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 57/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53-/- mice was more severe (p less than 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p less than 0.001), while expression of the marker of VSMCs had decreased (p less than 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p less than 0.001).p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.
|Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis. |
De Semir, D; Nosrati, M; Bezrookove, V; Dar, AA; Federman, S; Bienvenu, G; Venna, S; Rangel, J; Climent, J; Meyer Tamgüney, TM; Thummala, S; Tong, S; Leong, SP; Haqq, C; Billings, P; Miller, JR; Sagebiel, RW; Debs, R; Kashani-Sabet, M
Proceedings of the National Academy of Sciences of the United States of America 109 7067-72 2012
Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.
|Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells. |
Ip, J; Canham, P; Choo, KH; Inaba, Y; Jacobs, SA; Kalitsis, P; Mattiske, DM; Ng, J; Saffery, R; Wong, NC; Wong, LH; Mann, JR
PLoS genetics 8 e1002919 2012
Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age," given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.
|Evaluation of ITX 5061, a scavenger receptor B1 antagonist: resistance selection and activity in combination with other hepatitis C virus antivirals. |
Zhu, H; Wong-Staal, F; Lee, H; Syder, A; McKelvy, J; Schooley, RT; Wyles, DL
The Journal of infectious diseases 205 656-62 2012
ITX 5061 is a scavenger receptor B1 antagonist that has entered phase 1 clinical trials in hepatitis C virus (HCV)-infected humans. We evaluated ITX 5061 in combination with interferon-α, ribavirin, and HCV protease and polymerase inhibitors in a genotype 2a infectious virus system. ITX 5061 is a potent inhibitor of HCV replication and is additive to synergistic with interferon-α, ribavirin, BILN2061, VX950, VX1, and 2'-C-methyladenosine. Resistance selection experiments were performed using a Jc1-FEO virus co-culture system and intermittent ITX 5061 exposure under neomycin selection. We identified a mutant virus with a substitution of aspartic acid for asparagine at the highly conserved position 415 in E2 (N415D). Introduction of this mutation into wild-type virus conferred high-level resistance to ITX 5061. There was no cross-resistance between ITX 5061 and HCV protease inhibitors or interferon-α. These results suggest that ITX 5061 is a promising compound for study in combination with other HCV inhibitors.
|The level and distribution of the GABA(B)R1 and GABA(B)R2 receptor subunits in the rat's inferior colliculus. |
Jamal, L; Khan, AN; Butt, S; Patel, CR; Zhang, H
Frontiers in neural circuits 6 92 2012
The type B γ-aminobutyric acid receptor (GABA(B) receptor) is an important neurotransmitter receptor in the midbrain auditory structure, the inferior colliculus (IC). A functional GABA(B) receptor is a heterodimer consisting of two subunits, GABA(B)R1 and GABA(B)R2. Western blotting and immunohistochemical experiments were conducted to examine the expression of the two subunits over the IC including its central nucleus, dorsal cortex, and external cortex (ICc, ICd, and ICx). Results revealed that the two subunits existed in both cell bodies and the neuropil throughout the IC. The two subunits had similar regional distributions over the IC. The combined level of cell body and neuropil labeling was higher in the ICd than the other two subdivisions. Labeling in the ICc and ICx was stronger in the dorsal than the ventral regions. In spite of regional differences, no defined boundaries were formed between different areas. For both subunits, the regional distribution of immunoreactivity in the neuropil was parallel to that of combined immunoreactivity in the neuropil and cell bodies. The density of labeled cell bodies tended to be higher but sizes of cell bodies tended to be smaller in the ICd than in the other subdivisions. No systematic regional changes were found in the level of cell body immunoreactivity, except that GABA(B)R2-immunoreactive cell bodies in the ICd had slightly higher optic density (OD) than in other regions. Elongated cell bodies existed throughout the IC. Many labeled cell bodies along the outline of the IC were oriented in parallel to the outline. No strong tendency of orientation was found in labeled cell bodies in ICc. Regional distributions of the subunits in ICc correlated well with inputs to this subdivision. Our finding regarding the contrast in the level of neuropil immunoreactivity among different subdivisions is consistent with the fact that the GABA(B) receptor has different pre- and postsynaptic functions in different IC regions.
|ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK. |
Gao, D; Bambang, IF; Putti, TC; Lee, YK; Richardson, DR; Zhang, D
Laboratory investigation; a journal of technical methods and pathology 92 200-13 2012
Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58(IPK). In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, β(1)-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (Pless than 0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D(2) is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58(IPK) expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58(IPK) by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58(IPK) expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58(IPK) prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58(IPK) upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.
|Discovery and verification of panels of T-lymphocyte proteins as biomarkers of Parkinson's disease. |
Alberio, T; Pippione, AC; Zibetti, M; Olgiati, S; Cecconi, D; Comi, C; Lopiano, L; Fasano, M
Scientific reports 2 953 2012
The diagnosis of Parkinson's disease (PD) is currently based on the clinical evaluation of extrapyramidal signs with a considerable error rate. The identification of specific markers might allow PD diagnosis before the onset of classical motor symptoms. By two-dimensional electrophoresis we identified proteome alterations in T-lymphocytes of 17 control subjects and 15 PD patients. The observed changes were used to build predictive models that were verified by the leave-one-out cross-validation. We further built two functions able to stage the subjects. We chose to verify by Western blotting the identity of spots corresponding to β-fibrinogen and transaldolase, two recurrent proteins in six out of 20 spots. β-Fibrinogen levels are lowered in PD patients, whereas a heavy transaldolase set of isoforms was more abundant. Eventually, we identified a list of seven proteins showing different levels in early-onset with respect to late-onset PD patients.
|Mammalian α arrestins link activated seven transmembrane receptors to Nedd4 family e3 ubiquitin ligases and interact with β arrestins. |
Shea, FF; Rowell, JL; Li, Y; Chang, TH; Alvarez, CE
PloS one 7 e50557 2012
The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.
|Identification of novel human receptor activator of nuclear factor-kB isoforms generated through alternative splicing: implications in breast cancer cell survival and migration. |
Papanastasiou, AD; Sirinian, C; Kalofonos, HP
Breast cancer research : BCR 14 R112 2012
The receptor activator of nuclear factor-kB (NF-kB) (RANK)/receptor activator of NF-kB ligand (RANKL) axis emerges as a key regulator of breast cancer initiation, progression and metastasis. RANK receptor is a tumor necrosis superfamily member, which upon ligand binding transduces a variety of survival, proliferation, differentiation and migration signals. The majority of these intracellular cues merge through the NF-kB transcription machinery.TNFRSF11A (RANK) variants were identified and cloned in mammalian expression vectors. Their expression was analyzed using real time PCR on RNA from normal tissue, cell lines and breast cancer specimens. Western blot analysis and immunofluoresence stainings were used to study expression and localization of protein isoforms in a panel of breast cancer cell lines and in transfected 293T cells. Luciferase assays were employed to assess the contribution of each isoform alone or in combinations on NF-kB activation. Isoform effect on cell survival after doxorubicin treatment was analyzed through MTT assay. Wound healing and transwell assays were employed to evaluate the effect of TNFRSF11A isoforms on migration of MDA-MB-231 and 293T cells.We report the identification of three novel TNFRSF11A (RANK) variants, named TNFRSF11A_Δ9, TNFRSF11A_Δ8,9 and TNFRSF11A_Δ7,8,9 which result from the alternative splicing of exons 7 to 9. Interestingly, variant TNFRSF11A_Δ7,8,9 was found to be upregulated in breast cancer cells lines and its expression inversely correlated with tumor grade and proliferation index. TNFRSF11A_Δ7,8,9 encodes a 40-45 kDa protein, we named RANK-c, which lacks the transmembrane domain and most of the intracellular part of the wild type receptor. Furthermore, we showed that RANK-c could act as a dominant negative regulator of RANK-dependent NF-kB activation, affecting cell survival after apoptosis induction. In addition, RANK-c suppresses cell migration and represses the tumorigenic properties of invasive breast carcinoma cells.In this study, we provide evidence of a complex regulatory network of RANK receptor splice variants with a role in breast cancer. We identify that the RANK-c isoform is expressed in breast cancer samples and its expression reversely correlates with histological grade. Finally, isoform RANK-c seems to have the capacity to regulate signaling through wild type RANK and moreover to inhibit cell motility and migration of breast cancer cells.
|Myo1c regulates glucose uptake in mouse skeletal muscle. |
Toyoda, T; An, D; Witczak, CA; Koh, HJ; Hirshman, MF; Fujii, N; Goodyear, LJ
The Journal of biological chemistry 286 4133-40 2011
Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.
|Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites. |
Andersen, PL; Xu, F; Ziola, B; McGregor, WG; Xiao, W
Molecular biology of the cell 22 2373-83 2011
In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.
|Large scale RNAi screen reveals that the inhibitor of DNA binding 2 (ID2) protein is repressed by p53 family member p63 and functions in human keratinocyte differentiation. |
Wu, N; Castel, D; Debily, MA; Vigano, MA; Alibert, O; Mantovani, R; Iljin, K; Romeo, PH; Gidrol, X
The Journal of biological chemistry 286 20870-9 2011
The inhibitor of DNA binding 2, dominant negative helix-loop-helix protein, ID2, acts as an oncogene and elevated levels of ID2 have been reported in several malignancies. Whereas some inducers of the ID2 gene have been characterized, little is known regarding the proteins capable to repress its expression. We developed siRNA microarrays to perform a large scale loss-of-function screen in human adult keratinocytes engineered to express GFP under the control of the upstream region of ID2 gene. We screened the effect of siRNA-dependent inhibition of 220 cancer-associated genes on the expression of the ID2::GFP reporter construct. Three genes NBN, RAD21, and p63 lead to a repression of ID2 promoter activity. Strikingly NBN and RAD21 are playing on major role in cell cycle progression and mitosis arrest. These results underline the pregnant need to silence ID2 expression at transcript level to promote cell cycle exit. Central to this inhibitory mechanism we find p63, a key transcription factor in epithelial development and differentiation, which binds specific cis-acting sequence within the ID2 gene promoter both in vitro and in vivo. P63 would not suppress ID2 expression, but would rather prevent excessive expression of that protein to enable the onset of keratinocyte differentiation.
|Imprinted Rasgrf1 expression in neonatal mice affects olfactory learning and memory. |
N M Drake,L M DeVito,T A Cleland,P D Soloway
Genes, brain, and behavior 10 2011
Rasgrf1 is genomically imprinted; only the paternally inherited allele is expressed in the neonatal mouse brain until weaning, at which time expression becomes biallelic. Whereas Rasgrf1 has been implicated in learning and memory via knockout studies in adult mice, the effect of its normal imprinted expression on these phenotypes has not yet been examined. Neonatal mice with experimentally manipulated patterns of imprinted Rasgrf1 expression were assessed on an associative olfactory task. Neonates lacking the normally expressed wild-type paternal allele exhibited significant impairment in olfactory associative memory. Adult animals in which neonatal imprinting had been manipulated were also behaviorally assessed; while neonatal imprinting significantly affects body weight even into adulthood, no learning and memory phenotype attributable to imprinting was observed in adults. Additional analyses of neonates showed imprinted Rasgrf1 transcript selective to olfactory bulb even in mice that were null for Rasgrf1 in the rest of the brain and showed that Rasgrf1 affects Ras and Rac activation in the brain. Taken together, these results indicate that Rasgrf1 expression from the wild-type paternal allele contributes to learning and memory in neonatal mice.
|Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats. |
Sharma, N; Arias, EB; Bhat, AD; Sequea, DA; Ho, S; Croff, KK; Sajan, MP; Farese, RV; Cartee, GD
American journal of physiology. Endocrinology and metabolism 300 E966-78 2011
Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.
|The relationship between transcription initiation RNAs and CCCTC-binding factor (CTCF) localization. |
Taft, RJ; Hawkins, PG; Mattick, JS; Morris, KV
Epigenetics & chromatin 4 13 2011
Transcription initiation RNAs (tiRNAs) are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII) transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation.In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF) binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A). Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21 (p21alt) transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis.Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization.
|Lack of evidence for the direct activation of endothelial cells by adult female and microfilarial excretory-secretory products. |
Weinkopff, T; Lammie, P
PloS one 6 e22282 2011
Lymphangiectasia (dilation of the lymphatic vessel (LV)) is pathognomonic for lymphatic filariasis. In both infected humans and animal models of infection, lymphangiectasia is not restricted to the site of the worm nest, but is found along the infected vessel. These observations argue that soluble products secreted by the worm could be mediating this effect by activating the lymphatic endothelial cells (LEC) lining the vessel. We tested the ability of filarial Excretory-Secretory products to activate LECs, but were unable to detect a direct effect of the Excretory-Secretory products on the activation of LEC as assessed by a variety of approaches including cellular proliferation, cell surface molecule expression and cytokine and growth factor production (although other mediators used as positive controls did induce these effects). Collectively, these results do not support the hypothesis that Excretory-Secretory products directly activate LECs.
|17β-Estradiol-mediated increase in Cu/Zn superoxide dismutase expression in the brain: a mechanism to protect neurons from ischemia. |
Rao, AK; Dietrich, AK; Ziegler, YS; Nardulli, AM
The Journal of steroid biochemistry and molecular biology 127 382-9 2011
A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E(2), and that the E(2)-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E(2) reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E(2) and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E(2) treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E(2)-mediated neuroprotection may be conferred.
|Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP. |
Brinkworth, AJ; Malcolm, DS; Pedrosa, AT; Roguska, K; Shahbazian, S; Graham, JE; Hayward, RD; Carabeo, RA
Molecular microbiology 82 131-44 2011
Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹⁻²⁰⁰). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹⁻²⁰⁰ complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.
|Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities. |
Pastushok, L; Hanna, M; Xiao, W
Nucleic acids research 38 5047-58 2010
In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA.Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA.Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA.Ub resistance to DNA damage, nor did PCNA.Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.Artículo Texto completo
|Beta-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA. |
Stuenaes JT, Bolling A, Ingvaldsen A, Rommundstad C, Sudar E, Lin FC, Lai YC, Jensen J
Br J Pharmacol 160 116-29 2010
BACKGROUND AND PURPOSE: Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and beta-adrenergic stimulation in regulation of PKB phosphorylation. EXPERIMENTAL APPROACH: Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies. KEY RESULTS: Isoprenaline increased insulin-stimulated PKB Ser(473) and Thr(308) phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3beta Ser(9) phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (beta(1)-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (beta(2)-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser(16) phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N(6)-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N(6)-benzoyl-cAMP, but not 007, increased phospholamban Ser(16) phosphorylation. CONCLUSIONS AND IMPLICATIONS: beta-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during beta-adrenergic receptors stimulation.
|The beneficial effects of exercise in rodents are preserved after detraining: a phenomenon unrelated to GLUT4 expression. |
Lehnen, AM; Leguisamo, NM; Pinto, GH; Markoski, MM; De Angelis, K; Machado, UF; Schaan, B
Cardiovascular diabetology 9 67 2010
Although exercise training has well-known cardiorespiratory and metabolic benefits, low compliance with exercise training programs is a fact, and the harmful effects of physical detraining regarding these adaptations usually go unnoticed. We investigated the effects of exercise detraining on blood pressure, insulin sensitivity, and GLUT4 expression in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY).Studied animals were randomized into sedentary, trained (treadmill running/5 days a week, 60 min/day for 10 weeks), 1 week of detraining, and 2 weeks of detraining. Blood pressure (tail-cuff system), insulin sensitivity (kITT), and GLUT4 (Western blot) in heart, gastrocnemius and white fat tissue were measured.Exercise training reduced blood pressure (19%), improved insulin sensitivity (24%), and increased GLUT4 in the heart (+34%); gastrocnemius (+36%) and fat (+22%) in SHR. In WKY no change in either blood pressure or insulin sensitivity were observed, but there was an increase in GLUT4 in the heart (+25%), gastrocnemius (+45%) and fat (+36%) induced by training. Both periods of detraining did not induce any change in neither blood pressure nor insulin sensitivity in SHR and WKY. One-week detraining reduced GLUT4 in SHR (heart: -28%; fat: -23%) and WKY (heart: -19%; fat: -22%); GLUT4 in the gastrocnemius was reduced after a 2-week detraining (SHR: -35%; WKY: -25%). There was a positive correlation between GLUT4 (gastrocnemius) and the maximal velocity in the exercise test (r = 0.60, p = 0.004).The study findings show that in detraining, despite reversion of the enhanced GLUT4 expression, cardiorespiratory and metabolic beneficial effects of exercise are preserved.
|Research on promoting periodontal regeneration with human basic fibroblast growth factor-modified bone marrow mesenchymal stromal cell gene therapy. |
Zhen Tan, Qing Zhao, Ping Gong, Yang Wu, Na Wei, Quan Yuan, Chuan Wang, Dapeng Liao, Hua Tang
Cytotherapy 11 317-25 2009
BACKGROUND AIMS: Recently, it has been found that effective periodontal regeneration can be induced by bone marrow mesenchymal stromal cell (BMSC) transplantation or local application of basic fibroblast growth factor (bFGF). The aim of the present study was to assess, in dogs, the efficacy of periodontal regeneration via the delivery of BMSC transfected with bFGF to repair destruction of periodontal tissue. METHODS: BMSC from dogs were isolated, cultured and purified via density-gradient centrifugation. Polymerase chain reaction (PCR) was employed to clone bFGF cDNA from human periodontal cells, and the product was then ligated into the eukaryotic expression vector pDC316-IREs-EGFP. BMSC transfected with pDC316bFGF-IREs-EGFP were transplanted into root furcation defects of beagle dogs. After 6 weeks, regeneration in defects was assessed via clinical examination, X-ray, histologic observation and micro-CT analysis. RESULTS: DNA sequence analysis showed that the bFGF sequence of recombinant plasmid pDC316bFGF-IREs-EGFP was consistent with that reported by GeneBank. bFGF expression was detected with Western blotting, and active bFGF in supernatant was also observed. Our animal experiment proved that the regenerating speed of periodontal bone tissue in groups transplanted with BMSC containing the modified bFGF gene was higher than in those transplanted with BMSC alone. CONCLUSIONS: A successfully constructed eukaryotic expression vector containing human bFGF in pDC316bFGF-IREs-EGFP could produce bioactive bFGF in vitro. bFGF overexpression mediated by the recombinant plasmid pDC316bFGF- IREs-EGFP accelerated periodontal regeneration.
|Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C. |
Gildemeister OS, Sage JM, Knight KL
The Journal of biological chemistry 284 31945-52 2009
Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51-05-contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.
|In vivo verification of protein interactions in the inner ear by coimmunoprecipitation. |
Margaret C Harvey, Bernd H A Sokolowski
Methods in molecular biology (Clifton, N.J.) 493 299-310 2009
Genomics has provided us with vast amounts of data and thus, the challenge to identify and characterize gene products. Proteomics analysis, using methods such as yeast two-hybrid screenings, isoelectric focusing, and mass spectroscopy, generate potentially useful information. To determine functional relationships between and among proteins, however, the initial data for putative protein interactions must first be validated. One technique, which is considered the gold standard, is coimmunoprecipitation.
|Inhibitory effect of the extracts from Thai medicinal plants on iNOS expression in mouse macrophage RAW 264.7. |
Tomonori Nakamura, Naoko Kodama, Takuya Kumamoto, Yoshihiro Higuchi, Chaiyo Chaichantipyuth, Koichi Ueno, Tsutomu Ishikawa, Shingo Yano
Natural medicines = Shōyakugaku zasshi / The Japanese Society of Pharmacognosy 63 107-10 2009
In the present study, we screened the inhibitory effect of the extract from 50 Thai medicinal plants on an inducible-nitric oxide synthase (iNOS) expression induced by lipopolysaccharide (LPS) in mouse macrophages RAW 264.7. From this screening, the extracts of root bark of Clausena guillauminii, C. lunulata, and C. excavata (Rutaceae) were found as the extracts which showed potent inhibitory effect on the iNOS protein expression in concentration-dependent manner. Furthermore, hydrophobic active components may exist in C. guillauminii.
|Synergy of Eed and Tsix in the repression of Xist gene and X-chromosome inactivation. |
Shibata, S; Yokota, T; Wutz, A
The EMBO journal 27 1816-26 2008
X-chromosome inactivation (XCI) depends on the noncoding Xist gene. Xist transcription is negatively regulated by its antisense partner Tsix, whose disruption results in nonrandom XCI in females. However, males can maintain Xist in a repressed state without Tsix, indicating participation of additional factor(s) in the protection of the single male X from inactivation. Here, we provide evidence that the histone methyltransferase Eed is also involved in the process. Male embryonic stem cells with Eed-null and Tsix mutations (X(Delta)Y Eed-/-) showed Xist hyperactivation upon differentiation, whereas cells with either mutation alone did not. Impaired X-linked gene expression was observed in the X(Delta)Y Eed-/- ES cells at the onset of differentiation. The Xist promoter in the X(Delta)Y Eed-/- cells showed elevated histone H3-dimethyl lysine 4 modifications and lowered CpG methylation, which are characteristics of open chromatin. Hence, we identified Eed as an additional major player in the regulation of Xist expression. The synergy of Polycomb group proteins and antisense Tsix transcription in Xist gene regulation explains why males can repress Xist without Tsix.Artículo Texto completo
|P53-Rb signaling pathway is involved in tubular cell senescence in renal ischemia/reperfusion injury. |
Li Kailong, Xiaolan Du, He Yani, Zhao Lin, Yang Jvrong, Song Ruihua, Chen Lin
Biocell : official journal of the Sociedades Latinoamericanas de Microscopía Electronica ... et. al 31 213-23 2007
OBJECTIVE: To investigate the course of tubular cell senescence and expressions of p53, p21, and Rb during the late phase of ischemia/reperfusion (IRI) in the kidney, and assess the effects of the p53-Rb pathway on tubular cell senescence. METHODS: Experimental models of unilateral renal IRI were used in p53 (+/+) and p53 (-/-) mice. Histological changes at the tubular level, progress of cell senescence, and the expression of Rb, p21, and/or p53 proteins in tubular cells were studied at different moments in time after IRI. RESULTS: Chronic tubulointerstitial fibrosis was much more severe and widely distributed in IRI kidneys of p53 (+/+) mice in later stages than in earlier stages. Senescent tubular cells were significantly increased at 3 and 6 months after IRI. In contrast, in contralateral kidneys of p53 (+/+) mice and in both kidneys of p53 (-/-) mice, almost no senescent cells were observed at 1 and 3 months after IRI, and only a few senescent cells were detected in IRI kidneys of p53 (-/-) mice at 6 months. In mice of both genotypes, cell senescence was correlated with the expression levels of p53, p21, and Rb proteins. CONCLUSION: The IRI accelerated tubular cell senescence is presumed to be one of the mechanisms of the long-term effect of IRI. Furthermore, the activation of p53-Rb signaling pathway may play a vital role in tubular cell senescence induced by IRI.
|Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene. |
Dackor, RT; Fritz-Six, K; Dunworth, WP; Gibbons, CL; Smithies, O; Caron, KM
Molecular and cellular biology 26 2511-8 2006
Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl-/- pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl-/- embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl-/- embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM-/- phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl-/- and AM-/- embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.Artículo Texto completo
|Calcineurin-mediated slow-type fiber expression and growth in reloading condition. |
Mitsunori Miyazaki, Yoshiaki Hitomi, Takako Kizaki, Hideki Ohno, Toshihito Katsumura, Shukoh Haga, Tohru Takemasa
Medicine and science in sports and exercise 38 1065-72 2006
PURPOSE: Calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes and in muscle hypertrophy. Our aim was to investigate the functional role of CaN as a regulator of muscle growth and/or muscle fiber type under conditions of recovery from inactivity. METHODS: Female ICR mice (8 wk of age, 28-32 g) were used. To examine the effects of hindlimb suspension (HS) and reloading on skeletal muscle fiber size and muscle fiber type, animals were designated to 8 wk of HS and subsequent reloading for 4 wk. During reloading, animals were treated with pharmacological inhibitors for CaN (FK506) by intraperitoneal administration (3-5 mg.kg.d). After each experimental period, antigravitational soleus muscle was analyzed. RESULTS: HS treatment resulted in obvious muscle atrophy and slow-to-fast fiber-type transformation in the soleus muscle. Subsequent reloading for 4 wk following HS induced muscle regrowth and fiber-type reversion toward a slow profile. FK506 administration prevented this kind of reloading-induced transformation of muscle fiber type. Furthermore, it was confirmed that FK506 administration attenuated maintenance of fiber cross-sectional area and reloading-induced fiber regrowth, specifically in slow-type muscle fibers. CONCLUSION: Reloading-induced fiber-type reversion toward a slow profile is prevented by the pharmacological inhibition of CaN. Additionally, inhibition of CaN prevented maintenance and regrowth of slow-type muscle fibers. These results implicate that the CaN signaling pathway is required in the slow-type muscle fiber program under maintenance and suspension-reloading conditions.
|NADPH oxidase mediates hypersomnolence and brain oxidative injury in a murine model of sleep apnea. |
Zhan, G; Serrano, F; Fenik, P; Hsu, R; Kong, L; Pratico, D; Klann, E; Veasey, SC
American journal of respiratory and critical care medicine 172 921-9 2005
Persons with obstructive sleep apnea may have significant residual hypersomnolence, despite therapy. Long-term hypoxia/reoxygenation events in adult mice, simulating oxygenation patterns of moderate-severe sleep apnea, result in lasting hypersomnolence, oxidative injury, and proinflammatory responses in wake-active brain regions. We hypothesized that long-term intermittent hypoxia activates brain NADPH oxidase and that this enzyme serves as a critical source of superoxide in the oxidation injury and in hypersomnolence.We sought to determine whether long-term hypoxia/reoxygenation events in mice result in NADPH oxidase activation and whether NADPH oxidase is essential for the proinflammatory response and hypersomnolence.NADPH oxidase gene and protein responses were measured in wake-active brain regions in wild-type mice exposed to long-term hypoxia/reoxygenation. Sleep and oxidative and proinflammatory responses were measured in adult mice either devoid of NADPH oxidase activity (gp91phox-null mice) or in which NADPH oxidase activity was systemically inhibited with apocynin osmotic pumps throughout hypoxia/reoxygenation.Long-term intermittent hypoxia increased NADPH oxidase gene and protein responses in wake-active brain regions. Both transgenic absence and pharmacologic inhibition of NADPH oxidase activity throughout long-term hypoxia/reoxygenation conferred resistance to not only long-term hypoxia/reoxygenation hypersomnolence but also to carbonylation, lipid peroxidation injury, and the proinflammatory response, including inducible nitric oxide synthase activity in wake-active brain regions.Collectively, these findings strongly support a critical role for NADPH oxidase in the lasting hypersomnolence and oxidative and proinflammatory responses after hypoxia/reoxygenation patterns simulating severe obstructive sleep apnea oxygenation, highlighting the potential of inhibiting NADPH oxidase to prevent oxidation-mediated morbidities in obstructive sleep apnea.