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Sample records for protein ii differential

  1. DNA topoisomerase II is involved in regulation of cyst wall protein genes and differentiation in Giardia lamblia.

    PubMed

    Lin, Bo-Chi; Su, Li-Hsin; Weng, Shih-Che; Pan, Yu-Jiao; Chan, Nei-Li; Li, Tsai-Kun; Wang, Hsin-Chih; Sun, Chin-Hung

    2013-01-01

    The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.

  2. Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2)

    PubMed Central

    Xu, Zheng; Li, Weixin; Han, Jibo; Zou, Chunpeng; Huang, Weijian; Yu, Weihui; Shan, Xiaoou; Lum, Hazel; Li, Xiaokun; Liang, Guang

    2017-01-01

    Growing evidence indicates that angiotensin II (Ang II), a potent biologically active product of RAS, is a key regulator of renal inflammation and fibrosis. In this study, we tested the hypothesis that Ang II induces renal inflammatory injury and fibrosis through interaction with myeloid differentiation protein-2 (MD2), the accessory protein of toll-like receptor 4 (TLR4) of the immune system. Results indicated that in MD2−/− mice, the Ang II-induced renal fibrosis, inflammation and kidney dysfunction were significantly reduced compared to control Ang II-infused wild-type mice. Similarly, in the presence of small molecule MD2 specific inhibitor L6H21 or siRNA-MD2, the Ang II-induced increases of pro-fibrotic and pro-inflammatory molecules were prevented in tubular NRK-52E cells. MD2 blockade also inhibited activation of NF-κB and ERK. Moreover, MD2 blockade prevented the Ang II-stimulated formation of the MD2/TLR4/MyD88 signaling complex, as well as the increased surface binding of Ang II in NRK-52E cells. In addition, Ang II directly bound recombinant MD2 protein, rather than TLR4 protein. We conclude that MD2 is a significant contributor in the Ang II-induced kidney inflammatory injury in chronic renal diseases. Furthermore, MD2 inhibition could be a new and important therapeutic strategy for preventing progression of chronic renal diseases. PMID:28322341

  3. Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells

    SciTech Connect

    Sahyoun, N.; Wolf, M.; Besterman, J.; Hsieh, T.S.; Sander, M.; LeVine H. III; Chang, K.J.; Cuatrecasas, P.

    1986-03-01

    DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a K/sub m/ of about 100 nM, the reaction was rapid, occurring at 4/sup 0/C as well as at 30/sup 0/C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca/sup 2 +/ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), but not by the inactive analog o-AMSA.

  4. Differential protein expression of peroxiredoxin I and II by benzo(a)pyrene and quercetin treatment in 22Rv1 and PrEC prostate cell lines

    SciTech Connect

    Chaudhary, Amit; Pechan, Tibor; Willett, Kristine L. . E-mail: kwillett@olemiss.edu

    2007-04-15

    Mechanisms of benzo(a)pyrene (BaP)-mediated toxicity and chemopreventative potential of quercetin in prostate cancer are poorly understood. Two-dimensional gel electrophoresis was used to map the differences in protein expression in BaP (1 {mu}M)- and quercetin (5 {mu}M)-treated 22Rv1 human prostate cancer cells. As compared to DMSO, 26 proteins in BaP and 41 proteins in quercetin were found to be differentially expressed ({+-} 2-fold). Western blots confirmed that BaP increased peroxiredoxin (Prx) Prx I and decreased Prx II in 22Rv1 cells. Similar results were found in PrEC normal prostate epithelial cells. Quercetin (up to 10 {mu}M) upregulated Prx II without altering Prx I levels in 22Rv1 cells whereas in PrEC cells, it did not alter the constitutive protein expression of Prx I or II. The lack of quercetin-mediated changes in Prx expression suggests that quercetin does not interfere with H{sub 2}O{sub 2} levels, and thus may have no deleterious effect in normal prostate cells. Quercetin inhibited both BaP-mediated effects on Prx I and II in 22Rv1 cells. In PrEC cells, quercetin inhibited BaP-mediated upregulation of Prx I and had tendency to neutralize BaP-mediated downregulation of Prx II. Quercetin also inhibited BaP-induced concentrations of reactive oxygen species in both 22Rv1 and PrEC cells. These results suggest that Prx I and II may be involved in BaP-mediated toxicity and the potential chemopreventative mechanisms of quercetin.

  5. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    SciTech Connect

    Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  6. Presynaptic proteins complexin-I and complexin-II differentially influence cognitive function in early and late stages of Alzheimer's disease.

    PubMed

    Ramos-Miguel, Alfredo; Sawada, Ken; Jones, Andrea A; Thornton, Allen E; Barr, Alasdair M; Leurgans, Sue E; Schneider, Julie A; Bennett, David A; Honer, William G

    2017-03-01

    Progressive accumulation of Alzheimer's disease-related pathology is associated with cognitive dysfunction. Differences in cognitive reserve may contribute to individual differences in cognitive function in the presence of comparable neuropathology. The protective effects of cognitive reserve could contribute differentially in early versus late stages of the disease. We investigated presynaptic proteins as measures of brain reserve (a subset of total cognitive reserve), and used Braak staging to estimate the progression of Alzheimer's disease. Antemortem evaluations of cognitive function, postmortem assessments of pathologic indices, and presynaptic protein analyses, including the complexins I and II as respective measures of inhibitory and excitatory terminal function, were assayed in multiple key brain regions in 418 deceased participants from a community study. After covarying for demographic variables, pathologic indices, and overall synapse density, lower brain complexin-I and -II levels contributed to cognitive dysfunction (P < 0.01). Each complexin appeared to be dysregulated at a different Braak stage. Inhibitory complexin-I explained 14.4% of the variance in global cognition in Braak 0-II, while excitatory complexin-II explained 7.3% of the variance in Braak V-VI. Unlike other presynaptic proteins, complexins did not colocalize with pathologic tau within neuritic plaques, suggesting that these functional components of the synaptic machinery are cleared early from dystrophic neurites. Moreover, complexin levels showed distinct patterns of change related to memory challenges in a rat model, supporting the functional specificity of these proteins. The present results suggest that disruption of inhibitory synaptic terminals may trigger early cognitive impairment, while excitatory terminal disruption may contribute relatively more to later cognitive impairment.

  7. CCAAT/enhancer-binding protein β regulates the repression of type II collagen expression during the differentiation from proliferative to hypertrophic chondrocytes.

    PubMed

    Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Iwamoto, Yukihide

    2014-01-31

    CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation by stimulating type X collagen and matrix metalloproteinase 13 during chondrocyte differentiation. However, the effect of C/EBPβ on proliferative chondrocytes is unclear. Here, we investigated whether C/EBPβ represses type II collagen (COL2A1) expression and is involved in the regulation of sex-determining region Y-type high mobility group box 9 (SOX9), a crucial factor for transactivation of Col2a1. Endogenous expression of C/EBPβ in the embryonic growth plate and differentiated ATDC5 cells were opposite to those of COL2A1 and SOX9. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked repression of Col2a1. The expression of Sox9 mRNA and nuclear protein was also repressed, resulting in decreased binding of SOX9 to the Col2a1 enhancer as shown by a ChIP assay. Knockdown of C/EBPβ by lentivirus expressing shRNA caused significant stimulation of these genes in ATDC5 cells. Reporter assays demonstrated that C/EBPβ repressed transcriptional activity of Col2a1. Deletion and mutation analysis showed that the C/EBPβ core responsive element was located between +2144 and +2152 bp within the Col2a1 enhancer. EMSA and ChIP assays also revealed that C/EBPβ directly bound to this region. Ex vivo organ cultures of mouse limbs transfected with C/EBPβ showed that the expression of COL2A1 and SOX9 was reduced upon ectopic C/EBPβ expression. Together, these results indicated that C/EBPβ represses the transcriptional activity of Col2a1 both directly and indirectly through modulation of Sox9 expression. This consequently promotes the phenotypic conversion from proliferative to hypertrophic chondrocytes during chondrocyte differentiation.

  8. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  9. Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

    PubMed

    Marganski, William A; Gangopadhyay, Samudra S; Je, Hyun-Dong; Gallant, Cynthia; Morgan, Kathleen G

    2005-09-16

    Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

  10. Angiotensin II directly impairs adipogenic differentiation of human preadipose cells.

    PubMed

    Palominos, Marisol M; Dünner, Natalia H; Wabitsch, Martin; Rojas, Cecilia V

    2015-10-01

    Angiotensin II reduces adipogenic differentiation of preadipose cells present in the stroma-vascular fraction of human adipose tissue, which also includes several cell types. Because of the ability of non-adipose lineage cells in the stroma-vascular fraction to respond to angiotensin II, it is not possible to unequivocally ascribe the anti-adipogenic response to a direct effect of this hormone on preadipose cells. Therefore, we used the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain to investigate the consequences of angiotensin II treatment on adipogenic differentiation under serum-free conditions, by assessing expression of typical adipocyte markers perilipin and fatty acid-binding protein 4 (FABP4), at the transcript and protein level. Reverse transcription-polymerase chain reaction showed that perilipin and FABP4 transcripts were, respectively, reduced to 0.33 ± 0.07 (P < 0.05) and 0.41 ± 0.19-fold (P < 0.05) in SGBS cells induced to adipogenic differentiation in the presence of angiotensin II. Western Blot analysis corroborated reduction of the corresponding proteins to 0.23 ± 0.21 (P < 0.01) and 0.46 ± 0.30-fold (P < 0.01) the respective controls without angiotensin II. Angiotensin II also impaired morphological changes associated with early adipogenesis. Hence, we demonstrated that angiotensin II is able to directly reduce adipogenic differentiation of SGBS preadipose cells.

  11. Estrogen-Responsive Genes Encoding Egg Yolk Proteins Vitellogenin and Apolipoprotein II in Chicken are differentially regulated by Selective Estrogen Receptor Modulators

    PubMed Central

    Ratna, Warren N.; Bhatt, Vrushank D.; Chaudhary, Kawshik; Ariff, Ammar Bin; Bavadekar, Supriya A.; Ratna, Haran N.

    2015-01-01

    In a hen, large quantities of the egg yolk proteins apolipoprotein (apo) II and vitellogenin (VG), are expressed in the liver and transported to the oviduct during egg production. Estrogenic stimulation of the hepatic expression of apo II and VG is due to both transcriptional increase and mRNA stabilization. The nucleolytic degradation of apo II mRNA is prevented by estrogen-regulated mRNA stabilizing factor (E-RmRNASF). Gene-specific effects of a select panel of SERMs on the hepatic expression of the estrogen-responsive genes encoding apo II, VG and E-RmRNASF in the chicken liver were investigated. In the present study, 6-week-old roosters were treated with the vehicle, estrogen, the SERMs genistein, resveratrol, tamoxifen, pterostilbene, raloxifene, catechin and clomiphene or a combination of estrogen and a 200-fold excess of each of the SERMs. Results from mRNA stabilization studies, conducted to investigate the stimulation of expression of E-RmRNASF in the liver by these agents showed that the expression of E-RmRNASF in the liver, was stimulated by estrogen, and the SERMs genistein, resveratrol, tamoxifen, pterostilbene, and catechin, but not by the vehicle, clomiphene or raloxifene. The expression of apo II and VG from the above treatments was determined by Northern blot analysis, RNase protection assays and Western blot analysis. The transcription and protein expression of both apo II and VG genes were seen in response to treatment with estrogen but not with the SERMs or combinations of estrogen and each of the SERMs. The SERMs that stimulated the expression of E-RmRNASF, antagonized the stimulation of the expression of both apo II and VG by estrogen, demonstrating a gene-specific, selective regulation of the above genes in the chicken liver by the SERMs. The above panel of SERMs may likely have adverse effects on egg production. PMID:26452509

  12. Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: differential effects on nicotine-induced spinal and supraspinal antinociception in mice.

    PubMed

    Damaj, M Imad

    2007-10-15

    Recent studies have implicated the involvement of Ca(2+)-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

  13. Estrogen-responsive genes encoding egg yolk proteins vitellogenin and apolipoprotein II in chicken are differentially regulated by selective estrogen receptor modulators.

    PubMed

    Ratna, Warren N; Bhatt, Vrushank D; Chaudhary, Kawshik; Bin Ariff, Ammar; Bavadekar, Supriya A; Ratna, Haran N

    2016-02-01

    In a hen, large quantities of the egg yolk proteins, apolipoprotein II (apo-II) and vitellogenin (VG), are expressed in the liver and transported to the oviduct during egg production. Estrogenic stimulation of the hepatic expression of apo-II and VG is due to both transcriptional increase and mRNA stabilization. The nucleolytic degradation of apo-II messenger RNA (mRNA) is prevented by estrogen-regulated mRNA-stabilizing factor (E-RmRNASF). Gene-specific effects of a select panel of selective estrogen receptor modulators (SERMs) on the hepatic expression of the estrogen-responsive genes encoding apo-II, VG, and E-RmRNASF in the chicken liver were investigated. In the present study, 6-week-old roosters were treated with the vehicle, estrogen, the SERMs genistein, resveratrol, tamoxifen, pterostilbene, raloxifene, catechin, and clomiphene or a combination of estrogen and a 200-fold excess of each of the SERMs. Results from mRNA stabilization studies conducted to investigate the stimulation of expression of E-RmRNASF in the liver by these agents showed that the expression of E-RmRNASF in the liver was stimulated by estrogen and the SERMs genistein, resveratrol, tamoxifen, pterostilbene, and catechin but not by the vehicle, clomiphene or raloxifene. The expression of apo-II and VG from the aforementioned treatments was determined by Northern blot analysis, RNase protection assays, and Western blot analysis. The transcription and protein expression of both apo-II and VG genes were seen in response to treatment with estrogen but not with the SERMs or combinations of estrogen and each of the SERMs. The SERMs that stimulated the expression of E-RmRNASF antagonized the stimulation of the expression of both apo-II and VG by estrogen, demonstrating a gene-specific, selective regulation of the aforementioned genes in the chicken liver by the SERMs. The above panel of SERMs may likely have adverse effects on egg production.

  14. D1-protein dynamics in photosystem II: the lingering enigma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The D1/D2 heterodimer core dominates the photosystem II reaction center. A characteristic feature of this heterodimer is the differentially rapid, light-dependent degradation of the D1 protein. The D1 protein is possibly the most researched photosynthetic polypeptide, with aspects of structure–funct...

  15. Protein kinase CK2 in development and differentiation

    PubMed Central

    Götz, Claudia; Montenarh, Mathias

    2017-01-01

    Among the human kinomes, protein kinase CK2 (formerly termed casein kinase II) is considered to be essential, as it is implicated in the regulation of various cellular processes. Experiments with pharmacological inhibitors of the kinase activity of CK2 provide evidence that CK2 is essential for development and differentiation. Therefore, the present review addresses the role of CK2 during embryogenesis, neuronal, adipogenic, osteogenic and myogenic differentiation in established model cell lines, and in embryonic, neural and mesenchymal stem cells. CK2 kinase activity appears to be essential in the early stages of differentiation, as CK2 inhibition at early time points generally prevents differentiation. In addition, the present review reports on target proteins of CK2 in embryogenesis and differentiation. PMID:28357063

  16. Differential Precipitation and Solubilization of Proteins.

    PubMed

    Ryan, Barry J; Kinsella, Gemma K

    2017-01-01

    Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded proteins. Here, appropriate bioinformatics tools to aid in understanding a protein's propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein.

  17. Group II Introns and Their Protein Collaborators

    NASA Astrophysics Data System (ADS)

    Solem, Amanda; Zingler, Nora; Pyle, Anna Marie; Li-Pook-Than, Jennifer

    Group II introns are an abundant class of autocatalytic introns that excise themselves from precursor mRNAs. Although group II introns are catalytic RNAs, they require the assistance of proteins for efficient splicing in vivo. Proteins that facilitate splicing of organellar group II introns fall into two main categories: intron-encoded maturases and host-encoded proteins. This chapter will focus on the host proteins that group II introns recruited to ensure their function. It will discuss the great diversity of these proteins, define common features, and describe different strategies employed to achieve specificity. Special emphasis will be placed on DEAD-box ATPases, currently the best studied example of host-encoded proteins with a role in group II intron splicing. Since the exact mechanisms by which splicing is facilitated is not known for any of the host proteins, general mechanistic strategies for protein-mediated RNA folding are described and assessed for their potential role in group II intron splicing.

  18. Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.

    PubMed

    Stewart, C E; James, P L; Fant, M E; Rotwein, P

    1996-10-01

    Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

  19. Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin.

    PubMed

    Robison, A J; Bartlett, Ryan K; Bass, Martha A; Colbran, Roger J

    2005-11-25

    Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

  20. Protein S-palmitoylation in cellular differentiation

    PubMed Central

    Zhang, Mingzi M.

    2017-01-01

    Reversible protein S-palmitoylation confers spatiotemporal control of protein function by modulating protein stability, trafficking and activity, as well as protein–protein and membrane–protein associations. Enabled by technological advances, global studies revealed S-palmitoylation to be an important and pervasive posttranslational modification in eukaryotes with the potential to coordinate diverse biological processes as cells transition from one state to another. Here, we review the strategies and tools to analyze in vivo protein palmitoylation and interrogate the functions of the enzymes that put on and take off palmitate from proteins. We also highlight palmitoyl proteins and palmitoylation-related enzymes that are associated with cellular differentiation and/or tissue development in yeasts, protozoa, mammals, plants and other model eukaryotes. PMID:28202682

  1. Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation

    PubMed Central

    Mariani, John N.; Zhang, Chi; Sawai, Setsu; John, Gareth R.

    2017-01-01

    Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL) differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna) proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1) display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin Kpna4 is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. Kpna4 inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues. PMID:28107514

  2. Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation.

    PubMed

    Laitman, Benjamin M; Mariani, John N; Zhang, Chi; Sawai, Setsu; John, Gareth R

    2017-01-01

    Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL) differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna) proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1) display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin Kpna4 is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. Kpna4 inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.

  3. The MORPHEUS II protein crystallization screen

    SciTech Connect

    Gorrec, Fabrice

    2015-06-27

    MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  4. Differential effect of solution conditions on the conformation of the actinoporins Sticholysin II and Equinatoxin II.

    PubMed

    Fauth, Edson V F; Cilli, Eduardo M; Ligabue-Braun, Rodrigo; Verli, Hugo

    2014-12-01

    Actinoporins are a family of pore-forming proteins with hemolytic activity. The structural basis for such activity appears to depend on their correct folding. Such folding encompasses a phosphocholine binding site, a tryptophan-rich region and the activity-related N-terminus segment. Additionally, different solution conditions are known to be able to influence the pore formation by actinoporins, as for Sticholysin II (StnII) and Equinatoxin II (EqtxII). In this context, the current work intends to characterize the influence of distinct solution conditions in the conformational behavior of these proteins through molecular dynamics (MD) simulations. The obtained data offer structural insights into actinoporins dynamics in solution, characterizing its conformational behavior at the atomic level, in accordance with previous experimental data on StnII and EqtxII hemolytic activities.

  5. The MORPHEUS II protein crystallization screen.

    PubMed

    Gorrec, Fabrice

    2015-07-01

    High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  6. Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation.

    PubMed

    Zhao, Yuming; Xu, Pingxiang; Hu, Shengquan; Du, Libo; Xu, Zhiqing; Zhang, Huan; Cui, Wei; Mak, Shinghung; Xu, Daping; Shen, Jianggang; Han, Yifan; Liu, Yang; Xue, Ming

    2015-10-15

    Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3 nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIA's differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner.

  7. Angiotensin II induces differential insulin action in rat skeletal muscle.

    PubMed

    Surapongchai, Juthamard; Prasannarong, Mujalin; Bupha-Intr, Tepmanas; Saengsirisuwan, Vitoon

    2017-03-01

    Angiotensin II (ANGII) is reportedly involved in the development of skeletal muscle insulin resistance. The present investigation evaluated the effects of two ANGII doses on the phenotypic characteristics of insulin resistance syndrome and insulin action and signaling in rat skeletal muscle. Male Sprague-Dawley rats were infused with either saline (SHAM) or ANGII at a commonly used pressor dose (100 ng/kg/min; ANGII-100) or a higher pressor dose (500 ng/kg/min; ANGII-500) via osmotic minipumps for 14 days. We demonstrated that ANGII-100-infused rats exhibited the phenotypic features of non-obese insulin resistance syndrome, including hypertension, impaired glucose tolerance and insulin resistance of glucose uptake in the soleus muscle, whereas ANGII-500-treated rats exhibited diabetes-like symptoms, such as post-prandial hyperglycemia, impaired insulin secretion and hypertriglyceridemia. At the cellular level, insulin-stimulated glucose uptake in the soleus muscle of the ANGII-100 group was 33% lower (P < 0.05) than that in the SHAM group and was associated with increased insulin-stimulated IRS-1 Ser(307) and decreased Akt Ser(473) and AS160 Thr(642) phosphorylation and GLUT-4 expression. However, ANGII-500 infusion did not induce skeletal muscle insulin resistance or impair insulin signaling elements as initially anticipated. Moreover, we found that insulin-stimulated glucose uptake in the ANGII-500 group was accompanied by the enhanced expression of ACE2 and MasR proteins, which are the key elements in the non-classical pathway of the renin-angiotensin system. Collectively, this study demonstrates for the first time that chronic infusion with these two pressor doses of ANGII induced differential metabolic responses at both the systemic and skeletal muscle levels.

  8. Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix.

    PubMed

    Choi, You Hee; Choi, Jun-Ha; Oh, Jae-Wook; Lee, Kwang-Youl

    2013-03-08

    Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

  9. Altered Chondrocyte Differentiation and Extracellular Matrix Homeostasis in a Zebrafish Model for Mucolipidosis II

    PubMed Central

    Flanagan-Steet, Heather; Sias, Christina; Steet, Richard

    2009-01-01

    Mucolipidosis II (ML-II) is a pediatric disorder caused by defects in the biosynthesis of mannose 6-phosphate, the carbohydrate recognition signal responsible for targeting certain acid hydrolases to lysosomes. The mechanisms underlying the developmental defects of ML-II are largely unknown due in part to the lack of suitable animal models. To overcome these limitations, we developed a model for ML-II in zebrafish by inhibiting the expression of N-acetylglucosamine-1-phosphotransferase, the enzyme that initiates mannose 6-phosphate biosynthesis. Morphant embryos manifest craniofacial defects, impaired motility, and abnormal otolith and pectoral fin development. Decreased mannose phosphorylation of several lysosomal glycosidases was observed in morphant lysates, consistent with the reduction in phosphotransferase activity. Investigation of the craniofacial defects in the morphants uncovered striking changes in the timing and localization of both type II collagen and Sox9 expression, suggestive of an accelerated chondrocyte differentiation program. Accumulation of type II collagen was also noted within misshapen cartilage elements at later stages of development. Furthermore, we observed abnormal matrix formation and calcium deposition in morphant otoliths. Collectively, these data provide new insight into the developmental pathology of ML-II and suggest that altered production and/or homeostasis of extracellular matrix proteins are integral to the disease process. These findings highlight the potential of the zebrafish system in studying lysosomal disease pathogenesis. PMID:19834066

  10. IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop

    PubMed Central

    Ren, Hongxia; Yin, Ping; Duan, Cunming

    2008-01-01

    IGF-II stimulates both mitogenesis and myogenesis through its binding and activation of the IGF-I receptor (IGF-IR). How this growth factor pathway promotes these two opposite cellular responses is not well understood. We investigate whether local IGF binding protein-5 (IGFBP-5) promotes the myogenic action of IGF-II. IGFBP-5 is induced before the elevation of IGF-II expression during myogenesis. Knockdown of IGFBP-5 impairs myogenesis and suppresses IGF-II gene expression. IGF-II up-regulates its own gene expression via the PI3K-Akt signaling pathway. Adding IGF-II or constitutively activating Akt rescues the IGFBP-5 knockdown-caused defects. However, an IGF analogue that binds to the IGF-IR but not IGFBP has only a limited effect. When added with low concentrations of IGF-II, IGFBP-5 restores IGF-II expression and myogenic differentiation, whereas an IGF binding–deficient IGFBP-5 mutant has no effect. These findings suggest that IGFBP-5 promotes muscle cell differentiation by binding to and switching on the IGF-II auto-regulation loop. PMID:18762576

  11. Diverse roles of the nucleic acid binding protein KHSRP in cell differentiation and disease

    PubMed Central

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Bizzozzero, Nora Perrone; Gherzi, Roberto

    2015-01-01

    The single-stranded nucleic acid binding protein KHSRP (KH-Type Splicing Regulatory Protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer. PMID:26708421

  12. Diverse roles of the nucleic acid-binding protein KHSRP in cell differentiation and disease.

    PubMed

    Briata, Paola; Bordo, Domenico; Puppo, Margherita; Gorlero, Franco; Rossi, Martina; Perrone-Bizzozero, Nora; Gherzi, Roberto

    2016-01-01

    The single-stranded nucleic acid-binding protein KHSRP (KH-type splicing regulatory protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism, and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer.

  13. Differential geometry based solvation model II: Lagrangian formulation

    PubMed Central

    Chen, Zhan; Baker, Nathan A.; Wei, G. W.

    2010-01-01

    the purpose of computation, thanks to the equivalence of the Laplace-Beltrami operator in the two representations. The coupled partial differential equations (PDEs) are solved with an iterative procedure to reach a steady state, which delivers desired solvent-solute interface and electrostatic potential for problems of interest. These quantities are utilized to evaluate the solvation free energies and protein-protein binding affinities. A number of computational methods and algorithms are described for the interconversion of Lagrangian and Eulerian representations, and for the solution of the coupled PDE system. The proposed approaches have been extensively validated. We also verify that the mean curvature flow indeed gives rise to the minimal molecular surface (MMS) and the proposed variational procedure indeed offers minimal total free energy. Solvation analysis and applications are considered for a set of 17 small compounds and a set of 23 proteins. The salt effect on protein-protein binding affinity is investigated with two protein complexes by using the present model. Numerical results are compared to the experimental measurements and to those obtained by using other theoretical methods in the literature. PMID:21279359

  14. Differential geometry based solvation model II: Lagrangian formulation.

    PubMed

    Chen, Zhan; Baker, Nathan A; Wei, G W

    2011-12-01

    computation, thanks to the equivalence of the Laplace-Beltrami operator in the two representations. The coupled partial differential equations (PDEs) are solved with an iterative procedure to reach a steady state, which delivers desired solvent-solute interface and electrostatic potential for problems of interest. These quantities are utilized to evaluate the solvation free energies and protein-protein binding affinities. A number of computational methods and algorithms are described for the interconversion of Lagrangian and Eulerian representations, and for the solution of the coupled PDE system. The proposed approaches have been extensively validated. We also verify that the mean curvature flow indeed gives rise to the minimal molecular surface and the proposed variational procedure indeed offers minimal total free energy. Solvation analysis and applications are considered for a set of 17 small compounds and a set of 23 proteins. The salt effect on protein-protein binding affinity is investigated with two protein complexes by using the present model. Numerical results are compared to the experimental measurements and to those obtained by using other theoretical methods in the literature.

  15. Cyclical compressive stress induces differentiation of rat primary mandibular condylar chondrocytes through phosphorylated myosin light chain II.

    PubMed

    Liu, Limin; Chen, Lin; Mai, Zhihui; Peng, Zhuli; Yu, Kafung; Liu, Guanqi; Ai, Hong

    2016-11-01

    The role of myosin light chain II (MLC‑II) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a four‑point bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runt‑related transcription factor‑2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLC‑II to total MLC‑II was increased significantly (P<0.05). Silencing MLC‑II by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML‑7, reduced the CUCS‑associated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLC‑II.

  16. Interference with p53 protein inhibits hematopoietic and muscle differentiation

    PubMed Central

    1996-01-01

    The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant- negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity. PMID:8698814

  17. P(II) signal transduction proteins: nitrogen regulation and beyond.

    PubMed

    Huergo, Luciano F; Chandra, Govind; Merrick, Mike

    2013-03-01

    The P(II) proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P(II) proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described. Major advances have been made in structural studies of P(II) proteins, including the solution of the first structures of P(II) proteins complexed with their targets. We have also begun to gain insights into how the key effector molecules, 2-oxoglutarate and ATP/ADP, influence the activities of P(II) proteins. In this review, we have set out to summarize our current understanding of P(II) biology and to consider where future studies of these extraordinarily adaptable proteins might lead us.

  18. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  19. The MicroRNA 29 Family Promotes Type II Cell Differentiation in Developing Lung.

    PubMed

    Guo, Wei; Benlhabib, Houda; Mendelson, Carole R

    2016-08-15

    Lung alveolar type II cells uniquely synthesize surfactant, a developmentally regulated lipoprotein that is essential for breathing. Expression of the gene (SFTPA) encoding the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cyclic AMP (cAMP). cAMP induction of SP-A expression is repressed by transforming growth factor β (TGF-β) and by hypoxia. In this study, we found that expression of the microRNA 29 (miR-29) family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. miR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-β2, was coordinately decreased. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting their physiological relevance. This occurred through derepression of TGF-β signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor 1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells, and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in cotransfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-β signaling.

  20. The MicroRNA 29 Family Promotes Type II Cell Differentiation in Developing Lung

    PubMed Central

    Guo, Wei; Benlhabib, Houda

    2016-01-01

    Lung alveolar type II cells uniquely synthesize surfactant, a developmentally regulated lipoprotein that is essential for breathing. Expression of the gene (SFTPA) encoding the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cyclic AMP (cAMP). cAMP induction of SP-A expression is repressed by transforming growth factor β (TGF-β) and by hypoxia. In this study, we found that expression of the microRNA 29 (miR-29) family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. miR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-β2, was coordinately decreased. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting their physiological relevance. This occurred through derepression of TGF-β signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor 1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells, and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in cotransfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-β signaling. PMID:27215389

  1. Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

    SciTech Connect

    Johnson, Chassidy; Jahid, Sohail; Voelker, Dennis R.; Fan Hung

    2011-04-10

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.

  2. Regulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes

    PubMed Central

    Feng, Huanhuan; Zhang, Guojun; Liu, Guoyan; Yang, Can

    2016-01-01

    High mobility group box 1 protein (HMGB1) is a molecule related to the development of inflammation. Autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury. Our recent work focused on the relationship of HMGB1 and autophagy in 3T3-L1 cells. In vivo experimental results showed that, compared with the normal-diet group, the high-fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues. The expression levels of HMGB1 and LC3II also increased in epididymal adipose tissues in high-fat diet group compared to the normal-diet mice. The in vitro results indicated that HMGB1 protein treatment increased LC3II formation in 3T3-L1 preadipocytes in contrast to that in the control group. Furthermore, LC3II formation was inhibited through HMGB1 knockdown by siRNA. Treatment with the HMGB1 protein enhanced LC3II expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes. By contrast, FABP4 expression decreased on the fourth day and increased on the eighth day. Hence, the HMGB1 protein modulated autophagy-related proteins and lipid-metabolism-related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases. PMID:27843198

  3. Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

    PubMed Central

    Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

    2015-01-01

    The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

  4. In Vivo Probe of Lipid II-Interacting Proteins.

    PubMed

    Sarkar, Sourav; Libby, Elizabeth A; Pidgeon, Sean E; Dworkin, Jonathan; Pires, Marcos M

    2016-07-11

    β-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.

  5. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed Central

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor. Images PMID:8264628

  6. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

  7. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  8. Subcellular localization of mammalian type II membrane proteins.

    PubMed

    Aturaliya, Rajith N; Fink, J Lynn; Davis, Melissa J; Teasdale, Melvena S; Hanson, Kelly A; Miranda, Kevin C; Forrest, Alistair R R; Grimmond, Sean M; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-05-01

    Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

  9. Insulin and Insulin-like Growth Factor II Differentially Regulate Endocytic Sorting and Stability of Insulin Receptor Isoform A*

    PubMed Central

    Morcavallo, Alaide; Genua, Marco; Palummo, Angela; Kletvikova, Emilia; Jiracek, Jiri; Brzozowski, Andrzej M.; Iozzo, Renato V.; Belfiore, Antonino; Morrione, Andrea

    2012-01-01

    The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3–10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R−/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R−/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R−/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyrB26]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli. PMID:22318726

  10. Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro.

    PubMed

    Boden, S D; McCuaig, K; Hair, G; Racine, M; Titus, L; Wozney, J M; Nanes, M S

    1996-08-01

    Bone morphogenetic proteins (BMPs) induce cartilage and bone differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive proteins may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger bone nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to 10-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation.

  11. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    PubMed

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties.

  12. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  13. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  14. Life without double-headed non-muscle myosin II motor proteins

    NASA Astrophysics Data System (ADS)

    Betapudi, Venkaiah

    2014-07-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  15. Life without double-headed non-muscle myosin II motor proteins

    PubMed Central

    Betapudi, Venkaiah

    2014-01-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients' life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life. PMID:25072053

  16. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  17. The miR-200 family and its targets regulate type II cell differentiation in human fetal lung.

    PubMed

    Benlhabib, Houda; Guo, Wei; Pierce, Brianne M; Mendelson, Carole R

    2015-09-11

    Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-β. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-β2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-β2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-β. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-β, serve important roles in the developmental regulation of type II cell differentiation and function in HFL.

  18. Differential tissue-specific protein markers of vaginal carcinoma

    PubMed Central

    Hellman, K; Alaiya, A A; Becker, S; Lomnytska, M; Schedvins, K; Steinberg, W; Hellström, A-C; Andersson, S; Hellman, U; Auer, G

    2009-01-01

    The objective was to identify proteins differentially expressed in vaginal cancer to elucidate relevant cancer-related proteins. A total of 16 fresh-frozen tissue biopsies, consisting of 5 biopsies from normal vaginal epithelium, 6 from primary vaginal carcinomas and 5 from primary cervical carcinomas, were analysed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Of the 43 proteins identified with significant alterations in protein expression between non-tumourous and tumourous tissue, 26 were upregulated and 17 were downregulated. Some were similarly altered in vaginal and cervical carcinoma, including cytoskeletal proteins, tumour suppressor proteins, oncoproteins implicated in apoptosis and proteins in the ubiquitin–proteasome pathway. Three proteins were uniquely altered in vaginal carcinoma (DDX48, erbB3-binding protein and biliverdin reductase) and five in cervical carcinoma (peroxiredoxin 2, annexin A2, sarcomeric tropomyosin kappa, human ribonuclease inhibitor and prolyl-4-hydrolase beta). The identified proteins imply involvement of multiple different cellular pathways in the carcinogenesis of vaginal carcinoma. Similar protein alterations were found between vaginal and cervical carcinoma suggesting common tumourigenesis. However, the expression level of some of these proteins markedly differs among the three tissue specimens indicating that they might be useful molecular markers. PMID:19367286

  19. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  20. Differential Binding of Co(II) and Zn(II) to Metallo-beta-Lactamase Bla2 from Bacillus anthracis

    SciTech Connect

    Hawk, M.; Breece, R; Hajdin, C; Bender, K; Hu, Z; Costello, A; Bennett, B; Tierney, D; Crowder, M

    2009-01-01

    In an effort to probe the structure, mechanism, and biochemical properties of metallo-{beta}-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

  1. HEREDITARY DEPENDENCE IN THE THEORY OF DIFFERENTIAL EQUATIONS, PART II,

    DTIC Science & Technology

    A general class of differential equations with hereditary dependence is introduced which includes most equations of hereditary type encountered in...uniqueness of solutions and dependence on initial data and parameters are considered.

  2. Angiotensin II Induces a Region-Specific Hyperplasia of the Ascending Aorta Through Regulation of Inhibitor of Differentiation 3

    PubMed Central

    Owens, A. Phillip; Subramanian, Venkateswaran; Moorleghen, Jessica J.; Guo, Zhenheng; McNamara, Coleen A.; Cassis, Lisa A; Daugherty, Alan

    2010-01-01

    Rationale Angiotensin II (AngII) has diverse effects on smooth muscle cells. The diversity of effects may relate to the regional location of this cell type. Objective The aim of this study was to define whether AngII exerted divergent effects on smooth muscle cells (SMC) in the aorta and determine the role of blood pressure and specific oxidant mechanisms. Methods and Results AngII (1,000 ng/kg/min) infusion for 28 days into mice increased systolic blood pressure (SBP) and promoted medial expansion of equivalent magnitude throughout the entire aorta. Both effects were ablated by AT1a receptor deficiency. Similar increases in blood pressure by administration of norepinephrine promoted no changes in aortic medial thickness. Increased medial thickness was due to SMC expansion attributable to hypertrophy in most aortic regions, with the exception of hyperplasia of the ascending aorta. Deficiency of the p47phox component of NADPH oxidase ablated AngII-induced medial expansion in all aortic regions. Analysis of mRNA and protein throughout the aorta revealed a much higher abundance of the inhibitor of differentiation 3 (Id3) in the ascending aorta compared to all other regions. A functional role was demonstrated by Id3 deficiency inhibiting AngII-induced SMC hyperplasia of the ascending aorta. Conclusions In conclusion, AngII promotes both aortic medial hypertrophy and hyperplasia in a region-specific manner via an oxidant mechanism. The ascending aortic hyperplasia is dependent on Id3. PMID:20019328

  3. Mixed metal copper(II)-nickel(II) and copper(II)-zinc(II) complexes of multihistidine peptide fragments of human prion protein.

    PubMed

    Jószai, Viktória; Turi, Ildikó; Kállay, Csilla; Pappalardo, Giuseppe; Di Natale, Giuseppe; Rizzarelli, Enrico; Sóvágó, Imre

    2012-07-01

    Mixed metal copper(II)-nickel(II) and copper(II)-zinc(II) complexes of four peptide fragments of human prion protein have been studied by potentiometric, UV-vis and circular dichroism spectroscopic techniques. One peptide contained three histidyl residues: HuPrP(84-114) with H85 inside and H96, H111 outside the octarepeat domain. The other three peptides contained two histidyl residues; H96 and H111 for HuPrP(91-115) and HuPrP(84-114)H85A while HuPrP(84-114)H96A contained the histidyl residues at positions 85 and 111. It was found that both histidines of the latter peptides can simultaneously bind copper(II) and nickel(II) ions and dinuclear mixed metal complexes can exist in slightly alkaline solution. One molecule of the peptide with three histidyl residues can bind two copper(II) and one nickel(II) ions. H85 and H111 were identified as the major copper(II) and H96 as the preferred nickel(II) binding sites in mixed metal species. The studies on the zinc(II)-PrP peptide binary systems revealed that zinc(II) ions can coordinate to the 31-mer PrP peptide fragments in the form of macrochelates with two or three coordinated imidazol-nitrogens but the low stability of these complexes cannot prevent the hydrolysis of the metal ion in slightly alkaline solution. These data provide further support for the outstanding affinity of copper(II) ions towards the peptide fragments of prion protein but the binding of nickel(II) can significantly modify the distribution of copper(II) among the available metal binding sites.

  4. Differential protein labeling based on electrochemically generated reactive intermediates.

    PubMed

    Büter, Lars; Faber, Helene; Wigger, Tina; Vogel, Martin; Karst, Uwe

    2015-10-06

    A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as β-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments.

  5. Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

    PubMed

    Shetty, J; Diekman, A B; Jayes, F C; Sherman, N E; Naaby-Hansen, S; Flickinger, C J; Herr, J C

    2001-08-01

    The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

  6. Localization of organ of Corti protein II in the adult and developing gerbil cochlea.

    PubMed

    Yoho, E R; Thomopoulos, G N; Thalmann, I; Thalmann, R; Schulte, B A

    1997-02-01

    The distribution of organ of Corti protein II (OCP-II) was assessed in the developing and mature gerbil cochlea by light and electron microscopic immunohistochemistry. In the adult cochlea, OCP-II was expressed only in certain epithelial cells which included all supporting cells of the organ of Corti, inner and outer sulcus cells and interdental cells. Inner and outer hair cells lacked immunoreactivity. The highest gold particle labeling density was seen overlying intracellular regions devoid of organelles. In the developing inner ear, OCP-II was first detected at 2 days after birth (DAB) with the strongest staining in immature Deiters, inner phalangeal and pillar cells. Immunostaining intensity increased gradually in cells lying laterally and medially to the more centrally located supporting cells and reached adult levels in all reactive cell types around 18 DAB. The results demonstrated conclusively that OCP-II is a cytosolic protein and fail to support its role as a transcription factor postulated on the basis of its homology with p15 or a role in the control of the cycle as suggested by its near-identity with p19Skp1, a cyclin A/CDK2-associated protein. The continued high level of expression in the mature cochlea argues against OCP-II's involvement in regulating the development and differentiation of epithelial cells. The protein's unique distribution and its gradual increase in expression prior to and during the onset and maturation of hearing, however, support its potential function in the recycling of K+ effluxed from hair cells and neurons back to endolymph.

  7. A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

    PubMed Central

    Nielsen, Jacob; Christiansen, Jan; Lykke-Andersen, Jens; Johnsen, Anders H.; Wewer, Ulla M.; Nielsen, Finn C.

    1999-01-01

    Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development. PMID:9891060

  8. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  9. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  10. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    PubMed

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  11. Proteomic analysis reveals differentially expressed proteins in the rat frontal cortex after methamphetamine treatment.

    PubMed

    Faure, J J; Hattingh, S M; Stein, D J; Daniels, W M

    2009-12-01

    Methamphetamine (MA) is an addictive psycho-stimulant and the illicit use of the drug is escalating. In the present study, we examined protein expression profiles in the rat frontal cortex exposed to a total of eight MA injections (1 mg/kg, intraperitoneal) using 2-DE based proteomics. We investigated protein changes occurring in both the cytosolic fraction and the membrane fraction. 2-DE analysis resulted in 62 cytosolic and 44 membrane protein spots that were differentially regulated in the frontal cortex of rats exposed to MA when compared to control animals. Of these spots, 47 cytosolic and 42 membrane proteins were identified respectively, using ESI-Quad-TOF, which included ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), beta-synuclein, 78 kDa glucose-regulated protein (GRP 78), gamma-enolase, dihydropyrimidase-related protein 2 (DRP 2), complexin 2 and synapsin II. These proteins are associated with protein degradation, redox regulation, energy metabolism, cellular growth, cytoskeletal modifications and synaptic function. Proteomic research may be useful in exploring the complex underlying molecular mechanisms of MA dependence.

  12. Eigenvalues of singular differential operators by finite difference methods. II.

    NASA Technical Reports Server (NTRS)

    Baxley, J. V.

    1972-01-01

    Note is made of an earlier paper which defined finite difference operators for the Hilbert space L2(m), and gave the eigenvalues for these operators. The present work examines eigenvalues for higher order singular differential operators by using finite difference methods. The two self-adjoint operators investigated are defined by a particular value in the same Hilbert space, L2(m), and are strictly positive with compact inverses. A class of finite difference operators is considered, with the idea of application to the theory of Toeplitz matrices. The approximating operators consist of a good approximation plus a perturbing operator.

  13. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    PubMed

    Miranda, Alberto; Pericuesta, Eva; Ramírez, Miguel Ángel; Gutierrez-Adan, Alfonso

    2011-04-04

    Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  14. Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II.

    PubMed

    Takano, Tomomi; Nakano, Kenta; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2015-05-01

    Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed "viroporins". Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4'-disothiocyano-2,2'-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II.

  15. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

    PubMed

    Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

    2016-01-01

    During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells.

  16. Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells.

    PubMed

    Chiu, Li-Hsuan; Yeh, Tien-Shun; Huang, Huei-Mei; Leu, Sy-Jye; Yang, Charng-Bin; Tsai, Yu-Hui

    2012-06-01

    Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)β(1)/α(2)β(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)β(1) or α(2)β(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.

  17. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

    PubMed Central

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-01-01

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes. PMID:27383650

  18. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation.

    PubMed

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-07-07

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

  19. Activating types 1 and 2 angiotensin II receptors modulate the hypertrophic differentiation of chondrocytes☆

    PubMed Central

    Tsukamoto, Ichiro; Inoue, Shinji; Teramura, Takeshi; Takehara, Toshiyuki; Ohtani, Kazuhiro; Akagi, Masao

    2013-01-01

    A local tissue-specific renin–angiotensin system (local RAS) has been identified in many organs. However, no report has described the role of a local RAS in the hypertrophic differentiation of chondrocytes. To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes. Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2. PMID:23905010

  20. Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately

    PubMed Central

    1990-01-01

    The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins. PMID:2335560

  1. Differential diagnosis of food protein-induced enterocolitis syndrome

    PubMed Central

    Fiocchi, Alessandro; Claps, Alessia; Dahdah, Lamia; Brindisi, Giulia; Dionisi-Vici, Carlo; Martelli, Alberto

    2014-01-01

    Purpose of review To assess all the possible differential diagnosis of food protein-induced enterocolitis syndrome (FPIES), both in acute and chronic presentation, reviewing the data reported in published studies. Recent findings There is an increase of reported cases of FPIES in recent years. As the disease presents with nonspecific symptoms, it can be misunderstood in many ways. The differential diagnosis includes, in acute presentations, the following: sepsis, other infectious diseases, acute gastrointestinal episodes, surgical emergencies, food allergies. In its chronic forms, FPIES may mimic malabsorption syndromes, metabolic disorders, primary immunodeficiencies, neurological conditions, coagulation defects, and other types of non-IgE-mediated food allergy. Summary A thorough clinical evaluation, including symptoms, signs, and laboratory findings, is necessary to lead the clinicians toward the diagnosis of FPIES. The major reason for delayed diagnosis appears to be the lack of knowledge of the disease. PMID:24739227

  2. Differential electron scattering cross sections for the first optically forbidden and resonance transitions in Mg II, Zn II and Cd II

    NASA Technical Reports Server (NTRS)

    Williams, I. D.; Chutjian, A.; Mawhorter, R. J.

    1986-01-01

    Differential electron scattering cross sections have been measured for dipole-forbidden and resonance transitions in Mg II, Zn II and Cd II in the angular range theta = 4-17 deg at 50 eV. These provide the first recorded angular distributions for an optically forbidden transition. It is found that while the cross section for excitation of the 4s (2)S-3d(9)4s(2) (2)D transition in Zn II is small, those for the 3s (2)S-3d (2)D, 4s (2)S (unresolved lines) in Mg II, and the 5s (2)S-4d(9)5s(2) D in Cd II are comparable in magnitude with the cross sections for resonance excitation. In addition, for Cd II it is found that the allowed and forbidden transitions have very similar angular distributions, and it is proposed that excitation to the 2D state may be dominated by a virtual 'double-dipole' transition via the 2P state. Also, the total excitation cross section of the resonance 2P state in Cd II is a factor of four higher than that predicted by the Gaunt factor approximation, suggesting that the accepted value for the oscillator strength may be too low.

  3. Determination of protein-ligand interactions using differential scanning fluorimetry.

    PubMed

    Vivoli, Mirella; Novak, Halina R; Littlechild, Jennifer A; Harmer, Nicholas J

    2014-09-13

    A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

  4. Disorders of sexual differentiation: II. Diagnosis and treatment

    PubMed Central

    El-Sherbiny, Mohamed

    2013-01-01

    Objectives To provide a review and summary of recent advances in the diagnosis and management of disorder(s) of sexual differentiation (DSD), an area that has developed over recent years with implications for the management of children with DSD; and to assess the refinements in the surgical techniques used for genital reconstruction. Methods Recent publications (in the previous 10 years) were identified using PubMed, as were relevant previous studies, using following keywords; ‘diagnosis and management’, ‘ambiguous genitalia’, ‘intersex’, ‘disorders of sexual differentiation’, ‘genitogram’, ‘endocrine assessment’, ‘gender assignment’, ‘genitoplasty’, and ‘urogenital sinus’. The findings were reviewed. Results Arbitrary criteria have been developed to select patients likely to have DSD. Unnecessary tests, especially those that require anaesthesia or are associated with radiation exposure, should be limited to situations where a specific question needs to be answered. Laparoscopy is an important diagnostic tool in selected patients. The routine use of multidisciplinary diagnostic and expert surgical teams has become standard. Full disclosure of different therapeutic approaches and their timing is recommended. Conclusions Diagnostic tests should be tailored according to the available information. Parents and/or patients should be made aware of the paucity of well-designed studies, as these conditions are rare. Unnecessary irreversible surgery should be postponed until a multidisciplinary experienced team, with the parents’ and or patients’ approval, can make a well-judged decision. PMID:26579241

  5. Differential alleleic expression of the type II collagen gene (COL2A2) in osteoarthritic cartilage

    SciTech Connect

    Loughlin, J.; Irven, C.; Sykes, B.; Athanasou, N.; Carr, A.

    1995-05-01

    Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced <12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA. 31 refs., 4 figs., 3 tabs.

  6. Differential protein import deficiencies in human peroxisome assembly disorders

    PubMed Central

    1994-01-01

    Two peroxisome targeting signals (PTSs) for matrix proteins have been well defined to date. PTS1 comprises a COOH-terminal tripeptide, SKL, and has been found in several matrix proteins, whereas PTS2 has been found only in peroxisomal thiolase and is contained within an NH2- terminal cleavable presequence. We have investigated the functional integrity of the import routes for PTS1 and PTS2 in fibroblasts from patients suffering from peroxisome assembly disorders. Three of the five complementation groups tested showed a general loss of PTS1 and PTS2 import. Two complementation groups showed a differential loss of peroxisomal protein import: group I cells were able to import a PTS1- but not a PTS2- containing reporter protein into their peroxisomes, and group IV cells were able to import the PTS2 but not the PTS1 reporter into aberrant, peroxisomal ghostlike structures. The observation that the PTS2 import pathway is intact only in group IV cells is supported by the protection of endogenous thiolase from protease degradation in group IV cells and its sensitivity in the remaining complementation groups, including the partialized disorder of group I. The functionality of the PTS2 import pathway and colocalization of endogenous thiolase with the peroxisomal membranes in group IV cells was substantiated further using immunofluorescence, subcellular fractionation, and immunoelectron microscopy. The phenotypes of group I and IV cells provide the first evidence for differential import deficiencies in higher eukaryotes. These phenotypes are analogous to those found in Saccharomyces cerevisiae peroxisome assembly mutants. PMID:7910611

  7. Differential bicodon usage in lowly and highly abundant proteins

    PubMed Central

    2017-01-01

    Degeneracy in the genetic code implies that different codons can encode the same amino acid. Usage preference of synonymous codons has been observed in all domains of life. There is much evidence suggesting that this bias has a major role on protein elongation rate, contributing to differential expression and to co-translational folding. In addition to codon usage bias, other preference variations have been observed such as codon pairs. In this paper, I report that codon pairs have significant different frequency usage for coding either lowly or highly abundant proteins. These usage preferences cannot be explained by the frequency usage of the single codons. The statistical analysis of coding sequences of nine organisms reveals that in many cases bicodon preferences are shared between related organisms. Furthermore, it is observed that misfolding in the drug-transport protein, encoded by MDR1 gene, is better explained by a big change in the pause propensity due to the synonymous bicodon variant, rather than by a relatively small change in codon usage. These findings suggest that codon pair usage can be a more powerful framework to understand translation elongation rate, protein folding efficiency, and to improve protocols to optimize heterologous gene expression. PMID:28289571

  8. Osteogenic Differentiation of Mesenchymal Stem Cells in Defined Protein Beads

    PubMed Central

    Lund, Amanda W.; Bush, Jeff A.; Plopper, George E.; Stegemann, Jan P.

    2008-01-01

    There is a need to develop improved methods for directing and maintaining the differentiation of human mesenchymal stem cells (hMSC) for regenerative medicine. Here, we present a method for embedding cells in defined protein microenvironments for the directed osteogenic differentiation of hMSC. Composite matrices of collagen I and agarose were produced by emulsification and simultaneous polymerization in the presence of hMSC to produce 30–150 μm diameter hydrogel “beads.” The proliferation, morphology, osteogenic gene expression, and calcium deposition of hMSC in bead environments were compared to other two- and three-dimensional culture environments over 14–21 days in culture. Cells embedded within 40% collagen beads exhibited equivalent proliferation rates to those in gel disks, but showed upregulation of bone sialoprotein and increased calcium deposition over 2D controls. Osteocalcin gene expression was not changed in 3D beads and disks, while collagen type I gene expression was downregulated relative to cells in 2D culture. The hydrogel bead format allows controlled cell differentiation and is a cell delivery vehicle that may also enhance vascular invasion and host incorporation. Our results indicate that the application of such beads can be used to promote the osteogenic phenotype in hMSC, which is an important step toward using them in bone repair applications. PMID:18431753

  9. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  10. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  11. Colocalization of Polyphenol Oxidase and Photosystem II Proteins.

    PubMed

    Lax, A R; Vaughn, K C

    1991-05-01

    Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex.

  12. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  13. DIFFERENTIAL EFFECTS OF MINERALOCORTICOID AND ANGIOTENSIN II ON INCENTIVE AND MESOLIMBIC ACTIVITY

    PubMed Central

    Grafe, Laura A.; Flanagan-Cato, Loretta M.

    2016-01-01

    The controls of thirst and sodium appetite are mediated in part by the hormones aldosterone and angiotensin II (AngII). The present study examined the behavioral and neural mechanisms of altered effort-value in animals treated with systemic mineralocorticoids, intracerebroventricular AngII, or both. First, rats treated with mineralocorticoid and AngII were tested in the progressive ratio operant task. The willingness to work for sodium versus water depended on hormonal treatment. In particular, rats treated with both mineralocorticoid and AngII preferentially worked for access to sodium versus water compared with rats given only one of these hormones. Second, components of the mesolimbic dopamine pathway were examined for modulation by mineralocorticoids and AngII. Based on cFos immunohistochemistry, AngII treatment activated neurons in the ventral tegmental area and nucleus accumbens, with no enhancement by mineralocorticoid pretreatment. In contrast, western blot analysis revealed that combined hormone treatment increased levels of phospho-tyrosine hydroxylase in the ventral tegmental area. Thus, mineralocorticoid and AngII treatments differentially engaged the mesolimbic pathway based on tyrosine hydroxylase levels versus cFos activation. PMID:26730722

  14. The effect of type II collagen on MSC osteogenic differentiation and bone defect repair.

    PubMed

    Chiu, Li-Hsuan; Lai, Wen-Fu T; Chang, Shwu-Fen; Wong, Chin-Chean; Fan, Cheng-Yu; Fang, Chia-Lang; Tsai, Yu-Hui

    2014-03-01

    The function of type II collagen in cartilage is well documented and its importance for long bone development has been implicated. However, the involvement of type II collagen in bone marrow derived mesenchymal stem cell (BMSC) osteogenesis has not been well investigated. This study elucidated the pivotal role of type II collagen in BMSC osteogenesis and its potential application to bone healing. Type II collagen-coated surface was found to accelerate calcium deposition, and the interaction of osteogenic medium-induced BMSCs with type II collagen-coated surface was mainly mediated through integrin α2β1. Exogenous type II collagen directly activated FAK-JNK signaling and resulted in the phosphorylation of RUNX2. In a segmental defect model in rats, type II collagen-HA/TCP-implanted rats showed significant callus formation at the reunion site, and a higher SFI (sciatic function index) scoring as comparing to other groups were also observed at 7, 14, and 21 day post-surgery. Collectively, type II collagen serves as a better modulator during early osteogenic differentiation of BMSCs by facilitating RUNX2 activation through integrin α2β1-FAK-JNK signaling axis, and enhance bone defect repair through an endochondral ossification-like process. These results advance our understanding about the cartilaginous ECM-BMSC interaction, and provide perspective for bone defect repair strategies.

  15. Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

    PubMed Central

    Huang, Pai-Tsang; Chen, Chien-Ho; Hsu, I-Uen; Salim, Shaima’a Ahmad; Kao, Shu-Huei; Cheng, Chao-Wen; Lai, Chang-Hao; Lee, Cheng-Fan; Lin, Yung-Feng

    2015-01-01

    Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation. PMID:25671650

  16. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  17. Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

    PubMed

    He, Ying; Wang, Zaijie Jim

    2015-03-18

    As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

  18. TGF beta receptor II interacting protein-1, an intracellular protein has an extracellular role as a modulator of matrix mineralization

    PubMed Central

    Ramachandran, Amsaveni; Ravindran, Sriram; Huang, Chun-Chieh; George, Anne

    2016-01-01

    Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 μM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization. PMID:27883077

  19. A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

    PubMed Central

    Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

  20. Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

    PubMed Central

    Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

    2014-01-01

    Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

  1. Differential Use of Signal Peptides and Membrane Domains Is a Common Occurrence in the Protein Output of Transcriptional Units

    PubMed Central

    Davis, Melissa J; Hanson, Kelly A; Clark, Francis; Fink, J. Lynn; Zhang, Fasheng; Kasukawa, Takeya; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-01-01

    Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome. PMID:16683029

  2. Differential membrane association properties and regulation of class I and class II Arfs.

    PubMed

    Duijsings, Daniël; Lanke, Kjerstin H W; van Dooren, Sander H J; van Dommelen, Michiel M T; Wetzels, Roy; de Mattia, Fabrizio; Wessels, Els; van Kuppeveld, Frank J M

    2009-03-01

    ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.

  3. Monoclonal antibodies against type II rat brain protein kinase

    SciTech Connect

    Nakabayashi, C.H.; Huang, K.P.

    1987-05-01

    Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.

  4. Calcium/calmodulin-dependent protein kinase II expression in motor neurons: effect of axotomy.

    PubMed

    Lund, L M; McQuarrie, I G

    1997-11-20

    Although Ca2+/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIalpha and IIbeta are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIbeta antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIalpha, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIalpha immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIalpha is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIalpha as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIbeta down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIalpha appears to support axonal regrowth.

  5. Regulator of G protein signaling proteins differentially modulate signaling of μ and δ opioid receptors

    PubMed Central

    Xie, Zhihua; Li, Zhisong; Guo, Lei; Ye, Caiying; Li, Juan; Yu, Xiaoli; Yang, Huifen; Wang, Yulin; Chen, Chongguang; Zhang, Dechang; Liu-Chen, Lee-Yuan

    2009-01-01

    Effects of regulator of G protein signaling (RGS) proteins on μ and δ opioid receptors were investigated in HEK293 cells. Co-expression of RGS1, RGS2, RGS4, RGS9, RGS10 or RGS19 (Gα-interacting protein (GAIP)) significantly reduced [Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol]-Enkephalin (DAMGO)-induced inhibition of adenylyl cyclase (AC) mediated by μ opioid receptor, but only RGS9 decreased the effects of [Tyr-D-Pen-Gly-p-Chloro-Phe-D-Pen]-Enkephalin (DPDPE) mediated by δ opioid receptor. When C-tails of the receptors were exchanged (μ/δC and δ/μC chimeras), RGS proteins decreased δ/μC-mediated AC inhibition, but none had significant effects on that via μ/δC receptor. Thus, the C-terminal domains of the receptors are critical for the differential effects of RGS proteins, which may be due to differences in receptor - G protein - RGS protein interactions in signaling complexes. PMID:17433292

  6. Differential pattern of semantic memory organization between bipolar I and II disorders.

    PubMed

    Chang, Jae Seung; Choi, Sungwon; Ha, Kyooseob; Ha, Tae Hyon; Cho, Hyun Sang; Choi, Jung Eun; Cha, Boseok; Moon, Eunsoo

    2011-06-01

    Semantic cognition is one of the key factors in psychosocial functioning. The aim of this study was to explore the differences in pattern of semantic memory organization between euthymic patients with bipolar I and II disorders using the category fluency task. Study participants included 23 euthymic subjects with bipolar I disorder, 23 matched euthymic subjects with bipolar II disorder and 23 matched control subjects. All participants were assessed for verbal learning, recall, learning strategies, and fluency. The combined methods of hierarchical clustering and multidimensional scaling were used to compare the pattern of semantic memory organization among the three groups. Quantitative measures of verbal learning, recall, learning strategies, and fluency did not differ between the three groups. A two-cluster structure of semantic memory organization was identified for the three groups. Semantic structure was more disorganized in the bipolar I disorder group compared to the bipolar II disorder. In addition, patients with bipolar II disorder used less elaborate strategies of semantic memory organization than those of controls. Compared to healthy controls, strategies for categorization in semantic memory appear to be less knowledge-based in patients with bipolar disorders. A differential pattern of semantic memory organization between bipolar I and II disorders indicates a higher risk of cognitive abnormalities in patients with bipolar I disorder compared to patients with bipolar II disorder. Exploring qualitative nature of neuropsychological domains may provide an explanatory insight into the characteristic behaviors of patients with bipolar disorders.

  7. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

    PubMed

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  8. LHC II protein phosphorylation in leaves of Arabidopsis thaliana mutants deficient in non-photochemical quenching.

    PubMed

    Breitholtz, Hanna-Leena; Srivastava, Renu; Tyystjärvi, Esa; Rintamäki, Eevi

    2005-06-01

    Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b(6)f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin-like component, and H2O2 that inserts an inhibitory loop in the regulation of LHC II protein phosphorylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation.

  9. Quantitative Proteomics Reveals GIMAP Family Proteins 1 and 4 to Be Differentially Regulated during Human T Helper Cell Differentiation *S⃞

    PubMed Central

    Filén, Jan-Jonas; Filén, Sanna; Moulder, Robert; Tuomela, Soile; Ahlfors, Helena; West, Anne; Kouvonen, Petri; Kantola, Suvi; Björkman, Mari; Katajamaa, Mikko; Rasool, Omid; Nyman, Tuula A.; Lahesmaa, Riitta

    2009-01-01

    T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4+ cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation. PMID:18701445

  10. SIGNATURE OF DIFFERENTIAL ROTATION IN SUN-AS-A-STAR Ca II K MEASUREMENTS

    SciTech Connect

    Bertello, L.; Pietarila, A.; Pevtsov, A. A. E-mail: apietarila@nso.edu

    2012-12-10

    The characterization of solar surface differential rotation (SDR) from disk-integrated chromospheric measurements has important implications for the study of differential rotation and dynamo processes in other stars. Some chromospheric lines, such as Ca II K, are very sensitive to the presence of activity on the disk and are an ideal choice for investigating SDR in Sun-as-a-star observations. Past studies indicate that when the activity is low, the determination of Sun's differential rotation from integrated-sunlight measurements becomes uncertain. However, our study shows that using the proper technique, SDR can be detected from these type of measurements even during periods of extended solar minima. This paper describes results from the analysis of the temporal variations of Ca II K line profiles observed by the Integrated Sunlight Spectrometer during the declining phase of Cycle 23 and the rising phase of Cycle 24, and discusses the signature of SDR in the power spectra computed from time series of parameters derived from these profiles. The methodology described is quite general, and could be applied to photometric time series of other main-sequence stars for detecting differential rotation.

  11. Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase.

    PubMed

    Adroher, F J; Osuna, A; Lupiáñez, J A

    1990-04-18

    The activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11) and pyruvate kinase (ATP:pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4 microM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 microM. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60 mUnits/mg and 0.31 mM and for metacyclics 13.97 mUnits/mg and 0.16 mM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi.

  12. Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2.

    PubMed

    Forget, Diane; Lacombe, Andrée-Anne; Cloutier, Philippe; Lavallée-Adam, Mathieu; Blanchette, Mathieu; Coulombe, Benoit

    2013-08-01

    The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1-170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156-612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.

  13. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  14. Regulation of cardiomyocyte signaling by RGS proteins: differential selectivity towards G proteins and susceptibility to regulation.

    PubMed

    Hao, Jianming; Michalek, Christina; Zhang, Wei; Zhu, Ming; Xu, Xiaomei; Mende, Ulrike

    2006-07-01

    Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2-RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited Gq/11-mediated phospholipase C beta stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited Gq/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both Gq/11 and Gi/o signaling (carbachol-induced cAMP inhibition). Gs signaling was unaffected, and, contrary to reports in other cell lines, RGS2-RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced Gq/11 signaling (alpha1-adrenergic stimulation or Galphaq* overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of Gq/11 signaling (RGS3-RGS5) were unchanged. Endogenous RGS2 (but not RGS3-RGS5) expression was also up-regulated in cells with enhanced AC signaling (beta-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of Gq/11 signaling that could potentially become a promising

  15. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing.

    PubMed

    Rashmi, R N; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A

    2012-03-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

  16. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing

    PubMed Central

    Rashmi, R.N.; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A.

    2012-01-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing. PMID:22198684

  17. Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells.

    PubMed

    Hastie, Claire; Saxton, Malcolm; Akpan, Akunna; Cramer, Rainer; Masters, John R; Naaby-Hansen, Soren

    2005-09-01

    Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

  18. Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination

    PubMed Central

    Yamamoto, Yasusi; Hori, Haruka; Kai, Suguru; Ishikawa, Tomomi; Ohnishi, Atsuki; Tsumura, Nodoka; Morita, Noriko

    2013-01-01

    In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500–1,000 μmol photons m-2 s-1, and decreased at very high light intensity (1,500 μmol photons m-2 s-1). The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II. PMID:24194743

  19. Protein sorting within the MHC class II antigen-processing pathway.

    PubMed

    Marks, M S

    1998-01-01

    Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.

  20. Solar Differential Rotation in Calcium II K Line Spectra Supported with Spectroheliogram Analysis

    NASA Astrophysics Data System (ADS)

    Behm, Tyler; Keil, S. L.

    2013-07-01

    Two recent papers report on measuring differential rotation in data that views the Sun as a star. Unlike using tracers at different latitudes to measure the differential rotation, disk-integrated light averages over many latitudes and can only work if the features both exist at a dominate latitude that changes with the solar cycle and they persist long enough to affect the measured rotation rate. Bertello, Pevtsov, and Pietarila (2012, ApJ 761, pg 11) use disk-integrated Ca II K-line data from the SOLIS/ISS instrument to show that a change in rotation rate is clearly visible at the beginning of the current solar cycle in the disk-integrated K-line. Scargle, Keil, and Worden (2013, ApJ in press, arXiv:1303.6303) use the Sacramento Peak K-line series to look at the last current and previous three cycles with fairly strong evidence that the differential rotation is visible in cycle 22, but much harder to see in cycles 21 and 23. In order to understand the differences in the three cycles we report on solar differential rotation measurements in both the Sacramento Peak disk-integrated, Ca II K spectral time series (1977-2012) and full-disk, Ca II K spectroheliogram time series (1977-2002) observed at the Evans Solar Facility. The former data set is the same as used by Scargle et al (2013) and averages about 2-3 measurements per week. For the disk-integrated spectra, we use two interpolation schemes to fill in missing days (regression and singular value decomposition with proxy data sets) and use two methods (power spectra and autocorrelation) to find the rotation rates. We find a clear signature of solar differential rotation for solar cycle 21 and 22 and a partial signature for cycle 23. We test this result by measuring differential rotation using the Ca II K spectroheliograms using phase analysis between longitudinal bands. We have also explored the image features that lead to changes in the disk-integrated spectrum's signal-to-noise. The data analyzed in this

  1. Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27.

    PubMed

    Bunick, Christopher G; Presland, Richard B; Lawrence, Owen T; Pearton, David J; Milstone, Leonard M; Steitz, Thomas A

    2015-07-01

    The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein-protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.

  2. The Differential Response of Proteins to Macromolecular Crowding

    PubMed Central

    Candotti, Michela; Orozco, Modesto

    2016-01-01

    The habitat in which proteins exert their function contains up to 400 g/L of macromolecules, most of which are proteins. The repercussions of this dense environment on protein behavior are often overlooked or addressed using synthetic agents such as poly(ethylene glycol), whose ability to mimic protein crowders has not been demonstrated. Here we performed a comprehensive atomistic molecular dynamic analysis of the effect of protein crowders on the structure and dynamics of three proteins, namely an intrinsically disordered protein (ACTR), a molten globule conformation (NCBD), and a one-fold structure (IRF-3) protein. We found that crowding does not stabilize the native compact structure, and, in fact, often prevents structural collapse. Poly(ethylene glycol) PEG500 failed to reproduce many aspects of the physiologically-relevant protein crowders, thus indicating its unsuitability to mimic the cell interior. Instead, the impact of protein crowding on the structure and dynamics of a protein depends on its degree of disorder and results from two competing effects: the excluded volume, which favors compact states, and quinary interactions, which favor extended conformers. Such a viscous environment slows down protein flexibility and restricts the conformational landscape, often biasing it towards bioactive conformations but hindering biologically relevant protein-protein contacts. Overall, the protein crowders used here act as unspecific chaperons that modulate the protein conformational space, thus having relevant consequences for disordered proteins. PMID:27471851

  3. Differential expression of HLA class II antigens on human fetal and adult lymphocytes and macrophages.

    PubMed Central

    Edwards, J A; Jones, D B; Evans, P R; Smith, J L

    1985-01-01

    A panel of monoclonal antibodies to monomorphic determinants of the MHC class II subregion locus products: DP, DR and DQ, was used to investigate the expression of these antigens on early lymphocytes and macrophages from human fetal liver (13-20 weeks), placenta (16 weeks and term) and cord blood, in relation to the class II phenotype of cells from adult tonsil and peripheral blood. Fetal liver sections and cell suspensions showed differential expression of class II antigens. DP was expressed at a higher frequency (11.0% of nucleated cells) than DR on lymphoid cells and macrophages from fetal liver, and DQ was either absent or expressed on less than 0.3% of nucleated cells. Consistent with this finding, DP but not DR or DQ antigens were observed on vascular elements and macrophages in the villi of 16-week placenta. At term, all three subregion locus products were expressed. Adult tonsil and peripheral blood B lymphocytes expressed DP, DR and DQ antigens with similar frequency; however, DQ was expressed at a lower frequency than DP and DR on cord blood B lymphocytes. In contrast, 30-50% macrophages from cord blood and adult peripheral blood expressed DP and DR, but fewer (5% and 18%, respectively) expressed DQ. These data suggest that class II antigens are expressed in the sequence DP, DR, DQ on developing lymphocytes. A similar sequence is suggested for macrophages. Images Figure 1 Figure 2 Figure 3 PMID:3894221

  4. Interplay of matrix stiffness and protein tethering in stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Wen, Jessica H.; Vincent, Ludovic G.; Fuhrmann, Alexander; Choi, Yu Suk; Hribar, Kolin C.; Taylor-Weiner, Hermes; Chen, Shaochen; Engler, Adam J.

    2014-10-01

    Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

  5. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    SciTech Connect

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  6. Differential expression of secretogranin II and chromogranin A genes in the female rat pituitary through sexual maturation and estrous cycle

    SciTech Connect

    Anouar, Y.; Duval, J. )

    1991-03-01

    Secretogranin II (SgII) is a protein of pituitary secretory granules released by LHRH-stimulated gonadotrope cells. Estrogens and androgens are modulators of SgII release. Experiments were performed to determine the regulation of expression of the SgII gene in the female rat pituitary, during sexual maturation and according to the estrous cycle. Age- and cycle-related changes in SgII mRNA content were estimated through cytoplasmic slot blot; SgII content was determined by western blotting; maturation of the protein was controlled through (35S)sulfate labeling. Variations in chromogranin A (CgA), another protein of secretory granules, were analyzed in the same experimental conditions to assess the specificity of SgII regulation. The pituitary SgII concentration increased between days 7 and 21 (2.2-fold) and then declined to the initial 7-day-old value. Simultaneously, the CgA concentration went through a maximum between days 14 and 21 and then strongly dropped to barely detectable levels in the adult pituitary. The SgII mRNA concentration followed roughly the same pattern as the protein. Moreover, the sulfation level remained constant between days 14 and 60. These results demonstrated a regulatory mechanism operating, during sexual maturation, on the SgII gene and not on the protein processing or on storage/release steps. In the 4-day cycling females, the pituitary SgII mRNA and protein contents were the lowest during estrus. They then increased to their highest values in diestrus II. Moreover, the sulfation level of SgII was significantly higher during estrus than during any other stage. Due to its low content level, variations in pituitary CgA could not be demonstrated during the cycle.

  7. Protein palmitoylation regulates osteoblast differentiation through BMP-induced osterix expression.

    PubMed

    Leong, Wai Fook; Zhou, Tielin; Lim, Gek Liang; Li, Baojie

    2009-01-01

    Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53(-/-) osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

  8. rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

    2013-01-01

    Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

  9. Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Mallow, H; Trindl, A; Löffler, G

    2000-01-01

    During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.

  10. Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

    PubMed Central

    Crerar, M M; Leather, R; David, E; Pearson, M L

    1983-01-01

    To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation. Images PMID:6865946

  11. Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

    PubMed Central

    Lyn, Rodney K.; Hope, Graham; Sherratt, Allison R.; McLauchlan, John; Pezacki, John Paul

    2013-01-01

    Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. PMID:24223760

  12. Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

    PubMed Central

    Rouyez, M C; Boucheron, C; Gisselbrecht, S; Dusanter-Fourt, I; Porteu, F

    1997-01-01

    Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation. PMID:9271377

  13. Amide I'-II' 2D IR spectroscopy provides enhanced protein secondary structural sensitivity.

    PubMed

    Deflores, Lauren P; Ganim, Ziad; Nicodemus, Rebecca A; Tokmakoff, Andrei

    2009-03-11

    We demonstrate how multimode 2D IR spectroscopy of the protein amide I' and II' vibrations can be used to distinguish protein secondary structure. Polarization-dependent amide I'-II' 2D IR experiments on poly-l-lysine in the beta-sheet, alpha-helix, and random coil conformations show that a combination of amide I' and II' diagonal and cross peaks can effectively distinguish between secondary structural content, where amide I' infrared spectroscopy alone cannot. The enhanced sensitivity arises from frequency and amplitude correlations between amide II' and amide I' spectra that reflect the symmetry of secondary structures. 2D IR surfaces are used to parametrize an excitonic model for the amide I'-II' manifold suitable to predict protein amide I'-II' spectra. This model reveals that the dominant vibrational interaction contributing to this sensitivity is a combination of negative amide II'-II' through-bond coupling and amide I'-II' coupling within the peptide unit. The empirically determined amide II'-II' couplings do not significantly vary with secondary structure: -8.5 cm(-1) for the beta sheet, -8.7 cm(-1) for the alpha helix, and -5 cm(-1) for the coil.

  14. Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

    PubMed

    Alajem, Adi; Biran, Alva; Harikumar, Arigela; Sailaja, Badi Sri; Aaronson, Yair; Livyatan, Ilana; Nissim-Rafinia, Malka; Sommer, Andreia Gianotti; Mostoslavsky, Gustavo; Gerbasi, Vincent R; Golden, Daniel E; Datta, Arnab; Sze, Siu Kwan; Meshorer, Eran

    2015-03-31

    Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

  15. Rheumatoid Rescue of Misfolded Cellular Proteins by MHC Class II Molecules: A New Hypothesis for Autoimmune Diseases.

    PubMed

    Arase, Hisashi

    2016-01-01

    Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and thus are not transported outside cells. However, misfolded proteins in the endoplasmic reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by MHC class II molecules without being processed to peptides. Studies on the misfolded proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded proteins associated with MHC class II molecules and the autoimmune disease susceptibility conferred by each MHC class II allele. These new insights into MHC class II molecules suggest that misfolded proteins rescued from protein degradation by MHC class II molecules are recognized as "neo-self" antigens by immune system and are involved in autoimmune diseases as autoantibody targets.

  16. Arctic Micromonas uses protein pools and non-photochemical quenching to cope with temperature restrictions on Photosystem II protein turnover.

    PubMed

    Ni, Guangyan; Zimbalatti, Gabrielle; Murphy, Cole D; Barnett, Audrey B; Arsenault, Christopher M; Li, Gang; Cockshutt, Amanda M; Campbell, Douglas A

    2017-02-01

    Micromonas strains of small prasinophyte green algae are found throughout the world's oceans, exploiting widely different niches. We grew arctic and temperate strains of Micromonas and compared their susceptibilities to photoinactivation of Photosystem II, their counteracting Photosystem II repair capacities, their Photosystem II content, and their induction and relaxation of non-photochemical quenching. In the arctic strain Micromonas NCMA 2099, the cellular content of active Photosystem II represents only about 50 % of total Photosystem II protein, as a slow rate constant for clearance of PsbA protein limits instantaneous repair. In contrast, the temperate strain NCMA 1646 shows a faster clearance of PsbA protein which allows it to maintain active Photosystem II content equivalent to total Photosystem II protein. Under growth at 2 °C, the arctic Micromonas maintains a constitutive induction of xanthophyll deepoxidation, shown by second-derivative whole-cell spectra, which supports strong induction of non-photochemical quenching under low to moderate light, even if xanthophyll cycling is blocked. This non-photochemical quenching, however, relaxes during subsequent darkness with kinetics nearly comparable to the temperate Micromonas NCMA 1646, thereby limiting the opportunity cost of sustained downregulation of PSII function after a decrease in light.

  17. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    PubMed

    Prohaska, Thomas A; Wahlmüller, Felix C; Furtmüller, Margareta; Geiger, Margarethe

    2012-01-01

    The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

  18. Salmonella inhibits monocyte differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

    PubMed

    Rydström, Anna; Wick, Mary Jo

    2010-05-01

    Monocytes and DCs originate from a shared precursor in the bone marrow, and steady-state DCs in lymphoid organs develop directly from the precursor rather than via a monocyte intermediate. However, monocytes can differentiate into DCs in tissues such as the lung and gut mucosa and into macrophages in most tissues. As Ly6C hi monocytes accumulate in lymphoid organs during oral Salmonella infection, we investigated their ability to develop into potential DCs, identified as CD11c hi MHC-II hi cells, in infected hosts. Ly6C hi monocytes, isolated from the blood of Salmonella-infected mice, developed into CD11c hi MHC-II hi cells after culture with GM-CSF or Flt3L. In contrast, the same monocytes cultured in the presence of GM-CSF and heat-killed Salmonella did not differentiate into CD11c hi MHC-II hi cells. The bacteria-induced differentiation block was dependent on TLRs, as monocytes from MyD88-/- mice converted into CD11c hi MHC-II hi cells even in the presence of bacteria. We hypothesized that Salmonella-activated wild-type monocytes secreted mediators that inhibited differentiation of MyD88-/--derived monocytes. However, IL-6, IL-10, TNF-alpha, or IL-12p70 did not account for the inhibition. Finally, monocyte-derived CD11c hi MHC-II hi cells pulsed with OVA peptide or protein did not induce proliferation of antigen-specific CD4+ T cells but rather, suppressed the ability of DCs to activate CD4+ T cells. Overall, the data show that Ly6C hi monocytes from Salmonella-infected mice develop into CD11c hi MHC-II hi cells with poor antigen-presentation capacity when cultured ex vivo, and that monocyte exposure to Salmonella inhibits their differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

  19. Signaling of angiotensin II-induced vascular protein synthesis in conduit and resistance arteries in vivo

    PubMed Central

    Daigle, Christine; Martens, Fabrice MAC; Girardot, Daphné; Dao, Huy Hao; Touyz, Rhian M; Moreau, Pierre

    2004-01-01

    Background From in vitro studies, it has become clear that several signaling cascades are involved in angiotensin II-induced cellular hypertrophy. The aim of the present study was to determine some of the signaling pathways mediating angiotensin II (Ang II)-induced protein synthesis in vivo in large and small arteries. Methods Newly synthesized proteins were labeled during 4 hours with tritiated leucine in conscious control animals, or animals infused for 24 hours with angiotensin II (400 ng/kg/min). Hemodynamic parameters were measure simultaneously. Pharmacological agents affecting signaling cascades were injected 5 hours before the end of Ang II infusion. Results Angiotensin II nearly doubled the protein synthesis rate in the aorta and small mesenteric arteries, without affecting arterial pressure. The AT1 receptor antagonist Irbesartan antagonized the actions of Ang II. The Ang II-induced protein synthesis was associated with increased extracellular signal-regulated kinases (ERK)1/2 phosphorylation in aortic, but not in mesenteric vessels. Systemic administration of PD98059, an inhibitor of the ERK-1/2 pathway, produced a significant reduction of protein synthesis rate in the aorta, and only a modest decrease in mesenteric arteries. Rapamycin, which influences protein synthesis by alternative signaling, had a significant effect in both vessel types. Rapamycin and PD98059 did not alter basal protein synthesis and had minimal effects on arterial pressure. Conclusion ERK1/2 and rapamycin-sensitive pathways are involved in pressure-independent angiotensin II-induced vascular protein synthesis in vivo. However, their relative contribution may vary depending on the nature of the artery under investigation. PMID:15134586

  20. Heterochromatin protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II

    PubMed Central

    Kwon, So Hee; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2010-01-01

    Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II. PMID:20889714

  1. Differential effects of mutant SOD1 on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: role of chaperone network.

    PubMed

    Wei, Rochelle; Bhattacharya, Arunabh; Hamilton, Ryan T; Jernigan, Amanda L; Chaudhuri, Asish R

    2013-08-16

    Protein misfolding is considered to be a potential contributing factor for motor neuron and muscle loss in diseases like Amyotrophic lateral sclerosis (ALS). Several independent studies have demonstrated using over-expressed mutated Cu/Zn-superoxide dismutase (mSOD1) transgenic mouse models which mimic familial ALS (f-ALS), that both muscle and motor neurons undergo degeneration during disease progression. However, it is unknown whether protein conformation of skeletal muscle and spinal cord is equally or differentially affected by mSOD1-induced toxicity. It is also unclear whether heat shock proteins (Hsp's) differentially modulate skeletal muscle and spinal cord protein structure during ALS disease progression. We report three intriguing observations utilizing the f-ALS mouse model and cell-free in vitro system; (i) muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low level of soluble and absence of insoluble G93A protein aggregate, unlike in spinal cord, (ii) Hsp's levels are lower in muscle compared to spinal cord at any stage of the disease, and (iii) G93ASOD1 enzyme-induced toxicity selectively affects muscle protein conformation over spinal cord proteins. Together, these findings strongly suggest that differential chaperone levels between skeletal muscle and spinal cord may be a critical determinant for G93A-induced protein misfolding in ALS.

  2. Probing Proteins and Differentiating Their Native and Denatured States with Aggregation-Induced Emission Fluorogen

    NASA Astrophysics Data System (ADS)

    Leung, Chris Wai Tung; Hong, Yuning; Tang, Ben Zhong

    2013-06-01

    The tertiary 3D structures of proteins determine their unique functions. Perturbation of their native state including denaturation may cause loss of the protein functions. In this work, water-soluble tetraphenylethylene (TPE) fluorophore, sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (BSPOTPE), with aggregation-induced emission (AIE) characteristics is utilized as a fluorescent probe for protein detection and for differentiating their folding modes. Owing to hydrophobic interaction between the proteins and BSPOTPE, it provides a fast and simple method to differentiate the native and denatured states of the proteins through monitoring fluorescence change in solution and PAGE gels. Six proteins are chosen as model proteins in the study. Among them, cytochrome c shows distinctive behavior to other proteins due to the presence of heme group. A comprehensive study of cytochrome c and human serum albumin is carried out in this work.

  3. Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure.

    PubMed

    Soulages, Jose L; Kim, Kangmin; Arrese, Estela L; Walters, Christina; Cushman, John C

    2003-03-01

    Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation

  4. The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

    PubMed

    Lefebvre, Christophe; Largeau, Céline; Michelet, Xavier; Fourrage, Cécile; Maniere, Xavier; Matic, Ivan; Legouis, Renaud; Culetto, Emmanuel

    2016-04-01

    The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

  5. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination

    PubMed Central

    Yang, Hyun-Jeong; Vainshtein, Anna; Maik-Rachline, Galia; Peles, Elior

    2016-01-01

    While the formation of myelin by oligodendrocytes is critical for the function of the central nervous system, the molecular mechanism controlling oligodendrocyte differentiation remains largely unknown. Here we identify G protein-coupled receptor 37 (GPR37) as an inhibitor of late-stage oligodendrocyte differentiation and myelination. GPR37 is enriched in oligodendrocytes and its expression increases during their differentiation into myelin forming cells. Genetic deletion of Gpr37 does not affect the number of oligodendrocyte precursor cells, but results in precocious oligodendrocyte differentiation and hypermyelination. The inhibition of oligodendrocyte differentiation by GPR37 is mediated by suppression of an exchange protein activated by cAMP (EPAC)-dependent activation of Raf-MAPK-ERK1/2 module and nuclear translocation of ERK1/2. Our data suggest that GPR37 regulates central nervous system myelination by controlling the transition from early-differentiated to mature oligodendrocytes. PMID:26961174

  6. Collective Dynamics Differentiates Functional Divergence in Protein Evolution

    PubMed Central

    Glembo, Tyler J.; Farrell, Daniel W.; Gerek, Z. Nevin; Thorpe, M. F.; Ozkan, S. Banu

    2012-01-01

    Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function. PMID:22479170

  7. Differential expression of anti-Müllerian hormone (amh) and anti-Müllerian hormone receptor type II (amhrII) in the teleost medaka.

    PubMed

    Klüver, Nils; Pfennig, Frank; Pala, Irene; Storch, Katja; Schlieder, Marlen; Froschauer, Alexander; Gutzeit, Herwig O; Schartl, Manfred

    2007-01-01

    In mammals, the anti-Müllerian hormone (Amh) is responsible for the regression of the Müllerian ducts; therefore, Amh is an important factor of male sex differentiation. The amh gene has been cloned in various vertebrates, as well as in several teleost species. To date, all described species show a sexually dimorphic expression of amh during sex differentiation or at least in differentiated juvenile gonads. We have identified the medaka amh ortholog and examined its expression pattern. Medaka amh shows no sexually dimorphic expression pattern. It is expressed in both developing XY male and XX female gonads. In adult testes, amh is expressed in the Sertoli cells and in adult ovaries in granulosa cells surrounding the oocytes, like in mammals. To better understand the function of amh, we cloned the anti-Müllerian hormone receptor type II (amhrII) ortholog and compared its expression pattern with amh, aromatase (cyp19a1), and scp3. During gonad development, amhrII is coexpressed with medaka amh in somatic cells of the gonads and shows no sexually dimorphic expression. Only the expression level of the Amh type II receptor gene was decreased noticeably in adult female gonads. These results suggest that medaka Amh and AmhrII are involved in gonad formation and maintenance in both sexes.

  8. A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

    PubMed

    Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

    2015-12-21

    Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

  9. Breast Cancer Prevention by Fatty Acid Binding Protein MRG-Induced Pregnancy Like Mammary Gland Differentiation

    DTIC Science & Technology

    2005-08-01

    Annual Summary 3. DATES COVERED (From - To) 1 AUG 2004 - 31 JUL 2005 4. TITLE AND SUBTITLE Breast Cancer Prevention by Fatty Acid Binding Protein...differentiation. Overexpression of MRG in human breast cancer cells induced differentiation with changes in cellular morphology and a significant increase

  10. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.

    EPA Science Inventory

    Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. ...

  11. Visualization and Differential Analysis of Protein Expression Data Using R.

    PubMed

    Silva, Tomé S; Richard, Nadège

    2016-01-01

    Data analysis is essential to derive meaningful conclusions from proteomic data. This chapter describes ways of performing common data visualization and differential analysis tasks on gel-based proteomic datasets using a freely available statistical software package (R). A workflow followed is illustrated using a synthetic dataset as example.

  12. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    SciTech Connect

    Tan, Chye Ling; Gunaratne, Jayantha; Lai, Deborah; Carthagena, Laetitia; Wang, Qian; Xue, Yue Zhen; Quek, Ling Shih; Doorbar, John; Bachelerie, Francoise; Thierry, Francoise; Bellanger, Sophie

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  13. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry

    PubMed Central

    Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

    2016-01-01

    In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the

  14. Copper(II) binding to alpha-synuclein, the Parkinson's protein.

    PubMed

    Lee, Jennifer C; Gray, Harry B; Winkler, Jay R

    2008-06-04

    Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

  15. Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis.

    PubMed

    Cunha, A F; Sousa, M V; Silva, S P; Jesuíno, R S; Soares, C M; Felipe, M S

    1999-04-01

    Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

  16. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction

    PubMed Central

    Dumpala, Pradeep R.; Peterson, Brian C.; Lawrence, Mark L.; Karsi, Attila

    2015-01-01

    Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri. PMID:26168192

  17. Proposal of Dual Inhibitor Targeting ATPase Domains of Topoisomerase II and Heat Shock Protein 90

    PubMed Central

    Jun, Kyu-Yeon; Kwon, Youngjoo

    2016-01-01

    There is a conserved ATPase domain in topoisomerase II (topo II) and heat shock protein 90 (Hsp90) which belong to the GHKL (gyrase, Hsp90, histidine kinase, and MutL) family. The inhibitors that target each of topo II and Hsp90 are intensively studied as anti-cancer drugs since they play very important roles in cell proliferation and survival. Therefore the development of dual targeting anti-cancer drugs for topo II and Hsp90 is suggested to be a promising area. The topo II and Hsp90 inhibitors, known to bind to their ATP binding site, were searched. All the inhibitors investigated were docked to both topo II and Hsp90. Four candidate compounds as possible dual inhibitors were selected by analyzing the molecular docking study. The pharmacophore model of dual inhibitors for topo II and Hsp90 were generated and the design of novel dual inhibitor was proposed. PMID:27582553

  18. Differential Measurements of Oxidatively Modified Proteins in Colorectal Adenopolyps

    PubMed Central

    Mehrabi, Sharifeh; Wallace, Lashanale; Cohen, Shakeria; Yao, Xuebiao; Aikhionbare, Felix O.

    2015-01-01

    Introduction Adenopolyps patients have a three-fold higher risk of colon cancer over the general population, which increases to six-fold if the polyps are multiple and with lower survival among African American population. Currently, 6% of CRC can be ascribed to mutations in particular genes. Moreover, the optimal management of patients with colorectal adenopolyps depends on the accuracy of appropriate staging strategies because patients with similar colorectal adenocarcinoma architecture display heterogeneity in the course and outcome of the disease. Oxidative stress, due to an imbalance between reactive oxygen species (ROS) and antioxidant capacities as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids which promote tumor formation. Objective/Method This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and western blot analyses to assess the levels of oxidatively modified proteins in 41 pairs of primary colorectal tissues including normal/surrounding, adenopolyps (tubular, tubulovillous, villous, polypvillous) and carcinoma. Analysis of variance (ANOVA) and Student’s t-tests were utilized for the resulting data set. Results Our data showed that the levels of reactive protein carbonyl groups significantly increased as colorectal adenopolyps progresses to malignancy. No significant differences were found in the levels of carbonyl proteins between gender samples analyzed. For African American patients, there were, relative to Caucasians, 10% higher levels of reactive carbonyls in proteins of tubulovillous tissue samples (P < 0.05) and over 36% higher in levels in adenocarcinomas (P < 0.05). In normal tissues and tubular, there were no significant differences between the two groups in levels of protein carbonyls. Differences in the levels of protein carbonyl expression within individual patient samples with different number of tumor cells were notably

  19. Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

    PubMed

    Zhou, Qiu Gen; Peng, Xin; Hu, Li Li; Xie, Di; Zhou, Min; Hou, Fan Fan

    2010-10-01

    Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

  20. Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells.

    PubMed

    Schwind, Lisa; Wilhelm, Nadine; Kartarius, Sabine; Montenarh, Mathias; Gorjup, Erwin; Götz, Claudia

    2015-10-01

    CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.

  1. Alternatively spliced myeloid differentiation protein-2 (MD-2s) protein inhibits TLR4-mediated lung inflammation

    PubMed Central

    Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Jones, Heather D.; Chen, Shuang; Shimada, Kenichi; Crother, Timothy R.; Arditi, Moshe

    2014-01-01

    We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. Here we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intracheally (i.t.) an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid (BALF). Compared to Ad-EV control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, KC, and MIP-2. BALF from Ad-MD-2s mice transferred into lungs of naive mice before i.t. LPS challenge diminished pro-inflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on house dust mite (HDM)-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and house dust mite-triggered allergic lung inflammation. PMID:25576596

  2. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    PubMed Central

    Kim, Cy Hyun; Shin, Jin-Hong; Hwang, Sung Jun; Choi, Yung Hyun; Kim, Dae-Seong; Kim, Cheol Min

    2016-01-01

    Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

  3. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells.

    PubMed

    Re, Angela; Workman, Christopher T; Waldron, Levi; Quattrone, Alessandro; Brunak, Søren

    2014-09-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two programs. Functional analysis gathered insights in fate-specific candidates of interface functionalities. The non-transcriptionally regulated interface proteins were found to be highly regulated by post-translational ubiquitylation modification, which may synchronize the transition between cell proliferation and differentiation in ESCs.

  4. Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts*S⃞

    PubMed Central

    Yahi, Hakima; Fritsch, Lauriane; Philipot, Ophelie; Guasconi, Valentina; Souidi, Mouloud; Robin, Philippe; Polesskaya, Anna; Losson, Regine; Harel-Bellan, Annick; Ait-Si-Ali, Slimane

    2008-01-01

    Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1α, HP1β, and HP1γ play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1α and HP1β isoforms, but not HP1γ, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1α and HP1β, but not HP1γ, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner. PMID:18599480

  5. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    SciTech Connect

    Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  6. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

    PubMed

    Chow, Brian A; Donahue, Seth W; Vaughan, Michael R; McConkey, Brendan; Vijayan, Mathilakath M

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

  7. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  8. Protein Unfolding Coupled to Ligand Binding: Differential Scanning Calorimetry Simulation Approach

    ERIC Educational Resources Information Center

    Celej, Maria Soledad; Fidelio, Gerardo Daniel; Dassie, Sergio Alberto

    2005-01-01

    A comprehensive theoretical description of thermal protein unfolding coupled to ligand binding is presented. The thermodynamic concepts are independent of the method used to monitor protein unfolding but a differential scanning calorimetry is being used as a tool for examining the unfolding process.

  9. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide seq...

  10. Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide

    PubMed Central

    1991-01-01

    Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of

  11. Chronic antidepressants induce redistribution and differential activation of alphaCaM kinase II between presynaptic compartments.

    PubMed

    Barbiero, Valentina S; Giambelli, Roberto; Musazzi, Laura; Tiraboschi, Ettore; Tardito, Daniela; Perez, Jorge; Drago, Filippo; Racagni, Giorgio; Popoli, Maurizio

    2007-12-01

    Changes in synaptic plasticity are involved in pathophysiology of depression and in the mechanism of antidepressants. Ca(2+)/calmodulin (CaM) kinase II, a protein kinase involved in synaptic plasticity, has been previously shown to be a target of antidepressants. We previously found that antidepressants activate the kinase in hippocampal neuronal cell bodies by increasing phosphorylation at Thr(286), reduce the kinase phosphorylation in synaptic membranes, and in turn its phosphorylation-dependent interaction with syntaxin-1 and the release of glutamate from hippocampal synaptosomes. Here, we investigated the chronic effect of different antidepressants (fluoxetine, desipramine, and reboxetine) on the expression and function of the kinase in distinct subcellular compartments in order to dissect the different kinase pools affected. Acute treatments did not induce any change in the kinase. In total tissue extracts chronic drug treatments induced activation of the kinase; in hippocampus (HC), but not in prefrontal/frontal cortex, this was partially accounted for by increased Thr(286) phosphorylation, suggesting the involvement of different mechanisms of activation. In synaptosomes, all drugs reduced the kinase phosphorylation, particularly in HC where, upon fractionation of the synaptosomal particulate into synaptic vesicles and membranes, we found that the drugs induced a redistribution and differential activation of the kinase between membranes and vesicles. Furthermore, a large decrease in the level and phosphorylation of synapsin I located at synaptic membranes was consistent with the observed decrease of CaM kinase II. Overall, antidepressants induce a complex pattern of modifications in distinct subcellular compartments; at presynaptic level, these changes are in line with a dampening of glutamate release.

  12. A general model of invariant chain association with class II major histocompatibility complex proteins.

    PubMed Central

    Lee, C; McConnell, H M

    1995-01-01

    The binding of invariant chain to major histocompatibility complex (MHC) proteins is an important step in processing of MHC class II proteins and in antigen presentation. The question of how invariant chain can bind to all MHC class II proteins is central to understanding these processes. We have employed molecular modeling to predict the structure of class II-associated invariant chain peptide (CLIP)-MHC protein complexes and to ask whether the predicted mode of association could be general across all MHC class II proteins. CLIP fits identically into the MHC class II alleles HLA-DR3, I-Ak, I-Au, and I-Ad, with a consistent pattern of hydrogen bonds, contacts, and hydrophobic burial and without bad contacts. Our model predicts the burial of CLIP residues Met-91 and Met-99 in the deep P1 and P9 anchor pockets and other detailed interactions, which we have compared with available data. The predicted pattern of I-A allele-specific effects on CLIP binding is very similar to that observed experimentally by alanine-scanning mutations of CLIP. Together, these results indicate that CLIP may bind in a single, general way across products of MHC class II alleles. Images Fig. 1 Fig. 2 Fig. 3 PMID:7667280

  13. Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

    PubMed Central

    Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

    2015-01-01

    We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934

  14. A Hybrid Approach to Protein Differential Expression in Mass Spectrometry-Based Proteomics

    SciTech Connect

    Wang, Xuan; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2012-04-19

    Motivation: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein's associated spectral peaks. However, typical MS-based proteomics data sets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. Results: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of 'presence/ absence,' we enable the selection of proteins not typically amendable to quantitative analysis; e.g., 'one-state' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/ absence analysis of a given data set in a principled way, resulting in a single list of selected proteins with a single associated FDR.

  15. Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

    PubMed

    Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides.

  16. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1

    SciTech Connect

    Desprez, P.; Hara, E.; Bissell, M.J.

    1995-06-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

  17. Differential DNA binding properties of three human homeodomain proteins.

    PubMed Central

    Corsetti, M T; Briata, P; Sanseverino, L; Daga, A; Airoldi, I; Simeone, A; Palmisano, G; Angelini, C; Boncinelli, E; Corte, G

    1992-01-01

    The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes. Images PMID:1357628

  18. Fetal rat lung type II cell differentiation in serum-free isolated cell culture: modulation and inhibition.

    PubMed

    Fraslon, C; Lacaze-Masmonteil, T; Zupan, V; Chailley-Heu, B; Bourbon, J R

    1993-05-01

    Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

  19. Analysis of Differential Proteins in Two Wing-Type Females of Sogatella furcifera (Hemiptera: Delphacidae).

    PubMed

    Liang, Zi-Qiang; Song, Shao-Yun; Liang, Shi-Ke; Wang, Fang-Hai

    2016-01-01

    Sogatella furcifera(Horvath) is an important rice pest with the wing dimorphism, including macropterous and brachypterous morphs. The protein expression profiles in two wing-type adults and two wing-type disc fifth-instar nymphs were analyzed using two-dimensional gel protein electrophoresis and mass spectrometry. In adults and fifth-instar nymphs, 127 and 162 protein spots were detected, respectively. Fifty-five differentially expressed protein spots were identified between the long-winged adults and the short-winged adults, and 62 differentially expressed protein spots were found between the long-winged disc fifth-instar nymphs and short-winged disc fifth-instar nymphs. In long-winged and short-winged adults, six and seven specific protein spots were identified, respectively, with five and seven protein spots having more than threefold increased level, respectively. In long-winged and short-winged disc morph nymphs, 8 and 12 specific protein spots were identified, respectively, with 11 and 17 spots containing more than threefold increased level, respectively. Among the 16 identified proteins, five proteins are associated with muscle function, suggesting that muscle is a main tissue where the genes were differentially expressed between the two wing types. In addition, the content of a peptidase with an insulinase domain was higher (by 3.02 ± 0.59 fold) in the short-winged fifth-instar nymphs than in the long-winged fifth-instar nymphs, which suggests that this peptidase may be involved in wing differentiation by regulating insulin receptors. The results of this study provide some genetic clues for the wing differential development inS. furcifera and provide more references for future studies.

  20. Analysis of Differential Proteins in Two Wing-Type Females of Sogatella furcifera (Hemiptera: Delphacidae)

    PubMed Central

    Liang, Zi-Qiang; Song, Shao-Yun; Liang, Shi-Ke; Wang, Fang-Hai

    2016-01-01

    Sogatella furcifera (Horvath) is an important rice pest with the wing dimorphism, including macropterous and brachypterous morphs. The protein expression profiles in two wing-type adults and two wing-type disc fifth-instar nymphs were analyzed using two-dimensional gel protein electrophoresis and mass spectrometry. In adults and fifth-instar nymphs, 127 and 162 protein spots were detected, respectively. Fifty-five differentially expressed protein spots were identified between the long-winged adults and the short-winged adults, and 62 differentially expressed protein spots were found between the long-winged disc fifth-instar nymphs and short-winged disc fifth-instar nymphs. In long-winged and short-winged adults, six and seven specific protein spots were identified, respectively, with five and seven protein spots having more than threefold increased level, respectively. In long-winged and short-winged disc morph nymphs, 8 and 12 specific protein spots were identified, respectively, with 11 and 17 spots containing more than threefold increased level, respectively. Among the 16 identified proteins, five proteins are associated with muscle function, suggesting that muscle is a main tissue where the genes were differentially expressed between the two wing types. In addition, the content of a peptidase with an insulinase domain was higher (by 3.02 ± 0.59 fold) in the short-winged fifth-instar nymphs than in the long-winged fifth-instar nymphs, which suggests that this peptidase may be involved in wing differentiation by regulating insulin receptors. The results of this study provide some genetic clues for the wing differential development in S. furcifera and provide more references for future studies. PMID:27044649

  1. Site-specific protein glycosylation analysis with glycan isomer differentiation.

    PubMed

    Hua, Serenus; Nwosu, Charles C; Strum, John S; Seipert, Richard R; An, Hyun Joo; Zivkovic, Angela M; German, J Bruce; Lebrilla, Carlito B

    2012-05-01

    Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

  2. Iron(II) Initiation of Lipid and Protein Oxidation in Pork: The Role of Oxymyoglobin.

    PubMed

    Zhou, Feibai; Jongberg, Sisse; Zhao, Mouming; Sun, Weizheng; Skibsted, Leif H

    2016-06-08

    Iron(II), added as FeSO4·7H2O, was found to increase the rate of oxygen depletion as detected electrochemically in a pork homogenate from Longissimus dorsi through an initial increase in metmyoglobin formation from oxymyoglobin and followed by formation of primary and secondary lipid oxidation products and protein oxidation as detected as thiol depletion in myofibrillar proteins. Without added iron(II), under the same conditions at 37 °C, oxygen consumption corresponded solely to the slow oxymyoglobin autoxidation. Long-lived myofibrillar protein radicals as detected by ESR spectroscopy in the presence of iron(II) were formed subsequently to oxymyoglobin oxidation, and their level was increased by lipid oxidation when oxygen was completely depleted. Similarly, the time profile for formation of lipid peroxide indicated that oxymyoglobin oxidation initiates both protein oxidation and lipid oxidation.

  3. Serum CRP protein as a differential marker in cancer.

    PubMed

    Juan, Hu; Qijun, Wang; Junlan, Liu

    2012-11-01

    The objective of this study was to measure the serum concentrations of C-reactive protein (CRP) in cancer patients and compare with those of immune disease patients and healthy individuals for discriminatory analysis. For this purpose, automatic systems for special protein analysis (Type: Drcon Diognostica Tarbox) was used to measure serum CRP concentrations in 276 cancer patients (Group A), 110 immune disease patients (Group B), 161 phlogistic patients (Group C), and 125 age-matched healthy individuals (Group D). Our data show that serum CRP concentrations in Group A were significantly higher than those in Groups B and D, whereas CRP concentrations in Group B were higher than those in Group D. The differences of serum CRP concentrations between Groups A and B as well as between Groups B and D were significant (P < 0.01). We, therefore, concluded that the measurement of serum CRP concentrations was a fast and accurate method to distinguish between cancer and immune disease patients.

  4. Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation.

    PubMed

    Hansen, Jacob B; te Riele, Hein; Kristiansen, Karsten

    2004-06-01

    The differentiation of white and brown fat cells is controlled by a similar set of transcription factors, including PPARgamma and C/EBPalpha. However, despite many similarities between the two types of fat cells, they carry out essentially opposite functions in vivo, with white adipocytes being the major energy store and brown adipocytes being potent energy-dissipaters through thermogenesis. Yet, little is known about factors differentially regulating the formation of white and brown fat cells. Members of the retinoblastoma protein family (pRB, p107, p130) have been implicated in the regulation of adipocyte differentiation, and expression and phosphorylation of the three retinoblastoma family proteins oscillate in a characteristic manner during differentiation of the white preadipocyte cell line 3T3-L1. We have recently demonstrated a surprising function of the retinoblastoma protein in the regulation of white versus brown adipocyte differentiation in vitro and possibly in vivo. Here we summarize the current knowledge on the retinoblastoma protein in fat cells, with particular emphasis on its potential role in adipocyte lineage commitment and differentiation.

  5. Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function

    PubMed Central

    Okamura, Hirohiko; Yoshida, Kaya; Morimoto, Hiroyuki; Teramachi, Jumpei; Ochiai, Kazuhiko; Haneji, Tatsuji; Yamamoto, Akihito

    2017-01-01

    The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes. PMID:28241467

  6. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    SciTech Connect

    Walsh, Erica M.; Niu, MengMeng; Bergholz, Johann; Jim Xiao, Zhi-Xiong

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  7. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    PubMed

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  8. Differential Relationships between WISC-IV and WIAT-II Scales: An Evaluation of Potentially Moderating Child Demographics

    ERIC Educational Resources Information Center

    Konold, Timothy R.; Canivez, Gary L.

    2010-01-01

    Considerable debate exists regarding the accuracy of intelligence tests with members of different groups. This study investigated differential predictive validity of the "Wechsler Intelligence Scale for Children-Fourth Edition". Participants from the WISC-IV--WIAT-II standardization linking sample (N = 550) ranged in age from 6 through…

  9. Higher-Order Factor Structure of the Differential Ability Scales-II: Consistency across Ages 4 to 17

    ERIC Educational Resources Information Center

    Keith, Timothy Z.; Low, Justin A.; Reynolds, Matthew R.; Patel, Puja G.; Ridley, Kristen P.

    2010-01-01

    The recently published second edition of the Differential Abilities Scale (DAS-II) is designed to measure multiple broad and general abilities from Cattell-Horn-Carroll (CHC) theory. Although the technical manual presents information supporting the test's structure, additional research is needed to determine the constructs measured by the test and…

  10. A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry,.as both infect the ram’s horn snail, Plano...

  11. Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

    PubMed Central

    Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

    2015-01-01

    Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in

  12. The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

    PubMed Central

    Sundsten, Tea; Eberhardson, Michael; Göransson, Michael; Bergsten, Peter

    2006-01-01

    Background The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. Conclusion Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed. PMID:17163994

  13. Site specific chemoselective labelling of proteins with robust and highly sensitive Ru(II) bathophenanthroline complexes.

    PubMed

    Uzagare, Matthew C; Claussnitzer, Iris; Gerrits, Michael; Bannwarth, Willi

    2012-03-21

    The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(II) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies.

  14. Structural and functional characterization of endonexin II, a calcium- and phospholipid-binding protein

    SciTech Connect

    Schlaepfer, D.D.; Mehlman, T.; Burgess, W.H.; Haigler, H.T.

    1987-09-01

    A protein with an apparent M/sub r/ of 33,000 was previously purified from the EGTA eluate of a human placental particulate fraction. The authors now report the amino acid sequence of approximately one-third of this protein and show that it has extensive homology with a newly defined family of Ca/sup 2 +/-binding proteins termed annexins. The partial sequence of the placental protein could be aligned with the sequence of either lipocortin I or calpactin I such that 49% and 58%, respectively, of the residues were identical. A comparison of the partial sequences of the placental protein with the partial sequence of bovine endonexin revealed 74% sequence identity. Based on this close relationship, the placental protein was named endonexin II. Equilibrium dialysis showed that endonexin II bound Ca/sup 2 +/ and the affinity was increased by phosphatidylserine liposomes. In addition, endonexin II bound to phosphatidylserine- and phosphatidylethanolamine-containing liposomes in a Ca/sup 2 +/ -dependent manner, and the binding was cooperative with respect to Ca/sup 2 +/ concentration. The Ca/sup 2 +/- and phospholipid-binding properties of endonexin II raise the possibility that each of the four internally repeated sequences that have been demonstrated within this family of proteins contains a Ca/sup 2 +/-binding site

  15. Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin.

    PubMed

    Malaguti, Mattias; Nistor, Paul A; Blin, Guillaume; Pegg, Amy; Zhou, Xinzhi; Lowell, Sally

    2013-12-17

    Bone morphogenic protein (BMP) signalling contributes towards maintenance of pluripotency and favours mesodermal over neural fates upon differentiation, but the mechanisms by which BMP controls differentiation are not well understood. We report that BMP regulates differentiation by blocking downregulation of Cdh1, an event that accompanies the earliest stages of neural and mesodermal differentiation. We find that loss of Cdh1 is a limiting requirement for differentiation of pluripotent cells, and that experimental suppression of Cdh1 activity rescues the BMP-imposed block to differentiation. We further show that BMP acts prior to and independently of Cdh1 to prime pluripotent cells for mesoderm differentiation, thus helping to reinforce the block to neural differentiation. We conclude that differentiation depends not only on exposure to appropriate extrinsic cues but also on morphogenetic events that control receptivity to those differentiation cues, and we explain how a key pluripotency signal, BMP, feeds into this control mechanism. DOI: http://dx.doi.org/10.7554/eLife.01197.001.

  16. Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin

    PubMed Central

    Malaguti, Mattias; Nistor, Paul A; Blin, Guillaume; Pegg, Amy; Zhou, Xinzhi; Lowell, Sally

    2013-01-01

    Bone morphogenic protein (BMP) signalling contributes towards maintenance of pluripotency and favours mesodermal over neural fates upon differentiation, but the mechanisms by which BMP controls differentiation are not well understood. We report that BMP regulates differentiation by blocking downregulation of Cdh1, an event that accompanies the earliest stages of neural and mesodermal differentiation. We find that loss of Cdh1 is a limiting requirement for differentiation of pluripotent cells, and that experimental suppression of Cdh1 activity rescues the BMP-imposed block to differentiation. We further show that BMP acts prior to and independently of Cdh1 to prime pluripotent cells for mesoderm differentiation, thus helping to reinforce the block to neural differentiation. We conclude that differentiation depends not only on exposure to appropriate extrinsic cues but also on morphogenetic events that control receptivity to those differentiation cues, and we explain how a key pluripotency signal, BMP, feeds into this control mechanism. DOI: http://dx.doi.org/10.7554/eLife.01197.001 PMID:24347544

  17. Concentration-dependent Cu(II) binding to prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  18. Differential proteins of the optic ganglion in octopus vulgaris under methanol stress revealed using proteomics.

    PubMed

    Huang, Lin; Huang, Qing-Yu; Chen, Hai-Bin; Huang, Fu-Sheng; Huang, He-Qing

    2011-10-01

    An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

  19. Differentially expressed proteins in fluconazole-susceptible and fluconazole-resistant isolates of Candida glabrata.

    PubMed

    Shen, Yinzhong; Zhang, Lijun; Jia, Xiaofang; Zhang, Yongxin; Lu, Hongzhou

    2015-06-01

    The current study aimed to identify the differences presented in the proteome of fluconazole-susceptible isolates of Candida glabrata compared to those with fluconazole-resistant ones. Two-dimensional differential gel electrophoresis was applied to identify proteins that were differentially expressed in fluconazole-susceptible and fluconazole-resistant isolates of C. glabrata. Eight proteins including aspartyl-tRNA synthetase, translation elongation factor 3, 3-phosphoglycerate kinase, ribosomal protein L5, coproporphyrinogen III oxidase, pyruvate kinase, G-beta like protein, and F1F0-ATPase alpha subunit were found to be more abundantly represented, while four proteins including vitamin B12-(cobalamin)-independent isozyme of methionine synthase, microtubule-associated protein, adenylosuccinate synthetase, and aldose reductase were found to be less abundantly represented in fluconazole-resistant strains versus those with fluconazole-susceptible ones. These differentially expressed proteins were primarily associated with energy metabolism, stress response, and macromolecule synthesis. Proteins associated with energy metabolism, stress response, and macromolecule synthesis may play a role in the development of fluconazole resistance in the clinical isolates of C. glabrata. Multiple different mechanisms are involved in the development of fluconazole resistance in C. glabrata. These findings provide a scientific basis for discovering new genes and mechanisms associated with fluconazole resistance in C. glabrata.

  20. Detecting differential protein expression in large-scale population proteomics

    SciTech Connect

    Ryu, Soyoung; Qian, Weijun; Camp, David G.; Smith, Richard D.; Tompkins, Ronald G.; Davis, Ronald W.; Xiao, Wenzhong

    2014-06-17

    Mass spectrometry-based high-throughput quantitative proteomics shows great potential in clinical biomarker studies, identifying and quantifying thousands of proteins in biological samples. However, methods are needed to appropriately handle issues/challenges unique to mass spectrometry data in order to detect as many biomarker proteins as possible. One issue is that different mass spectrometry experiments generate quite different total numbers of quantified peptides, which can result in more missing peptide abundances in an experiment with a smaller total number of quantified peptides. Another issue is that the quantification of peptides is sometimes absent, especially for less abundant peptides and such missing values contain the information about the peptide abundance. Here, we propose a Significance Analysis for Large-scale Proteomics Studies (SALPS) that handles missing peptide intensity values caused by the two mechanisms mentioned above. Our model has a robust performance in both simulated data and proteomics data from a large clinical study. Because varying patients’ sample qualities and deviating instrument performances are not avoidable for clinical studies performed over the course of several years, we believe that our approach will be useful to analyze large-scale clinical proteomics data.

  1. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    NASA Astrophysics Data System (ADS)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS – which was released from scaffolds quickly – significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  2. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    PubMed Central

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-01-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS – which was released from scaffolds quickly – significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA. PMID:28256634

  3. Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast.

    PubMed

    Yuan, Zhuning; Chen, Yaosheng; Zhang, Xumeng; Zhou, Xingyu; Li, Mingsen; Chen, Hu; Wu, Ming; Zhang, Ying; Mo, Delin

    2017-03-11

    Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

  4. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)*

    PubMed Central

    Rieger, Megan E.; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-hong; Minoo, Parviz; Crandall, Edward D.; Brody, Steven L.; Kahn, Michael; Borok, Zea

    2016-01-01

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  5. Proteomic analysis of differentially expressed proteins in the two developmental stages of Ichthyophthirius multifiliis.

    PubMed

    Yao, Jia-Yun; Xu, Yang; Yuan, Xue-Mei; Yin, Wen-Lin; Yang, Gui-Lian; Lin, Ling-Yun; Pan, Xiao-Yi; Wang, Chun-Feng; Shen, Jin-Yu

    2017-02-01

    Ichthyophthirius is a severe disease of farmed freshwater fish caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich). This disease can lead to considerable economic loss, but the protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from trophonts and theronts of Ich were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2300 proteins were identified in the two developmental stages, of which 1520 proteins were differentially expressed. Among them, 84 proteins were uniquely expressed in the theronts stage, while 656 proteins were expressed only in trophonts. The differentially expressed proteins were catalogued (assorted) to various functions of Ich life cycle, including biological process, cellular component, and molecular function that occur at distinct stages. Using a 1.5-fold change in expression as a physiologically significant benchmark, a lot of differentially expressed proteins were reliably quantified by iTRAQ analysis. Two hundred forty upregulated and 57 downregulated proteins in the trophonts stage were identified as compared with theronts. The identified proteins were involved in various functions of the I. multifiliis life cycle, including binding, catalytic activity, structural molecule activity, and transporter activity. Further investigation of the transcriptional levels of periplasmic immunogenic protein, transketolase, zinc finger, isocitrate dehydrogenase, etc., from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. This work provides an effective resource to further our understanding of Ich biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

  6. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    PubMed Central

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  7. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    PubMed

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  8. Changes in erythroid membrane proteins during erythropoietin-mediated terminal differentiation.

    PubMed

    Koury, M J; Bondurant, M C; Rana, S S

    1987-12-01

    Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of

  9. Structure of catabolite activator protein with cobalt(II) and sulfate

    SciTech Connect

    Rao, Ramya R.; Lawson, Catherine L.

    2014-04-15

    The crystal structure of E. coli catabolite activator protein with bound cobalt(II) and sulfate ions at 1.97 Å resolution is reported. The crystal structure of cyclic AMP–catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP–DNA complex structures.

  10. Integral and differential form of the protein folding problem

    NASA Astrophysics Data System (ADS)

    Tramontano, Anna

    2004-07-01

    The availability of the complete genomic sequences of many species, including human, has raised enormous expectations in medicine, pharmacology, ecology, biotechnology and forensic sciences. However, knowledge is only a first step toward understanding, and we are only at the early stage of a scientific process that might lead us to satisfy all the expectations raised by the genomic projects. In this review I will discuss the present status of computational methods that attempt to infer the unique three-dimensional structure of proteins from their amino acid sequences. Although this problem has been defined as the “holy grail” of biology, it represents only one of the many hurdles in our path towards the understanding of life at a molecular level.

  11. Excess manganese differentially inhibits photosystem I versus II in Arabidopsis thaliana

    PubMed Central

    Alberdi, M.

    2013-01-01

    The effects of exposure to increasing manganese concentrations (50–1500 µM) from the start of the experiment on the functional performance of photosystem II (PSII) and photosystem I (PSI) and photosynthetic apparatus composition of Arabidopsis thaliana were compared. In agreement with earlier studies, excess Mn caused minimal changes in the PSII photochemical efficiency measured as Fv/Fm, although the characteristic peak temperature of the S2/3QB – charge recombinations was shifted to lower temperatures at the highest Mn concentration. SDS-PAGE and immunoblot analyses also did not exhibit any significant change in the relative abundance of PSII-associated polypeptides: PSII reaction centre protein D1, Lhcb1 (major light-harvesting protein of LHCII complex), and PsbO (OEC33, a 33kDa protein of the oxygen-evolving complex). In addition, the abundance of Rubisco also did not change with Mn treatments. However, plants grown under excess Mn exhibited increased susceptibility to PSII photoinhibition. In contrast, in vivo measurements of the redox transients of PSI reaction centre (P700) showed a considerable gradual decrease in the extent of P700 photooxidation (P700+) under increased Mn concentrations compared to control. This was accompanied by a slower rate of P700+ re-reduction indicating a downregulation of the PSI-dependent cyclic electron flow. The abundance of PSI reaction centre polypeptides (PsaA and PsaB) in plants under the highest Mn concentration was also significantly lower compared to the control. The results demonstrate for the first time that PSI is the major target of Mn toxicity within the photosynthetic apparatus of Arabidopsis plants. The possible involvement mechanisms of Mn toxicity targeting specifically PSI are discussed. PMID:23183256

  12. Excess manganese differentially inhibits photosystem I versus II in Arabidopsis thaliana.

    PubMed

    Millaleo, R; Reyes-Díaz, M; Alberdi, M; Ivanov, A G; Krol, M; Hüner, N P A

    2013-01-01

    The effects of exposure to increasing manganese concentrations (50-1500 µM) from the start of the experiment on the functional performance of photosystem II (PSII) and photosystem I (PSI) and photosynthetic apparatus composition of Arabidopsis thaliana were compared. In agreement with earlier studies, excess Mn caused minimal changes in the PSII photochemical efficiency measured as F(v)/F(m), although the characteristic peak temperature of the S(2/3)Q(B) (-) charge recombinations was shifted to lower temperatures at the highest Mn concentration. SDS-PAGE and immunoblot analyses also did not exhibit any significant change in the relative abundance of PSII-associated polypeptides: PSII reaction centre protein D1, Lhcb1 (major light-harvesting protein of LHCII complex), and PsbO (OEC33, a 33 kDa protein of the oxygen-evolving complex). In addition, the abundance of Rubisco also did not change with Mn treatments. However, plants grown under excess Mn exhibited increased susceptibility to PSII photoinhibition. In contrast, in vivo measurements of the redox transients of PSI reaction centre (P700) showed a considerable gradual decrease in the extent of P700 photooxidation (P700(+)) under increased Mn concentrations compared to control. This was accompanied by a slower rate of P700(+) re-reduction indicating a downregulation of the PSI-dependent cyclic electron flow. The abundance of PSI reaction centre polypeptides (PsaA and PsaB) in plants under the highest Mn concentration was also significantly lower compared to the control. The results demonstrate for the first time that PSI is the major target of Mn toxicity within the photosynthetic apparatus of Arabidopsis plants. The possible involvement mechanisms of Mn toxicity targeting specifically PSI are discussed.

  13. Cooperative binding modes of Cu(II) in prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  14. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  15. HNF4α Regulates Claudin-7 Protein Expression during Intestinal Epithelial Differentiation

    PubMed Central

    Farkas, Attila E.; Hilgarth, Roland S.; Capaldo, Christopher T.; Gerner-Smidt, Christian; Powell, Doris R.; Vertino, Paula M.; Koval, Michael; Parkos, Charles A.; Nusrat, Asma

    2016-01-01

    The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. PMID:26216285

  16. Muscle Lim Protein isoform negatively regulates striated muscle actin dynamics and differentiation

    PubMed Central

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A.; Papalouka, Vasiliki; Terzis, Gerasimos; Roumeliotis, Theodoros I.; Spengos, Konstantinos; Garbis, Spiros D.; Manta, Panagiota; Kranias, Evangelia G.; Sanoudou, Despina

    2015-01-01

    Muscle Lim Protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, while aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/CFL2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components including α-actinin, T-cap and MLP. Our findings unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, and in differentiated striated muscles as a contributor to sarcomeric integrity. PMID:24860983

  17. A simple theory of motor protein kinetics and energetics. II.

    PubMed

    Qian, H

    2000-01-10

    A three-state stochastic model of motor protein [Qian, Biophys. Chem. 67 (1997) pp. 263-267] is further developed to illustrate the relationship between the external load on an individual motor protein in aqueous solution with various ATP concentrations and its steady-state velocity. A wide variety of dynamic motor behavior are obtained from this simple model. For the particular case of free-load translocation being the most unfavorable step within the hydrolysis cycle, the load-velocity curve is quasi-linear, V/Vmax = (cF/Fmax-c)/(1-c), in contrast to the hyperbolic relationship proposed by A.V. Hill for macroscopic muscle. Significant deviation from the linearity is expected when the velocity is less than 10% of its maximal (free-load) value--a situation under which the processivity of motor diminishes and experimental observations are less certain. We then investigate the dependence of load-velocity curve on ATP (ADP) concentration. It is shown that the free load Vmax exhibits a Michaelis-Menten like behavior, and the isometric Fmax increases linearly with ln([ATP]/[ADP]). However, the quasi-linear region is independent of the ATP concentration, yielding an apparently ATP-independent maximal force below the true isometric force. Finally, the heat production as a function of ATP concentration and external load are calculated. In simple terms and solved with elementary algebra, the present model provides an integrated picture of biochemical kinetics and mechanical energetics of motor proteins.

  18. TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

    PubMed Central

    Batrakou, Dzmitry G.; de las Heras, Jose I.; Czapiewski, Rafal; Mouras, Rabah; Schirmer, Eric C.

    2015-01-01

    Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity. PMID:26024229

  19. Knockdown of Lingo1b protein promotes myelination and oligodendrocyte differentiation in zebrafish.

    PubMed

    Yin, Wu; Hu, Bing

    2014-01-01

    Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the central nervous system (CNS), resulting in myelin sheath damage and axonal loss. Leucine-rich repeat and immunoglobulin domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1 (LINGO-1) have been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1 inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. In this study, we cloned the zebrafish homolog of the human LINGO-1 and found that lingo1b regulated myelination and oligodendrocyte differentiation. The expression of lingo1b started 1 (mRNA) and 2 (protein) days post-fertilization (dpf) in the CNS. Morpholino oligonucleotide knockdown of lingo1b resulted in developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord. The lack of lingo1b enhanced myelination and oligodendrocyte differentiation during embryogenesis. Furthermore, immunohistochemistry and movement analysis showed that lingo1b was involved in the axon development of primary motor neurons. These results suggested that Lingo1b protein functions as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development.

  20. Profiling of Proteins Regulated by Venlafaxine during Neural Differentiation of Human Cells

    PubMed Central

    Doh, Mi Sook; Han, Dal Mu Ri; Oh, Dong Hoon; Kim, Seok Hyeon

    2015-01-01

    Objective Antidepressants are known to positively influence several factors in patients with depressive disorders, resulting in increased neurogenesis and subsequent relief of depressive disorders. To study the effects of venlafaxine during neural differentiation at the cellular level, we looked at its effect on protein expression and regulation mechanisms during neural differentiation. Methods After exposing NCCIT cell-derived EBs to venlafaxine during differentiation (1 day and 7 days), changes in protein expression were analyzed by 2-DE and MALDI-TOF MS analysis. Gene levels of proteins regulated by venlafaxine were analyzed by real-time RT-PCR. Results Treatment with venlafaxine decreased expression of prolyl 4-hydroxylase (P4HB), ubiquitin-conjugating enzyme E2K (HIP2) and plastin 3 (T-plastin), and up-regulated expression of growth factor beta-3 (TGF-β3), dihydropyrimidinase-like 3 (DPYSL3), and pyruvate kinase (PKM) after differentiation for 1 and 7 days. In cells exposed to venlafaxine, the mRNA expression patterns of HIP2 and PKM, which function as negative and positive regulators of differentiation and neuronal survival, respectively, were consistent with the observed changes in protein expression. Conclusion Our findings may contribute to improve understanding of molecular mechanism of venlafaxine. PMID:25670950

  1. Differential expressed protein in developing stages of Nepenthes gracilis Korth. pitcher.

    PubMed

    Pinthong, Krit; Chaveerach, Arunrat; Tanee, Tawatchai; Sudmoon, Runglawan; Mokkamul, Piya

    2009-03-15

    Nepenthes gracilis Korth. is a member of carnivorous plants in family Nepenthaceae. The plants have beautiful and economically important pitchers. It is interesting to study the protein(s) correlated with the pitcher. Crude proteins were extracted from leaf, leaf with developing pitcher and developed pitcher of the same plant and analyzed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Two protein bands with molecular weights of 42.7 and 38 kDa were obtained from young leaf and leaf with developing pitcher, respectively. The 42.7 kDa protein was identified as phosphoglycerate kinase (PGK) by Liquid Chromatography Mass Spectrometry (LC-MS/MS), but the 38 kDa band is an unknown protein. Both proteins were differentially expressed in each developing stage of the pitcher, thus may be powerful candidates play role in development pathway of leaf and pitcher.

  2. Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus.

    PubMed

    Walliwalagedara, Chamari; Atkinson, Ian; van Keulen, Harry; Cutright, Teresa; Wei, Robert

    2010-09-01

    The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.

  3. Angiotensin II-Activated Protein Kinase D Mediates Acute Aldosterone Secretion

    PubMed Central

    Shapiro, Brian A.; Olala, Lawrence; Arun, Senthil Nathan; Parker, Peter M.; George, Mariya V.; Bollag, Wendy B.

    2009-01-01

    Summary Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24 hours) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively-active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion. PMID:19961896

  4. Differential expression of Prx I and II in mouse testis and their up-regulation by radiation.

    PubMed

    Lee, Keesook; Park, Ji-Sun; Kim, Yun-Jeong; Soo Lee, Yong Soo; Sook Hwang, Tae Sook; Kim, Dae-Joong; Park, Eun-Mi; Park, Young-Mee

    2002-08-16

    Testis is one of the most sensitive organs to ionizing radiation. The present study was designed to unravel the possible role of antioxidant proteins, peroxiredoxin I and II (Prx I and II) in the testis. Our results show that Prx I and II are constitutively expressed in the testis and their expression levels are decreased to some extent as the testis develops. Interestingly, immunohistochemical analysis revealed a preferential expression of Prx I and II in Leydig and Sertoli cells, respectively. Neither Prx I nor Prx II expression was obvious in the testicular germ cells including spermatogonia and spermatocytes. Ionizing radiation exerted oxidative stress on the testis and induced apoptosis primarily in the germ cells. When the irradiated testis was examined, the Prx system was found to be transiently up-regulated. Taken together, we suggest that the relative radiation-resistance of Leydig and Sertoli cells could be attributed in part to the antioxidant function of the Prx system in these cells.

  5. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  6. The desmosomal protein Desmoglein 1 aids recovery of epidermal differentiation after acute ultraviolet light exposure

    PubMed Central

    Johnson, Jodi L.; Koetsier, Jennifer L.; Sirico, Anna; Agidi, Ada T.; Antonini, Dario; Missero, Caterina; Green, Kathleen J.

    2014-01-01

    Epidermal structure is damaged by exposure to ultraviolet (UV) light but the molecular mechanisms governing structural repair are largely unknown. UVB (290-320 nm wavelengths) exposure prior to induction of differentiation reduced expression of differentiation-associated proteins, including Desmoglein 1 (Dsg1), Desmocollin 1 (Dsc1) and Keratins 1 and 10 (K1/K10) in a dose-dependent manner in normal human epidermal keratinocytes (NHEKs). The UVB- induced reduction in both Dsg1 transcript and protein was associated with reduced binding of the p63 transcription factor to previously unreported enhancer regulatory regions of the Dsg1 gene. Since Dsg1 promotes epidermal differentiation in addition to participating in cell-cell adhesion, the role of Dsg1 in aiding differentiation after UVB damage was tested. Compared to controls, depleting Dsg1 via shRNA resulted in further reduction of Dsc1 and K1/K10 expression in monolayer NHEK cultures and in abnormal epidermal architecture in organotypic skin models recovering from UVB exposure. Ectopic expression of Dsg1 in keratinocyte monolayers rescued the UVB-induced differentiation defect. Treatment of UVB-exposed monolayer or organotypic cultures with Trichostatin A, a histone deacetylase inhibitor, partially restored differentiation marker expression, suggesting a potential therapeutic strategy for reversing UV-induced impairment of epidermal differentiation after acute sun exposure. PMID:24594668

  7. The desmosomal protein desmoglein 1 aids recovery of epidermal differentiation after acute UV light exposure.

    PubMed

    Johnson, Jodi L; Koetsier, Jennifer L; Sirico, Anna; Agidi, Ada T; Antonini, Dario; Missero, Caterina; Green, Kathleen J

    2014-08-01

    Epidermal structure is damaged by exposure to UV light, but the molecular mechanisms governing structural repair are largely unknown. UVB (290-320 nm wavelengths) exposure before induction of differentiation reduced expression of differentiation-associated proteins, including desmoglein 1 (Dsg1), desmocollin 1 (Dsc1), and keratins 1 and 10 (K1/K10), in a dose-dependent manner in normal human epidermal keratinocytes (NHEKs). The UVB-induced reduction in both Dsg1 transcript and protein was associated with reduced binding of the p63 transcription factor to previously unreported enhancer regulatory regions of the Dsg1 gene. As Dsg1 promotes epidermal differentiation in addition to participating in cell-cell adhesion, the role of Dsg1 in aiding differentiation after UVB damage was tested. Compared with controls, depleting Dsg1 via short hairpin RNA resulted in further reduction of Dsc1 and K1/K10 expression in monolayer NHEK cultures and in abnormal epidermal architecture in organotypic skin models recovering from UVB exposure. Ectopic expression of Dsg1 in keratinocyte monolayers rescued the UVB-induced differentiation defect. Treatment of UVB-exposed monolayer or organotypic cultures with trichostatin A, a histone deacetylase inhibitor, partially restored differentiation marker expression, suggesting a potential therapeutic strategy for reversing UV-induced impairment of epidermal differentiation after acute sun exposure.

  8. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-09

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  9. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  10. Differential Toxicity of Antibodies to the Prion Protein

    PubMed Central

    Hornemann, Simone; Herrmann, Uli S.; Arand, Michael; Hawke, Simon; Aguzzi, Adriano

    2016-01-01

    Antibodies against the prion protein PrPC can antagonize prion replication and neuroinvasion, and therefore hold promise as possible therapeutics against prion diseases. However, the safety profile of such antibodies is controversial. It was originally reported that the monoclonal antibody D13 exhibits strong target-related toxicity, yet a subsequent study contradicted these findings. We have reported that several antibodies against certain epitopes of PrPC, including antibody POM1, are profoundly neurotoxic, yet antibody ICSM18, with an epitope that overlaps with POM1, was reported to be innocuous when injected into mouse brains. In order to clarify this confusing situation, we assessed the neurotoxicity of antibodies D13 and ICSM18 with dose-escalation studies using diffusion-weighted magnetic resonance imaging and various histological techniques. We report that both D13 and ICSM18 induce rapid, dose-dependent, on-target neurotoxicity. We conclude that antibodies directed to this region may not be suitable as therapeutics. No such toxicity was found when antibodies against the flexible tail of PrPC were administered. Any attempt at immunotherapy or immunoprophylaxis of prion diseases should account for these potential untoward effects. PMID:26821311

  11. Differentially expressed proteins underlying childhood cortical dysplasia with epilepsy identified by iTRAQ proteomic profiling

    PubMed Central

    Liu, Shiyong; Liu, Yi; Yang, Yixuan; Yang, Hui; Chen, Yangmei; Chen, Lifen

    2017-01-01

    Cortical dysplasia accounts for at least 14% of epilepsy cases, and is mostly seen in children. However, the understanding of molecular mechanisms and pathogenesis underlying cortical dysplasia is limited. The aim of this cross-sectional study is to identify potential key molecules in the mechanisms of cortical dysplasia by screening the proteins expressed in brain tissues of childhood cortical dysplasia patients with epilepsy using isobaric tags for relative and absolute quantitation-based tandem mass spectrometry compared to controls, and several differentially expressed proteins that are not reported to be associated with cortical dysplasia previously were selected for validation using real-time polymerase chain reaction, immunoblotting and immunohistochemistry. 153 out of 3340 proteins were identified differentially expressed between childhood cortical dysplasia patients and controls. And FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, and FABP3 were selected for validation and identified to be increased in childhood cortical dysplasia patients, while PRDX6 and PSAP were identified decreased. This is the first report on differentially expressed proteins in childhood cortical dysplasia. We identified differential expression of FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, FABP3, PRDX6 and PSAP in childhood cortical dysplasia patients, these proteins are involved in various processes and have various function. These results may provide new directions or targets for the research of childhood cortical dysplasia, and may be helpful in revealing molecular mechanisms and pathogenesis and/or pathophysiology of childhood cortical dysplasia if further investigated. PMID:28222113

  12. Bare lymphocyte syndrome. Consequences of absent class II major histocompatibility antigen expression for B lymphocyte differentiation and function.

    PubMed Central

    Clement, L T; Plaeger-Marshall, S; Haas, A; Saxon, A; Martin, A M

    1988-01-01

    The bare lymphocyte syndrome is a rare combined immunodeficiency disorder associated with the absence of class I and/or class II major histocompatibility (MHC) antigens. Although it has been inferred that the immune deficiency is a consequence of disordered MHC-restricted interactions among otherwise normal cells, the biological capabilities and differentiation of B lymphocytes deficient in class II MHC antigens have not been rigorously analyzed. We have examined the phenotypic and functional attributes of B cells with absent class II MHC antigens. Our data demonstrate that these B cells are intrinsically defective in their responses to membrane-mediated activation stimuli. In addition, virtually all the B cells had phenotypic evidence of arrested differentiation at an immature stage. Finally, these B cells also failed to express the C3d-EBV receptor normally present on all B lymphocytes. These data indicate that class II MHC molecules are vital participants in early events of the B cell activation cascade, and that other non-MHC membrane molecules may also be absent as a consequence of either arrested differentiation or as a result of the basic defect affecting the expression of MHC membrane antigens. PMID:3257764

  13. Crystal structure of the sweet-tasting protein thaumatin II at 1.27 A

    SciTech Connect

    Masuda, Tetsuya; Ohta, Keisuke; Tani, Fumito; Mikami, Bunzo; Kitabatake, Naofumi

    2011-07-08

    Highlights: {yields} X-ray crystallographic structure of sweet-tasting protein, thaumatin II, was determined at a resolution of 1.27 A. {yields} The overall structure of thaumatin II is similar to that of thaumatin I, but a slight shift of the C{alpha} atom of G96 in thaumatin II was observed. {yields} The side chain of two critical residues, 67 and 82, for sweetness was modeled in two alternative conformations. {yields} The flexibility and fluctuation of side chains at 67 and 82 seems to be suitable for interaction of thaumatin molecules with sweet receptors. -- Abstract: Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 A. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the C{alpha} atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.

  14. A structural transition in class II major histocompatibility complex proteins at mildly acidic pH

    PubMed Central

    1996-01-01

    Peptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen- presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment- sensitive fluorescent probe ANS (8-anilino-1-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IAd conformation was found to change at a higher pH than IEd, IEk, or IAk, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules. PMID:8551215

  15. Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

    PubMed

    Cerrada, Alejandro; de la Torre, Paz; Grande, Jesús; Haller, Thomas; Flores, Ana I; Pérez-Gil, Jesús

    2014-01-01

    Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

  16. The Novel Small Leucine-rich Protein Chondroadherin-like (CHADL) Is Expressed in Cartilage and Modulates Chondrocyte Differentiation*

    PubMed Central

    Tillgren, Viveka; Ho, James C. S.; Önnerfjord, Patrik; Kalamajski, Sebastian

    2015-01-01

    The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix. PMID:25451920

  17. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    PubMed

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  18. Smooth Muscle Cell Deletion of LDL Receptor Related Protein-1 Augments AngII-Induced Superior Mesenteric Arterial and Ascending Aortic Aneurysms

    PubMed Central

    Davis, Frank M.; Rateri, Debra L.; Balakrishnan, Anju; Howatt, Deborah A.; Strickland, Dudley K.; Muratoglu, Selen C.; Haggerty, Christopher M.; Fornwalt, Brandon K.; Cassis, Lisa A.; Daugherty, Alan

    2015-01-01

    Objective LRP1, a multifunctional protein involved in endocytosis and cell signaling pathways, leads to a range of vascular pathologies when deleted in vascular smooth muscle cells (SMCs). The purpose of this study was to determine whether LRP1 deletion in SMCs influenced AngII-induced arterial pathologies. Approach and Results LRP1 protein abundance was equivalent in selected arterial-regions, but SMC-specific LRP1 depletion had no effect on abdominal and ascending aortic diameters in young mice. To determine the effects of LRP1 deficiency on AngII vascular responses, SMC-specific LRP1 (smLRP1) +/+ and -/- mice were infused with saline, AngII, or norepinephrine (NE). Several smLRP-/- mice died of superior mesenteric arterial (SMA) rupture during AngII infusion. In surviving mice, AngII profoundly augmented SMA dilation in smLRP1-/- mice. SMA dilation was blood pressure-dependent as demonstrated by a similar response during NE infusion. SMA dilation was also associated with profound macrophage accumulation, but minimal elastin fragmentation. AngII infusion led to no significant differences in abdominal aorta diameters between smLRP1+/+ and -/- mice. In contrast, ascending aortic dilation was exacerbated markedly in AngII-infused smLRP1-/- mice, but NE had no significant effect on either aortic region. Ascending aortas of smLRP1-/- mice infused with AngII had minimal macrophage accumulation but significantly increased elastin fragmentation and mRNA abundance of several LRP1 ligands including MMP-2 and uPA. Conclusions smLRP1 deficiency had no effect on AngII-induced abdominal aortic aneurysm formation. Conversely, AngII infusion in smLRP1-/- mice exacerbated SMA and ascending aorta dilation. Dilation in these two regions had differential association with blood pressure and divergent pathologic characteristics. PMID:25395615

  19. Purification of a protein phosphatase from Acanthamoeba that dephosphorylates and activates myosin II.

    PubMed

    McClure, J A; Korn, E D

    1983-12-10

    The actin-activated ATPase activity of myosin II from Acanthamoeba castellanii is inhibited by phosphorylation of 3 serine residues near the carboxyl end of the heavy chain of the molecule. We have purified a protein phosphatase from Acanthamoeba using myosin II as a substrate. This phosphatase has a molecular weight of 39,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point in urea of 5.2. The enzyme also is active against other phosphoserine protein substrates such as turkey gizzard smooth muscle myosin light chain, but not against a synthetic phosphotyrosine protein substrate. It does not hydrolyze ATP or p-nitrophenol phosphate. No effector has been found to increase substantially the activity of the enzyme as isolated, but it is inhibited by ATP, pyrophosphate, and NaF. This inhibition is reduced in the presence of MnCl2. The Mg2+-dependent actin-activated ATPase of myosin II is activated by dephosphorylation of phosphorylated myosin II by the phosphatase. Its broad substrate specificity, molecular weight, and response to protein phosphatase inhibitors suggest that the Acanthamoeba protein phosphatase is a type 2A phosphatase (Cohen, P. (1982) Nature (Lond.) 206, 613-620).

  20. Degradation of antenna chlrophyll-binding protein CP43 during photoinhibition of photosystem II

    SciTech Connect

    Yamamoto, Yasusi; Akasaka, Tatsuya

    1995-07-18

    When photosystem II (PS II) membranes from spinach were treated with Tris (0.8 M, pH 9.0) and illuminated with white light (5000 {mu}E m{sup -2} s{sup -1}) under aerobic conditions at 25{degrees}C, not only were the reaction center-forming D1 and D2 proteins degraded but the antenna chlorophyll-binding protein CP43 was also degraded. Three products of the degradation of CP43, with molecular masses of 17.0, 15.5, and 14 kDa, respectively, were identified by sodium dodecyl sulfate/urea polyacrylamide gel electrophoresis and Western blotting with a specific antibody. Degradation products of another antenna chlorophyll-binding protein of PS II, CP47, were not detected under the same conditions. Concomitant with the damage to the D1 and D2 proteins and CP43, cross-linked products of the D1 protein, CP43, and CP47 were formed. These products were identified as slow-moving smeared bands in the higher molecular weight range of the gel during electrophoresis. Both the degradation and the cross-linking of these proteins were prevented by the addition of electron donors to PS II, a result that suggests that these processes were caused by the donor-side mechanism of photoinhibition. The photoinduced degradation of CP43 and the cross-linking among the D1 protein, CP43, and CP47 were less obvious in the PS II membranes that had been treated with hydroxylamine rather than Tris and in the membranes that had been treated with Tris and reconstituted by addition of an extrinsic 33-kDa protein (OEC33). These results indicate that removal of OEC33, which is closely associated with CP43, from the PS II complex accelerates the degradation and cross-linking of CP43 during photoinhibition. It is suggested that OEC33 is involved in the stabilization of the antenna chlorophyll-binding proteins in PS II during photoinhibition. 55 refs., 7 figs.

  1. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila.

    PubMed

    Carney, Travis D; Struck, Adam J; Doe, Chris Q

    2013-10-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS.

  2. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila

    PubMed Central

    Carney, Travis D.; Struck, Adam J.; Doe, Chris Q.

    2013-01-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS. PMID:24026126

  3. Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA

    SciTech Connect

    Elton, T.S.; Dion, L.D.; Bost, K.L.; Oparil, S.; Blalock, J.E.

    1988-04-01

    The authors have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. They demonstrate that the antibody competes specifically with /sup 125/I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, they show this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, they demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of M/sub r/ 66,000 that specifically binds radiolabeled AII.

  4. Protein kinase C beta II suppresses colorectal cancer by regulating IGF-1 mediated cell survival

    PubMed Central

    Dowling, Catríona M.; Phelan, James; Callender, Julia A.; Cathcart, Mary Clare; Mehigan, Brian; McCormick, Paul; Dalton, Tara; Coffey, John C.; Newton, Alexandra C.; O'sullivan, Jacintha; Kiely, Patrick A.

    2016-01-01

    Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n = 166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II results in a dramatic downregulation of IGF-I-induced AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome. PMID:26989024

  5. Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients.

    PubMed

    Ruhaak, L Renee; Kim, Kyoungmi; Stroble, Carol; Taylor, Sandra L; Hong, Qiuting; Miyamoto, Suzanne; Lebrilla, Carlito B; Leiserowitz, Gary

    2016-03-04

    Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n = 84) and matched healthy controls (n = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis.

  6. Dynamic association of p300 with the promoter of the G protein-coupled rat delta opioid receptor gene during NGF-induced neuronal differentiation.

    PubMed

    Chen, Yulong L; Monteith, Nancy; Law, Ping-Y; Loh, Horace H

    2010-05-28

    The G protein-coupled delta opioid receptor (DOR) plays a critical role in pain control. Emerging evidence shows that DOR also plays a role in neuronal differentiation and survival. Nerve growth factor (NGF) is known to be critical for the development and maintenance of the central and peripheral nervous systems. Our previous studies have shown that sustained activation of NGF/PI3K/Akt/NF-kappaB signaling is essential for NGF-induced dor gene expression during neuronal differentiation and that the epigenetic modifications at histone 3 lysine 9 temporally correlate with the dor gene transcription. In this study, we cloned the rat dor gene promoter and identified an NGF-responsive region similar to that from the mouse dor gene promoter. We further identified p300, a known NF-kappaB binding partner with intrinsic histone acetyltransferase activity, to be dynamically associated with the dor gene. We also found that assembling of RNA polymerase II (Pol II) at the promoter took place before NGF stimulation, indicating that p300 could only interact with preassembled Pol II at the promoter after NGF stimulation. Taken together, these results implicate that preassembly of the Pol II preinitiation complex, sustained activation of PI3K/Akt/NF-kappaB signaling, and dynamic p300 association at the promoters sequentially is one of the mechanisms of induction of the late phase genes during NGF-induced neuronal differentiation.

  7. High resolution structures of the bone morphogenetic protein type II receptor in two crystal forms: Implications for ligand binding

    SciTech Connect

    Mace, Peter D.; Cutfield, John F.; Cutfield, Sue M. . E-mail: sue.cutfield@otago.ac.nz

    2006-12-29

    BMPRII is a type II TGF-{beta} serine threonine kinase receptor which is integral to the bone morphogenetic protein (BMP) signalling pathway. It is known to bind BMP and growth differentiation factor (GDF) ligands, and has overlapping ligand specificity with the activin type II receptor, ActRII. In contrast to activin and TGF-{beta} type ligands, BMPs bind to type II receptors with lower affinity than type I receptors. Crystals of the BMPRII ectodomain were grown in two different forms, both of which diffracted to high resolution. The tetragonal form exhibited some disorder, whereas the entire polypeptide was seen in the orthorhombic form. The two structures retain the basic three-finger toxin fold of other TGF-{beta} receptor ectodomains, and share the main hydrophobic patch used by ActRII to bind various ligands. However, they present different conformations of the A-loop at the periphery of the proposed ligand-binding interface, in conjunction with rearrangement of a disulfide bridge within the loop. This particular disulfide (Cys94-Cys117) is only present in BMPRII and activin receptors, suggesting that it is important for their likely shared mode of binding. Evidence is presented that the two crystal forms represent ligand-bound and free conformations of BMPRII. Comparison with the solved structure of ActRII bound to BMP2 suggests that His87, unique amongst TGF-{beta} receptors, may play a key role in ligand recognition.

  8. ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis.

    PubMed

    Konopacki, Filip A; Wong, Hovy Ho-Wai; Dwivedy, Asha; Bellon, Anaïs; Blower, Michael D; Holt, Christine E

    2016-04-01

    Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS.

  9. Cellular misfolded proteins rescued from degradation by MHC class II molecules are possible targets for autoimmune diseases.

    PubMed

    Arase, Noriko; Arase, Hisashi

    2015-11-01

    The major function of major histocompatibility complex (MHC) class II molecules is the presentation of peptide antigens to helper T cells. However, when misfolded proteins are associated with MHC class II molecules in the endoplasmic reticulum, they are transported to the cell surface by MHC class II molecules without processing to peptides. Of note, misfolded proteins complexed with MHC class II molecules are specifically recognized by autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins rescued from degradation by MHC class II molecules may be recognized as 'neo-self' antigens by the immune system and be involved in the pathogenicity of autoimmune diseases.

  10. Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation.

    PubMed

    Young, Brent M; Nguyen, Elaine; Chedrawe, Matthew A J; Rainey, Jan K; Dupré, Denis J

    2017-02-24

    G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated β-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.

  11. A new function of Nell-1 protein in repressing adipogenic differentiation.

    PubMed

    James, Aaron W; Pan, Angel; Chiang, Michael; Zara, Janette N; Zhang, Xinli; Ting, Kang; Soo, Chia

    2011-07-22

    A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.

  12. Differentially expressed proteins of soybean (Glycine max) pulvinus in light and dark conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean leaves (Glycine max) both track and avoid the sun through turgor changes of the pulvinus tissue at the base of leaves. Pulvinar response is known to be affected by both diurnally varying environmental factors and circadian patterns. Differential expression of the proteins between light and d...

  13. Modulation of neurotransmitter receptors and synaptic differentiation by proteins containing complement-related domains.

    PubMed

    Nakayama, Minoru; Hama, Chihiro

    2011-02-01

    Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits.

  14. Differential protein levels and post-translational modifications in spinal cord injury of the rat.

    PubMed

    Afjehi-Sadat, Leila; Brejnikow, Mika; Kang, Sung Ung; Vishwanath, Vinay; Walder, Nadja; Herkner, Kurt; Redl, Heinz; Lubec, Gert

    2010-03-05

    Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

  15. Differential proteomic analysis of bovine conceptus fluid proteins in pregnancies generated by assisted reproductive technologies.

    PubMed

    Riding, George A; Hill, Jonathan R; Jones, Alun; Holland, Michael K; Josh, Peter F; Lehnert, Sigrid A

    2008-07-01

    Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.

  16. Plant Protein and Animal Proteins: Do They Differentially Affect Cardiovascular Disease Risk?12

    PubMed Central

    Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

    2015-01-01

    Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat. PMID:26567196

  17. Plant protein and animal proteins: do they differentially affect cardiovascular disease risk?

    PubMed

    Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

    2015-11-01

    Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat.

  18. Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells

    PubMed Central

    Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A.; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

    2017-01-01

    Novel APOBEC1 target 1 (Nat1) (also known as “p97,” “Dap5,” and “Eif4g2”) is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil–containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1. Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation. PMID:28003464

  19. Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling

    PubMed Central

    Klebanow, Lindsey R.; Peshel, Emanuela C.; Schuster, Andrew T.; De, Kuntal; Sarvepalli, Kavitha; Lemieux, Madeleine E.; Lenoir, Jessica J.; Moore, Adrian W.; McDonald, Jocelyn A.

    2016-01-01

    The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. PMID:27317808

  20. Roles of an unconventional protein kinase and myosin II in amoeba osmotic shock responses.

    PubMed

    Betapudi, Venkaiah; Egelhoff, Thomas T

    2009-12-01

    The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. Green fluorescent protein (GFP)-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild-type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa.

  1. Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation

    PubMed Central

    Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Hernandez, Joseph; Potluri, Vani; Wang, Liguo; Li, Wei; Lemay, Danielle G.

    2013-01-01

    Background Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. Methodology/Principal Findings Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. Conclusions/Significance A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the

  2. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

    PubMed Central

    Bonin, Sawyer R.; Gibeault, Sabrina; De Repentigny, Yves; Kothary, Rashmi

    2016-01-01

    Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic

  3. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

    NASA Astrophysics Data System (ADS)

    Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

    2014-03-01

    During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

  4. Differential dehydration effects on globular proteins and intrinsically disordered proteins during film formation.

    PubMed

    Yoneda, Juliana Sakamoto; Miles, Andew J; Araujo, Ana Paula Ulian; Wallace, B A

    2017-04-01

    Globular proteins composed of different secondary structures and fold types were examined by synchrotron radiation circular dichroism spectroscopy to determine the effects of dehydration on their secondary structures. They exhibited only minor changes upon removal of bulk water during film formation, contrary to previously reported studies of proteins dehydrated by lyophilization (where substantial loss of helical structure and gain in sheet structure was detected). This near lack of conformational change observed for globular proteins contrasts with intrinsically disordered proteins (IDPs) dried in the same manner: the IDPs, which have almost completely unordered structures in solution, exhibited increased amounts of regular (mostly helical) secondary structures when dehydrated, suggesting formation of new intra-protein hydrogen bonds replacing solvent-protein hydrogen bonds, in a process which may mimic interactions that occur when IDPs bind to partner molecules. This study has thus shown that the secondary structures of globular and intrinsically disordered proteins behave very differently upon dehydration, and that films are a potentially useful format for examining dehydrated soluble proteins and assessing IDPs structures.

  5. Differential dehydration effects on globular proteins and intrinsically disordered proteins during film formation

    PubMed Central

    Yoneda, Juliana Sakamoto; Miles, Andew J.; Araujo, Ana Paula Ulian

    2017-01-01

    Abstract Globular proteins composed of different secondary structures and fold types were examined by synchrotron radiation circular dichroism spectroscopy to determine the effects of dehydration on their secondary structures. They exhibited only minor changes upon removal of bulk water during film formation, contrary to previously reported studies of proteins dehydrated by lyophilization (where substantial loss of helical structure and gain in sheet structure was detected). This near lack of conformational change observed for globular proteins contrasts with intrinsically disordered proteins (IDPs) dried in the same manner: the IDPs, which have almost completely unordered structures in solution, exhibited increased amounts of regular (mostly helical) secondary structures when dehydrated, suggesting formation of new intra‐protein hydrogen bonds replacing solvent‐protein hydrogen bonds, in a process which may mimic interactions that occur when IDPs bind to partner molecules. This study has thus shown that the secondary structures of globular and intrinsically disordered proteins behave very differently upon dehydration, and that films are a potentially useful format for examining dehydrated soluble proteins and assessing IDPs structures. PMID:28097742

  6. Involvement of RNA binding proteins AUF1 in mammary gland differentiation

    SciTech Connect

    Nagaoka, Kentaro . E-mail: akenaga@mail.ecc.u-tokyo.ac.jp; Tanaka, Tetsuya; Imakawa, Kazuhiko; Sakai, Senkiti

    2007-08-01

    The expression of many genes, such as {beta}-casein, c-myc, and cyclin D1, is altered by lactogenic hormone stimulation during mammary epithelial cell differentiation. Here, we demonstrate that post-transcriptional regulation plays an important role to establish gene expression required to initiate milk production as well as transcriptional control. AUF1 protein, a member of the AU-rich element (ARE)-binding protein family, plays a role in ARE-mRNA turnover by regulating mRNA stability and/or translational control. Cytoplasmic localization of AUF1 protein is critically linked to function. We show that as the mammary gland differentiates, AUF1 protein moves from the cytoplasm to the nucleus. Moreover, in mammary gland epithelial cells (HC11), stimulation by lactogenic hormone decreased cytoplasmic and increased nuclear AUF1 levels. Direct binding of AUF1 protein was observed on c-myc mRNA, but not {beta}-casein or cyclin D1 mRNA. AUF1 downregulation in HC11 cells increased the expression of {beta}-casein mRNA and decreased the expression of c-myc mRNA by lactogenic hormone. Conversely, overexpression of AUF1 inhibited these effects of lactogenic hormone stimulation in HC11 cells. These results suggest that AUF1 participates in mammary gland differentiation processes under the control of lactogenic hormone signals.

  7. Wnt5a through Noncanonical Wnt/JNK or Wnt/PKC Signaling Contributes to the Differentiation of Mesenchymal Stem Cells into Type II Alveolar Epithelial Cells In Vitro

    PubMed Central

    Cai, Shixia; Dong, Liang; Liu, Le; Yang, Yi; Guo, Fengmei; Lu, Xiaomin; He, Hongli; Chen, Qihong; Hu, Shuling; Qiu, Haibo

    2014-01-01

    The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and Wnt signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical Wnt pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical Wnt pathway in this process is unclear. It was disclosed in this study that noncanonical Wnt signaling in mouse bone marrow–derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-12 cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when Wnt5a was added to activate noncanonical Wnt signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of Wnt5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the Wnt5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of Wnt5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, Wnt5a through Wnt/JNK signaling alone or both Wnt/JNK and Wnt/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through Wnt/PKC signaling, Wnt5a increased the survival of mMSCs after H2O2 exposure in vitro. PMID:24658098

  8. Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

    PubMed

    Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

    2017-03-01

    Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA.

  9. Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses

    PubMed Central

    Tian, Tian; Yang, Mifang; Li, Zhongming; Ping, Fengfeng; Fan, Weixin

    2016-01-01

    Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days). Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease. PMID:26752403

  10. Differential regulation of genomic imprinting by TET proteins in embryonic stem cells

    PubMed Central

    Liu, Lizhi; Mao, Shi-Qing; Ray, Chelsea; Zhang, Yu; Bell, Fong T.; Ng, Sheau-Fang; Xu, Guo-Liang; Li, Xiajun

    2015-01-01

    TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs. PMID:26397890

  11. A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives.

    PubMed

    Keil-Dlouha, V; Paulin, D; Bagilet, L K; Keil, B

    1980-03-27

    Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3) were compared by two-dimensional polyacrylamide gel electrophoresis after selective iodination with 125I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major 125I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14--15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.

  12. Cyclooxygenase-2 Inhibition Limits Angiotensin II-Induced DNA Oxidation and Protein Nitration in Humans

    PubMed Central

    Pialoux, Vincent; Poulin, Marc J.; Hemmelgarn, Brenda R.; Muruve, Daniel A.; Chirico, Erica N.; Faes, Camille; Sola, Darlene Y.; Ahmed, Sofia B.

    2017-01-01

    Compared to other cyclooxygenase-2 inhibitors, celecoxib is associated with a lower cardiovascular risk, though the mechanism remains unclear. Angiotensin II is an important mediator of oxidative stress in the pathophysiology of vascular disease. Cyclooxygenase-2 may modify the effects of angiotensin II though this has never been studied in humans. The purpose of the study was to test the effects of selective cyclooxygenase-2 inhibition on plasma measures of oxidative stress, the vasoconstrictor endothelin-1, and nitric oxide metabolites, both at baseline and in respose to Angiotensin II challenge in healthy humans. Measures of 8-hydroxydeoxyguanosine, advanced oxidation protein products, nitrotyrosine, endothelin-1, and nitric oxide metabolites were assessed from plasma samples drawn at baseline and in response to graded angiotensin II infusion (3 ng/kg/min × 30 min, 6 ng/kg/min × 30 min) before and after 14 days of cyclooxygenase-2 inhibition in 14 healthy subjects (eight male, six female) in high salt balance, a state of maximal renin angiotensin system suppression. Angiotensin II infusion significantly increased plasma oxidative stress compared to baseline (8-hydroxydeoxyguanosine; +17%; advanced oxidation protein products; +16%), nitrotyrosine (+76%). Furthermore, levels of endothelin-1 levels were significantly increased (+115%) and nitric oxide metabolites were significantly decreased (−20%). Cycloxygenase-2 inhibition significantly limited the increase in 8-hydroxydeoxyguanosine, nitrotyrosine and the decrease in nitric oxide metabolites induced by angiotensin II infusion, though no changes in advanced oxidation protein products and endothelin-1 concentrations were observed. Cyclooxygenase-2 inhibition with celecoxib partially limited the angiotensin II-mediated increases in markers of oxidative stress in humans, offering a potential physiological pathway for the improved cardiovascular risk profile of this drug. PMID:28344559

  13. Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

    PubMed

    Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-08-01

    We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

  14. Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

    SciTech Connect

    Maganga, Richard; Giles, Natalie; Adcroft, Katharine; Unni, Ambili; Keeney, Diane; Wood, Fiona; Fear, Mark Dharmarajan, Arunasalam

    2008-12-12

    The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

  15. Protein Arginine Methyltransferase 1 Interacts with and Activates p38α to Facilitate Erythroid Differentiation

    PubMed Central

    Hua, Wei-Kai; Chang, Yuan-I; Yao, Chao-Ling; Hwang, Shiaw-Min; Chang, Chung-Yi; Lin, Wey-Jinq

    2013-01-01

    Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34+ cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38β did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38β-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and

  16. Discovery and verification of serum differential expression proteins for pulmonary tuberculosis.

    PubMed

    Li, Cuiping; He, Xiao; Li, Hongtao; Zhou, Yi; Zang, Ning; Hu, Shuixiu; Zheng, Yanyan; He, Min

    2015-09-01

    Pulmonary tuberculosis (PTB) is a chronic disease and has remained a severe threat to public health. Valuable biomarkers for improving the detection rate are crucial for controlling this disease. The purpose of this study was to discover potential biomarkers in sera from PTB patients compared with pneumonia patients and normal healthy controls. A total of 336 human serum specimens were enrolled in this study. Differentially expressed proteins were identified using iTRAQ method combining with MALDI-TOF-MS. Data was analyzed using relative bioinformatics methods. Potential biomarkers were further validated by IHC, ELISA and Western blot. As a result, 489 non-redundant proteins were identified in the sera, and 159 of which could be quantified by calculating their iTRAQ ratios. Compared to the controls, 26 differentially expressed proteins were recognized among PTB patients, including 16 overexpressed proteins and 10 downregulated proteins. Analysis of their functional interactions revealed that 12 proteins appeared in the center of the functional network. One of these key proteins, sex hormone binding globulin (SHBG), was found to be significantly elevated among PTB patients as compared with the controls examined by IHC, ELISA and Western blot. This result was consistent with the iTRAQ result. An independent blinded testing set to examine serum SHBG by ELISA achieved an accuracy of 78.74%, sensitivity of 75.6% and specificity of 91.5% in diagnosing PTB. In summary, iTRAQ in combination with MALDI-TOF-MS technology can efficiently screen differentially expressed proteins in sera from the PTB patients. SHBG is suggested to be a possible and novel serum biomarker for PTB.

  17. Protein arginine methyltransferase 1 (PRMT1) represses MHC II transcription in macrophages by methylating CIITA

    PubMed Central

    Fan, Zhiwen; Li, Jianfei; Li, Ping; Ye, Qing; Xu, Huihui; Wu, Xiaoyan; Xu, Yong

    2017-01-01

    Efficient presentation of alien antigens triggers activation of T lymphocytes and robust host defense against invading pathogens. This pathophysiological process relies on the expression of major histocompatibility complex (MHC) molecules in antigen presenting cells such as macrophages. Aberrant MHC II transactivation plays a crucial role in the pathogenesis of atherosclerosis. Class II transactivator (CIITA) mediates MHC II induction by interferon gamma (IFN-γ). CIITA activity can be fine-tuned at the post-translational level, but the mechanisms are not fully appreciated. We investigated the role of protein arginine methyltransferase 1 (PRMT1) in this process. We report here that CIITA interacted with PRMT1. IFN-γ treatment down-regulated PRMT1 expression and attenuated PRMT1 binding on the MHC II promoter. Over-expression of PRMT1 repressed MHC II promoter activity while PRMT1 depletion enhanced MHC II transactivation. Mechanistically, PRMT1 methylated CIITA and promoted CIITA degradation. Therefore, our data reveal a previously unrecognized role for PRMT1 in suppressing CIITA-mediated MHC II transactivation. PMID:28094290

  18. Specific Microbiota-Induced Intestinal Th17 Differentiation Requires MHC II but not GALT and Mesenteric Lymph Nodes

    PubMed Central

    Geem, Duke; Medina-Contreras, Oscar; McBride, Michelle; Newberry, Rodney D.; Koni, Pandelakis A.; Denning, Timothy L.

    2014-01-01

    Interleukin (IL)-17 expressing CD4+ T lymphocytes (Th17 cells) naturally reside in the intestine where specific cytokines and microbiota, such as segmented filamentous bacteria (SFB), promote their differentiation. Intestinal Th17 cells are believed to initially differentiate in the GALT and/or mesenteric lymph nodes (mLN) upon antigen encounter and subsequently home to the lamina propria (LP) where they mediate effector functions. However, whether GALT and/or mLN are required for intestinal Th17 differentiation, and how microbiota containing SFB regulate antigen-specific intestinal Th17 cells remain poorly defined. Here we observed that naïve CD4+ T cells were abundant in the intestinal LP prior to weaning and that the accumulation of Th17 cells in response to microbiota containing SFB occurred in the absence of lymphotoxin (LT)-dependent lymphoid structures and the spleen. Furthermore, the differentiation of intestinal Th17 cells in the presence of microbiota containing SFB was dependent on MHC II expression by CD11c+ cells. Lastly, the differentiation of antigen-specific Th17 cells required both the presence of cognate antigen and microbiota containing SFB. These findings suggest that microbiota containing SFB create an intestinal milieu that may induce antigen-specific Th17 differentiation against food and/or bacterial antigens directly in the intestinal LP. PMID:24899505

  19. Differential abundance of egg white proteins in laying hens treated with corticosterone.

    PubMed

    Kim, Jimin; Choi, Yang-Ho

    2014-12-24

    Stressful environments can affect not only egg production and quality but also gene and protein abundance in the ovary and oviduct in laying hens. The oviductal magnum of laying hens is the organ responsible for the synthesis and secretion of egg white proteins. The objective of this study was to investigate the effects of dietary corticosterone as a stress model on the abundance of proteins in the egg white and of mRNA and proteins in the magnum in laying hens. After a 14-day acclimation, 40 laying hens were divided into two groups which were provided for the next 14 days with either control (Control) or corticosterone (Stress) diet containing at 30 mg/kg. Corticosterone treatment resulted in increased feed intake (P ≤ 0.05) and decreased egg production. Two-dimensional electrophoresis (2DE) with MALDI-TOF/TOF MS/MS using eggs obtained on days 0 and 5 revealed differential abundance of egg white proteins by Stress: transiently expressed in neural precursors (TENP), hemopexin (HPX), IgY-Fcυ3-4, and extracellular fatty acid-binding protein (Ex-FABP) were decreased while ovoinhibitor and ovalbumin-related protein X (OVAX) were increased on days 5 vs 0 (P ≤ 0.05). Expression of mRNAs and proteins was also significantly modulated in the magnum of hens in Stress on day 14 (P ≤ 0.05). In conclusion, the current study provides the first evidence showing that dietary corticosterone modulates protein abundance in the egg white in laying hens, and it suggests that environmental stress can differentially modify expression of egg white proteins in laying hens.

  20. Collagen type II, alpha 1 protein: a bioactive component of shark cartilage.

    PubMed

    Merly, Liza; Smith, Sylvia L

    2013-02-01

    Previous studies have shown that extracts of shark cartilage induce a cytokine response in human leukocytes, but the nature of the bioactive component(s) is unknown. Extracts treated with proteases lost 80% of their cytokine-inducing property, suggesting that the active component(s) was likely a complex protein. The aim of the present study was to determine the nature of the bioactive molecule(s). Solid phase extraction followed by ion exchange chromatography and electrophoretic separation were used to partially purify a bioactive preparation from commercial shark cartilage that has been identified as a small glycoprotein. LC-MS analysis yielded peptides with 100% molecular identity with collagen type II, alpha I protein from the lesser spotted catshark, Scyliorhinus canicula. The implications for the consumption of shark cartilage as a dietary supplement are discussed given the presence of collagen type II, alpha 1 protein in extracts.

  1. Yes associated protein is a poor prognostic factor in well-differentiated lung adenocarcinoma.

    PubMed

    Kim, Mi Hyun; Kim, Young Keum; Shin, Dong Hoon; Lee, Hyun Jeong; Shin, Nari; Kim, Arong; Lee, Jung Hee; Choi, Kyung Un; Kim, Jee Yeon; Lee, Chang Hun; Sol, Mee Young

    2015-01-01

    The Hippo pathway is a highly conserved potent regulator of cell growth and apoptosis including large tumor suppressor (LATS) and Yes-associated protein (YAP). LATS has been regarded as a tumor suppressor gene and YAP as either of a tumor suppressor gene or an oncogene. We investigated their expression in lung adenocarcinoma. YAP and LATS protein expression was assessed in 167 surgically resected lung adenocarcinomas and compared with clinicopathologic factors. Disease free survival and overall survival were also evaluated. YAP expression was noted in cytoplasm (48 cases; 28.7%), nuclear (34; 20.4%) and both locations (4; 2.4%). The nuclear expression was typically observed in well differentiated adenocarcinoma. LATS was expressed in cytoplasm when its signal is weak. Perinuclear expression of LATS was observed when it is strongly expressed. While cytoplasmic and nuclear YAP expressions were inversely related. In well differentiated adenocarcinoma patients, YAP nuclear expression was related with more frequent relapse. Both of nuclear YAP and LATS expression were more frequently observed in well differentiated adenocarcinoma. Furthermore, YAP expression exhibited more frequent relapse in well differentiated adenocarcinoma group. We suggest that YAP may act as an oncogene and predict poorer prognosis in well differentiated lung adenocarcinoma.

  2. Alteration of protein prenylation promotes spermatogonial differentiation and exhausts spermatogonial stem cells in newborn mice

    PubMed Central

    Diao, Fan; Jiang, Chen; Wang, Xiu-Xing; Zhu, Rui-Lou; Wang, Qiang; Yao, Bing; Li, Chao-Jun

    2016-01-01

    Spermatogenesis in adulthood depends on the successful neonatal establishment of the spermatogonial stem cell (SSC) pool and gradual differentiation during puberty. The stage-dependent changes in protein prenylation in the seminiferous epithelium might be important during the first round of spermatogenesis before sexual maturation, but the mechanisms are unclear. We have previous found that altered prenylation in Sertoli cells induced spermatogonial apoptosis in the neonatal testis, resulting in adult infertility. Now we further explored the role of protein prenylation in germ cells, using a conditional deletion of geranylgeranyl diphosphate synthase (Ggpps) in embryonic stage and postmeiotic stage respectively. We observed infertility of Ggpps−/− Ddx4-Cre mice that displayed a Sertoli-cell-only syndrome phenotype, which resulted from abnormal spermatogonial differentiation and SSC depletion during the prepubertal stage. Analysis of morphological characteristics and cell-specific markers revealed that spermatogonial differentiation was enhanced from as early as the 7th postnatal day in the first round of spermatogenesis. Studies of the molecular mechanisms indicated that Ggpps deletion enhanced Rheb farnesylation, which subsequently activated mTORC1 and facilitated spermatogonial differentiation. In conclusion, the prenylation balance in germ cells is crucial for spermatogonial differentiation fate decision during the prepubertal stage, and the disruption of this process results in primary infertility. PMID:27374985

  3. Expression of osterix inhibits bone morphogenetic protein-induced chondrogenic differentiation of mesenchymal progenitor cells.

    PubMed

    Tominaga, Hiroyuki; Maeda, Shingo; Miyoshi, Hiroyuki; Miyazono, Kohei; Komiya, Setsuro; Imamura, Takeshi

    2009-01-01

    Osteoblasts and chondrocytes arise from common bipotential mesenchymal progenitor cells. Although the differentiation of these two cell lineages can be induced by treatment with bone morphogenetic proteins (BMPs), the responses of mesenchymal progenitors to BMP differ from cell line to cell line. Here we demonstrate that C3H/10T1/2 cells preferred chondrogenic differentiation, primary bone marrow stroma cells (MSCs) tended to convert to osteoblasts, and ST-2 cells differentiated into both the osteoblastic and chondrocytic lineages simultaneously, suggesting that a molecular switch functions to select cell fate. Osterix, the secondary master regulator of osteoblastogenesis, was induced by BMP at high and low levels in MSCs and ST-2 cells, respectively; in contrast, C3H/10T1/2 cells demonstrated only faint expression. As osterix has been suggested as a negative regulator of chondrogenesis, we hypothesized that the intense chondrocyte differentiation of C3H/10T1/2 cells may have resulted from an absence of osterix. We therefore restored osterix gene expression in C3H/10T1/2 cells using an adenovirus vector. Following BMP treatment, infection with an osterix-encoding virus dramatically inhibited the chondrocytic differentiation of C3H/10T1/2 cells, resulting instead in prominent osteoblast differentiation. These results indicate the chondrogenic potential of C3H/10T1/2 cells was abrogated by osterix expression. Chondrocyte differentiation of MSCs, however, was not enhanced by silencing the osterix gene using lentivirus-mediated shRNA, despite successful suppression of osteoblast differentiation. These results suggest that the low levels of osterix expression remaining after knockdown are sufficient to block chondrogenesis, whereas higher expression may be required to promote osteoblastic differentiation.

  4. Annotated differentially expressed salivary proteins of susceptible and insecticide-resistant mosquitoes of Anopheles stephensi.

    PubMed

    Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

    2015-01-01

    Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito-parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins

  5. Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

    PubMed Central

    Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Chi-shing Cho, William; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

    2017-01-01

    We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. PMID:28216608

  6. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    PubMed

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis.

  7. HIVed, a knowledgebase for differentially expressed human genes and proteins during HIV infection, replication and latency

    PubMed Central

    Li, Chen; Ramarathinam, Sri H.; Revote, Jerico; Khoury, Georges; Song, Jiangning; Purcell, Anthony W.

    2017-01-01

    Measuring the altered gene expression level and identifying differentially expressed genes/proteins during HIV infection, replication and latency is fundamental for broadening our understanding of the mechanisms of HIV infection and T-cell dysfunction. Such studies are crucial for developing effective strategies for virus eradication from the body. Inspired by the availability and enrichment of gene expression data during HIV infection, replication and latency, in this study, we proposed a novel compendium termed HIVed (HIV expression database; http://hivlatency.erc.monash.edu/) that harbours comprehensive functional annotations of proteins, whose genes have been shown to be dysregulated during HIV infection, replication and latency using different experimental designs and measurements. We manually curated a variety of third-party databases for structural and functional annotations of the protein entries in HIVed. With the goal of benefiting HIV related research, we collected a number of biological annotations for all the entries in HIVed besides their expression profile, including basic protein information, Gene Ontology terms, secondary structure, HIV-1 interaction and pathway information. We hope this comprehensive protein-centric knowledgebase can bridge the gap between the understanding of differentially expressed genes and the functions of their protein products, facilitating the generation of novel hypotheses and treatment strategies to fight against the HIV pandemic. PMID:28358052

  8. Conserved lamin A protein expression in differentiated cells in the earthworm Eudrilus eugeniae.

    PubMed

    Kalidas, Ramamoorthy M; Raja, Subramanian Elaiya; Mydeen, Sheik Abdul Kader Nagoor Meeran; Samuel, Selvan Christyraj Johnson Retnaraj; Durairaj, Selvan Christyraj Jackson; Nino, Gopi D; Palanichelvam, Karuppaiah; Vaithi, Arumugaswami; Sudhakar, Sivasubramaniam

    2015-09-01

    Lamin A is an intermediate filament protein found in most of the differentiated vertebrate cells but absent in stem cells. It shapes the skeletal frame structure beneath the inner nuclear membrane of the cell nucleus. As there are few studies of the expression of lamin A in invertebrates, in the present work, we have analyzed the sequence, immunochemical conservation and expression pattern of lamin A protein in the earthworm Eudrilus eugeniae, a model organism for tissue regeneration. The expression of lamin A has been confirmed in E. eugeniae by immunoblot. Its localization in the nuclear membrane has been observed by immunohistochemistry using two different rabbit anti-sera raised against human lamin A peptides, which are located at the C-terminus of the lamin A protein. These two antibodies detected 70 kDa lamin A protein in mice and a single 65 kDa protein in the earthworm. The Oct-4 positive undifferentiated blastemal tissues of regenerating earthworm do not express lamin A, while the Oct-4 negative differentiated cells express lamin A. This pattern was also confirmed in the earthworm prostate gland. The present study is the first evidence for the immunochemical identification of lamin A and Oct-4 in the earthworm. Along with the partial sequence obtained from the earthworm genome, the present results suggest that lamin A protein and its expression pattern is conserved from the earthworm to humans.

  9. A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿

    PubMed Central

    Xu, Hongbin; Chater, Keith F.; Deng, Zixin; Tao, Meifeng

    2008-01-01

    Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development. PMID:18487344

  10. Protein differential expression induced by endocrine disrupting compounds in a terrestrial isopod.

    PubMed

    Lemos, Marco F L; Esteves, Ana Cristina; Samyn, Bart; Timperman, Isaak; van Beeumen, Jozef; Correia, António; van Gestel, Cornelis A M; Soares, Amadeu M V M

    2010-04-01

    Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist.

  11. TET family proteins and their role in stem cell differentiation and transformation

    PubMed Central

    Cimmino, Luisa; Abdel-Wahab, Omar; Levine, Ross L.; Aifantis, Iannis

    2011-01-01

    One of the main regulators of gene expression during embryonegesis and stem cell differentiation is DNA methylation. The recent identification of hydroxymethylcytosine (5hmC) as a novel epigenetic mark sparked an intense effort to characterize its specialized enzymatic machinery and to understand the biological significance of 5hmC. The recent discovery of recurrent deletions and somatic mutations in the TET gene family, which includes proteins that can hydroxylate methylcytosine (5mC), in a large fraction of myeloid malignancies further suggested a key role for dynamic DNA methylation changes in the regulation of stem cell differentiation and transformation. PMID:21885017

  12. Protein Kinase D2 Is an Essential Regulator of Murine Myoblast Differentiation

    PubMed Central

    Pusapati, Ganesh V.; Armacki, Milena; Müller, Martin; Tümpel, Stefan; Illing, Anett; Hartmann, Daniel; Brunner, Cornelia; Liebau, Stefan; Rudolph, Karl L.; Adler, Guido; Seufferlein, Thomas

    2011-01-01

    Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration. PMID:21298052

  13. Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

    PubMed Central

    Jönsson, Peter; Southcombe, Jennifer H.; Santos, Ana Mafalda; Huo, Jiandong; Fernandes, Ricardo A.; McColl, James; Lever, Melissa; Evans, Edward J.; Hudson, Alexander; Chang, Veronica T.; Hanke, Tomáš; Godkin, Andrew; Dunne, Paul D.; Horrocks, Mathew H.; Palayret, Matthieu; Screaton, Gavin R.; Petersen, Jan; Rossjohn, Jamie; Fugger, Lars; Dushek, Omer; Xu, Xiao-Ning; Davis, Simon J.; Klenerman, David

    2016-01-01

    The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm2. This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2–20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination. PMID:27114505

  14. Rtp1p is a karyopherin-like protein required for RNA polymerase II biogenesis.

    PubMed

    Gómez-Navarro, Natalia; Peiró-Chova, Lorena; Rodriguez-Navarro, Susana; Polaina, Julio; Estruch, Francisco

    2013-05-01

    The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex.

  15. Bone Morphogenetic Protein-9 Induces PDLSCs Osteogenic Differentiation through the ERK and p38 Signal Pathways

    PubMed Central

    Ye, Guo; Li, Conghua; Xiang, Xuerong; Chen, Chu; Zhang, Ruyi; Yang, Xia; Yu, Xuesong; Wang, Jinhua; Wang, Lan; Shi, Qiong; Weng, Yaguang

    2014-01-01

    Periodontal ligament stem cells (PDLSCs) with bone morphogenic ability are used to treat diseases such as periodontitis. Their treatment potential is increased when used in combination with proteins that induce osteogenic differentiation. For example, bone morphogenetic protein-9 (BMP9) has been found to have potent osteogenic activity. In the present study, PDLSCs were isolated from human periodontal membrane and infected with recombinant adenoviruses expressing BMP9 (Ad-BMP9). Levels of osteogenic markers such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) as well as mineralization ability were measured. The results showed that BMP9 promoted bone formation of PDLSCs. In other experiments, SB203580 and PD98059, which are inhibitors of p38 and ERK1/2, respectively, were used to determine if these kinases are involved in the osteogenic differentiation process. The resulting protein expression profiles and osteogenic markers of PDLSCs revealed that the mitogen-activated protein kinase (MAPK) signaling pathway might play an important role in the process of BMP9-induced osteogenic differentiation of PDLSCs. PMID:25136261

  16. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

    PubMed

    Del Campo, Mark; Tijerina, Pilar; Bhaskaran, Hari; Mohr, Sabine; Yang, Quansheng; Jankowsky, Eckhard; Russell, Rick; Lambowitz, Alan M

    2007-10-12

    The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  17. The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

    PubMed

    Wittmann, Sina; Renner, Max; Watts, Beth R; Adams, Oliver; Huseyin, Miles; Baejen, Carlo; El Omari, Kamel; Kilchert, Cornelia; Heo, Dong-Hyuk; Kecman, Tea; Cramer, Patrick; Grimes, Jonathan M; Vasiljeva, Lidia

    2017-04-03

    Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.

  18. MET1 Is a Thylakoid-Associated TPR Protein Involved in Photosystem II Supercomplex Formation and Repair in Arabidopsis

    PubMed Central

    Bhuiyan, Nazmul H.; Friso, Giulia; Poliakov, Anton; Ponnala, Lalit

    2015-01-01

    Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts. PMID:25587003

  19. The role of unfolded protein response in differentiation of mammary epithelial cells.

    PubMed

    Tsuchiya, Megumi; Koizumi, Yumiko; Hayashi, Satoko; Hanaoka, Miyuki; Tokutake, Yukako; Yonekura, Shinichi

    2017-03-18

    The accumulation of misfolded proteins in the ER provokes ER stress by increasing the demand for energy, chaperones, and other proteins that are needed to fold client proteins or to degrade unfoldable secretory cargo. This stress activates a signaling network called the unfolded protein response (UPR). However, recent accumulated data suggested that the UPR also provides important signals for regulating cell differentiation and maturation. However, the relationship between UPR and mammary gland development has not been fully elucidated. To define the involvement of the UPR in mammary gland development, mammary glands were collected from non-pregnant mice, at days 5, 10 and 15 of pregnancy, at days 1 and 7 of lactation, and the expression patterns of UPR-related genes were determined by real-time PCR. We found that the mRNA expression of ATF4 and XBP1 significant increased during pregnancy. Moreover, we found that both ATF4 and XBP1 proteins are expressed in mammary epithelial cells by immunohistological analysis. In order to know the role of ATF4 and XBP1 in the differentiation of mammary epithelial cell, we performed gene knockdown experiment using HC11 cells. We found that ATF4 or XBP1 knockdown suppressed the mRNA expression of beta-casein and lactogenic hormone receptor in differentiating HC11 cells. Our results demonstrate that XBP1 and ATF4, which are UPR-related transcription factors, directly or indirectly participate in cell differentiation mechanisms through the regulation of the expression of lactogenic hormone receptors in mouse mammary epithelial cells.

  20. Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms.

    PubMed

    Oliveira, Katia C; Carvalho, Mariana L P; Bonatto, José Matheus C; Schechtman, Debora; Verjovski-Almeida, Sergio

    2016-02-01

    Schistosoma mansoni and its vertebrate host have a complex and intimate connection in which several molecular stimuli are exchanged and affect both organisms. Human tumor necrosis factor alpha (hTNF-α), a pro-inflammatory cytokine, is known to induce large-scale gene expression changes in the parasite and to affect several parasite biological processes such as metabolism, egg laying, and worm development. Until now, the molecular mechanisms for TNF-α activity in worms are not completely understood. Here, we aimed at exploring the effect of hTNF-α on S. mansoni protein phosphorylation by 2D gel electrophoresis followed by a quantitative analysis of phosphoprotein staining and protein identification by mass spectrometry. We analyzed three biological replicates of adult male worms exposed to hTNF-α and successfully identified 32 protein spots with a statistically significant increase in phosphorylation upon in vitro exposure to hTNF-α. Among the differentially phosphorylated proteins, we found proteins involved in metabolism, such as glycolysis, galactose metabolism, urea cycle, and aldehyde metabolism, as well as proteins related to muscle contraction and to cytoskeleton remodeling. The most differentially phosphorylated protein (30-fold increase in phosphorylation) was 14-3-3, whose function is known to be modulated by phosphorylation, belonging to a signal transduction protein family that regulates a variety of processes in all eukaryotic cells. Further, 75% of the identified proteins are known in mammals to be related to TNF-α signaling, thus suggesting that TNF-α response may be conserved in the parasite. We propose that this work opens new perspectives to be explored in the study of the molecular crosstalk between host and pathogen.

  1. Plasmatic retinol-binding protein 4 and glial fibrillary acidic protein as biomarkers to differentiate ischemic stroke and intracerebral hemorrhage.

    PubMed

    Llombart, Víctor; García-Berrocoso, Teresa; Bustamante, Alejandro; Giralt, Dolors; Rodriguez-Luna, David; Muchada, Marian; Penalba, Anna; Boada, Cristina; Hernández-Guillamon, Mar; Montaner, Joan

    2016-01-01

    A rapid differentiation of acute ischemic stroke and intracerebral hemorrhage (ICH) is essential for an adequate treatment and to promote a better outcome. Our aim was to identify new plasma biomarkers to differentiate stroke subtypes and to combine their diagnostic ability with other biomarkers already described for this clinical indication. Plasma samples of ischemic stroke patients (36) and ICH patients (10) were screened using a 177 antibodies library, and 11 showed different concentrations among stroke subtypes (p < 0.05), mainly chemokines, growth factors and angiogenic factors. Five proteins were selected for replication in 16 ischemic stroke patients and 16 ICH patients, and retinol-binding protein 4 (RPB4), apolipoprotein B100 and pigment epithelial-derived factor were replicated (p < 0.05). These proteins, together with glial fibrillary acidic protein (GFAP) and receptor for advanced glycation end product, were tested in 38 ischemic stroke and 28 ICH samples. Finally, RBP4 >61 μg/mL and GFAP <0.07 ng/mL showed a specificity of 100% for both subtypes. Moreover, after multivariate logistic regression analysis, RBP4 >48.75 μg/mL (ORadj : 6.09 (1.3-28.57), p = 0.02) and GFAP <0.07 ng/mL (ORadj : 0.03 (0.003-0.31), p = 0.003) resulted in independent predictors of stroke subtype, improving discrimination by 29% (p < 0.0001). Both biomarkers might be useful as diagnostic biomarkers to differentiate ischemic stroke and ICH. A rapid differentiation of ischemic stroke from intracerebral hemorrhage is essential to provide the appropriate treatment. We describe the discovery and subsequent replications of RBP4 and its combination with circulating GFAP as plasmatic biomarkers for hyperacute stroke subtype differentiation. The combination of these biomarkers and others might aid to speed up the discrimination of both stroke subtypes improving the outcome of patients.

  2. Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix

    PubMed Central

    Templeton, Catherine M.; Ostovar pour, Saeideh; Hobbs, Jeanette R.; Blanch, Ewan W.; Munger, Steven D.

    2011-01-01

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding. PMID:21343241

  3. p38 Mitogen-Activated Protein Kinase Pathway Regulates Genes during Proliferation and Differentiation in Oligodendrocytes

    PubMed Central

    Haines, Jeffery D.; Fulton, Debra L.; Richard, Stephane; Almazan, Guillermina

    2015-01-01

    We have previously shown that p38 mitogen-activated protein kinase (p38 MAPK) is important for oligodendrocyte (OLG) differentiation and myelination. However, the precise cellular mechanisms by which p38 regulates OLG differentiation remain largely unknown. To determine whether p38 functions in part through transcriptional events in regulating OLG identity, we performed microarray analysis on differentiating oligodendrocyte progenitors (OLPs) treated with a p38 inhibitor. Consistent with a role in OLG differentiation, pharmacological inhibition of p38 down-regulated the transcription of genes that are involved in myelin biogenesis, transcriptional control and cell cycle. Proliferation assays showed that OLPs treated with the p38 inhibitor retained a proliferative capacity which could be induced upon application of mitogens demonstrating that after two days of p38-inhibition OLGs remained poised to continue mitosis. Together, our results suggest that the p38 pathway regulates gene transcription which can coordinate OLG differentiation. Our microarray dataset will provide a useful resource for future studies investigating the molecular mechanisms by which p38 regulates oligodendrocyte differentiation and myelination. PMID:26714323

  4. Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion.

    PubMed

    Atshaves, B P; Foxworth, W B; Frolov, A; Roths, J B; Kier, A B; Oetama, B K; Piedrahita, J A; Schroeder, F

    1998-03-01

    The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.

  5. Purification and Characterization of the Manganese(II) Oxidizing Protein from Erythrobacter sp. SD-21

    NASA Astrophysics Data System (ADS)

    Nakama, K. R.; Lien, A.; Johnson, H. A.

    2013-12-01

    The manganese(II) oxidizing protein (Mop) found in the alpha-proteobacterium Erythrobacter sp. SD-21 catalyzes the formation of insoluble Mn(III/IV) oxides from soluble Mn(II). These Mn(III/IV) oxides formed are one of the strongest naturally occurring oxides, next to oxygen, and can be used to adsorb and oxidize toxic chemicals from the surrounding environment. Because of the beneficial use in the treatment of contaminated sources, the mechanism and biochemical properties of this novel enzyme are being studied. Due to low expression levels in the native host strain, purification of Mop has been problematic. To overcome this problem the gene encoding Mop, mopA, was cloned from the native host into a C-terminal histidine tag vector and expressed in Escherichia coli cells. Affinity chromatography under denaturing conditions have been applied in attempts to purify an active Mop. Western blots have confirmed that the protein is being expressed and is at the expected size of 250 kDa. Preliminary characterization on crude extract containing Mop has shown a Km and vmax value of 2453 uM and 0.025 uM min-1, respectively. Heme and pyrroloquinoline quinone can stimulate Mn(II) oxidizing activity, but hydrogen peroxide does not affect activity, despite the sequence similarity to animal heme peroxidase proteins. Research has been shown that calcium is essential for Mop activity. Purifying an active Mn(II) oxidizing protein will allow for a better understanding behind the enigmatic process of Mn(II) oxidation.

  6. Evolutionary model selection and parameter estimation for protein-protein interaction network based on differential evolution algorithm

    PubMed Central

    Huang, Lei; Liao, Li; Wu, Cathy H.

    2016-01-01

    Revealing the underlying evolutionary mechanism plays an important role in understanding protein interaction networks in the cell. While many evolutionary models have been proposed, the problem about applying these models to real network data, especially for differentiating which model can better describe evolutionary process for the observed network urgently remains as a challenge. The traditional way is to use a model with presumed parameters to generate a network, and then evaluate the fitness by summary statistics, which however cannot capture the complete network structures information and estimate parameter distribution. In this work we developed a novel method based on Approximate Bayesian Computation and modified Differential Evolution (ABC-DEP) that is capable of conducting model selection and parameter estimation simultaneously and detecting the underlying evolutionary mechanisms more accurately. We tested our method for its power in differentiating models and estimating parameters on the simulated data and found significant improvement in performance benchmark, as compared with a previous method. We further applied our method to real data of protein interaction networks in human and yeast. Our results show Duplication Attachment model as the predominant evolutionary mechanism for human PPI networks and Scale-Free model as the predominant mechanism for yeast PPI networks. PMID:26357273

  7. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  8. Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.

    PubMed

    Hensley, M Patrick; Gunasekera, Thusitha S; Easton, J Allen; Sigdel, Tara K; Sugarbaker, Stacy A; Klingbeil, Lindsey; Breece, Robert M; Tierney, David L; Crowder, Michael W

    2012-06-01

    RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.

  9. Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart.

    PubMed

    Stepanova, Anna; Shurubor, Yevgeniya; Valsecchi, Federica; Manfredi, Giovanni; Galkin, Alexander

    2016-09-01

    Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10(-8)M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2±6.0% and 56.4±5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

  10. Pravastatin activates activator protein 2 alpha to argument the angiotensin II-induced abdominal aortic aneurysms.

    PubMed

    Ma, Hui; Liang, Wen-Jing; Shan, Mei-Rong; Wang, Xue-Qing; Zhou, Sheng-Nan; Chen, Yuan; Guo, Tao; Li, Peng; Yu, Hai-Ya; Liu, Chao; Yin, Ya-Ling; Wang, Yu-Lin; Dong, Bo; Pang, Xin-Yan; Wang, Shuang-Xi

    2017-02-04

    We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.

  11. Heterologous protein expression in Trichoderma reesei using the cbhII promoter.

    PubMed

    Meng, Fanju; Wei, Dongzhi; Wang, Wei

    2013-09-01

    To express homologous or heterologous proteins in fungi, a protein expression system using the promoter of cellobiohydrolase II gene (cbhII) was constructed by generating an expression vector called pWEIIF00. The obtained vector possesses the left and right borders, a hygromycin phosphotransferase B selective marker and a strong promoter and terminator of cbhII from Trichoderma reesei. It can easily undergo random recombination. The applicability of the vector was tested by red fluorescent protein gene (DsRed2) expression detection in T. reesei Rut C30. Using this system, a recombinant Cel5A variant, N342R (Qin et al., 2008), was then selected to express in Rut-C30. Compared to that of the parent strain, integration of the N342R gene resulted in 31.09% increased carboxymethyl-cellulose-degrading (CMCase) activity at pH 5.0 and 56.06% increased activity at pH 6.0. The increased CMCase activity of the recombinant strains would be beneficial for its application uses in multiple industries. The vector constructed in this study can used in fungi to produce industrial proteins.

  12. In vitro degradation of the 32kDa PS II reaction centre protein

    SciTech Connect

    Eckenswiller, L.C.; Greenberg, B.M. )

    1989-04-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components.

  13. Igf Binding Proteins Protect Undifferentiated Spermatogonia in the Zebrafish Testis Against Excessive Differentiation.

    PubMed

    Safian, Diego; Morais, Roberto D V S; Bogerd, Jan; Schulz, Rüdiger W

    2016-11-01

    IGF binding proteins (IGFBPs) modulate the availability of IGFs for their cognate receptors. In zebrafish testes, IGF3 promotes the proliferation and differentiation of type A undifferentiated (Aund) spermatogonia, and igf3 expression is strongly elevated by FSH but also responds to T3. Here we report the effects of FSH and T3 on igfbp transcript levels in adult zebrafish testis. We then examined T3 and FSH effects on zebrafish spermatogenesis and explored the relevance of IGFBPs in modulating these T3 or FSH effects, using a primary tissue culture system for adult zebrafish testis. T3 up-regulated igfbp1a and igfbp3 expression, whereas FSH reduced igfbp1a transcript levels. To quantify effects on spermatogenesis, we determined the mitotic index and relative section areas occupied by Aund, type A differentiating, or type B spermatogonia. In general, T3 and FSH stimulated spermatogonial proliferation and increased the areas occupied by spermatogonia, suggesting that both self-renewal and differentiating divisions were stimulated. Preventing IGF/IGFBP interaction by NBI-31772 further increased T3- or FSH-induced spermatogonial proliferation. However, under these conditions the more differentiated type A differentiating and B spermatogonia occupied larger surface areas at the expense of the area held by Aund spermatogonia. Clearly decreased nanos2 transcript levels are in agreement with this finding, and reduced amh expression may have facilitated spermatogonial differentiation. We conclude that elevating IGF3 bioactivity by blocking IGFBPs shifted T3- or FSH-induced signaling from stimulating spermatogonial self-renewal as well as differentiation toward predominantly stimulating spermatogonial differentiation, which leads to a depletion of type Aund spermatogonia.

  14. The role of prolyl hydroxylase domain protein (PHD) during rosiglitazone-induced adipocyte differentiation.

    PubMed

    Kim, Juyoung; Kwak, Hyun Jeong; Cha, Ji-Young; Jeong, Yun-Seung; Rhee, Sang Dahl; Cheon, Hyae Gyeong

    2014-01-31

    Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor γ (PPARγ). Previous two-dimensional proteomics studies using C3H10T1/2 murine mesenchymal pluripotent stem cells revealed that prolyl hydroxylase domain protein (PHD) levels significantly increased during rosiglitazone-induced adipocyte differentiation (RIAD). In this study, we investigated the functional role played by PHD during RIAD. Three PHD isoforms (PHD1, 2, and 3) were found to be up-regulated in C3H10T1/2 cells during RIAD, whereas PHD knockdown and treatment with PHD inhibitors (dimethyloxalyl glycine or ethyl-3,4-dihydroxybenzoate) blocked RIAD. PHD inhibition was found to be associated with increases in the levels of anti-adipogenic proteins such as GATA-3, KLF-2, and transcriptional coactivator with PDZ binding motif (TAZ), with their reduced ubiquitination, suggesting that PHDs evoke the ubiquitination/proteasomal degradation of anti-adipogenic proteins. On the other hand, MG-132 (a proteasomal inhibitor) prevented the degradation of anti-adipogenic proteins and retarded RIAD. PPARγ antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPARγ binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPARγ activation. Consistent with in vitro results, oral administration of rosiglitazone to ob/ob mice for 2 weeks increased adipose PHD levels and decreased anti-adipogenic protein levels by increasing their ubiquitination. These results suggest that rosiglitazone increases PHD expression in a PPARγ-dependent manner and that this leads to the commitment of anti-adipogenic proteins to the ubiquitination-proteasomal pathway and to the subsequent induction of adipocyte differentiation.

  15. Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression.

    PubMed

    Wang, Shuai; Yang, Yanbei; Zhao, Yulin; Zhao, Honghai; Bai, Jingwen; Chen, Jianqing; Zhou, Yonghui; Wang, Chang; Li, Yanhua

    2016-01-01

    Streptococcus suis (S.suis) is an important zoonotic pathogen that causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections that are difficult to treat. In this study, the effect of tylosin on biofilm formation of S. suis was investigated. 1/2 minimal inhibitory concentration (MIC) and 1/4 MIC of tylosin were shown to inhibit S. suis biofilm formation in vitro. By using the iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment and with no treatment. A total of 1501 proteins were identified by iTRAQ. Ninety-six differentially expressed proteins were identified (Ratio > ±1.5, p < 0.05). Several metabolism proteins (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) were found to be involved in biofilm formation. Our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth with sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that may provide a foundation for future research into mechanisms and targets.

  16. Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression

    PubMed Central

    Wang, Shuai; Yang, Yanbei; Zhao, Yulin; Zhao, Honghai; Bai, Jingwen; Chen, Jianqing; Zhou, Yonghui; Wang, Chang; Li, Yanhua

    2016-01-01

    Streptococcus suis (S.suis) is an important zoonotic pathogen that causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections that are difficult to treat. In this study, the effect of tylosin on biofilm formation of S. suis was investigated. 1/2 minimal inhibitory concentration (MIC) and 1/4 MIC of tylosin were shown to inhibit S. suis biofilm formation in vitro. By using the iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment and with no treatment. A total of 1501 proteins were identified by iTRAQ. Ninety-six differentially expressed proteins were identified (Ratio > ±1.5, p < 0.05). Several metabolism proteins (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) were found to be involved in biofilm formation. Our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth with sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that may provide a foundation for future research into mechanisms and targets. PMID:27065957

  17. Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

    PubMed Central

    Munro, Kathryn M.; Kennedy, Matthew J.; Gunnersen, Jenny M.

    2014-01-01

    In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available. PMID:24561550

  18. Simultaneous determination of trace Cd(II), Pb(II) and Cu(II) by differential pulse anodic stripping voltammetry using a reduced graphene oxide-chitosan/poly-l-lysine nanocomposite modified glassy carbon electrode.

    PubMed

    Guo, Zhuo; Li, Dong-di; Luo, Xian-Ke; Li, Ya-Hui; Zhao, Qi-Nai; Li, Meng-Meng; Zhao, Yang-Ting; Sun, Tian-Shuai; Ma, Chi

    2017-03-15

    The reduced graphene oxide (RGO) and Chitosan (CS) hybrid matrix RGO-CS were coated onto the glassy carbon electrode (GCE) surface, then, poly-l-lysine films (PLL) were prepared by electropolymerization with cyclic voltammetry (CV) method to prepare RGO-CS/PLL modified glassy carbon electrode (RGO-CS/PLL/GCE) for the simultaneous electrochemical determination of heavy metal ions Cd(II), Pb(II) and Cu(II). Combining the advantageous features of RGO and CS, RGO and CS are used together because the positively charged CS can interact with the negatively changed RGO to prevent their aggregation. Furthermore, CS has many amino groups along its macromolecular chains and possessed strongly reactive with metal ions. Moreover, PLL modified electrodes have good stability, excellent permselectivity, more active sites and strong adherence to electrode surface, which enhanced electrocatalytic activity. The RGO-CS/PLL/GCE was characterized voltammetrically using redox couples (Fe(CN)6(3-/4-)), complemented with electrochemical impedance spectroscopy (EIS). Differential pulse anodic stripping voltammetry (DPASV) has been used for the detection of Cd(II), Pb(II) and Cu(II). The detection limit of RGO-CS/PLL/GCE toward Cd(II), Pb(II) and Cu(II) is 0.01μgL(-1), 0.02μgL(-1) and 0.02μgL(-1), respectively. The electrochemical parameters that exert influence on deposition and stripping of metal ions, such as supporting electrolytes, pH value, deposition potential, and deposition time, were carefully studied.

  19. Recombinant expressions of sweet plant protein mabinlin II in Escherichia coli and food-grade Lactococcus lactis.

    PubMed

    Gu, Wenliang; Xia, Qiyu; Yao, Jing; Fu, Shaoping; Guo, Jianchun; Hu, Xinwen

    2015-04-01

    Sweet plant proteins, which are safe, natural, low-calorie sweeteners, may be suitable replacements for sugars in the food and beverage industries. Mabinlin II, a sweet plant protein, shows the most pronounced heat stability and acid resistance of any of the six known types of plant sweet proteins. However, mabinlin II is difficult to extract from the Capparis masaikai plant, which is itself becoming increasingly scarce. This limits the use of naturally acquired mabinlin II. In this study, recombinant mabinlin II proteins were expressed and purified in Escherichia coli and in food-grade Lactococcus lactis. Recombinant mabinlin II proteins MBL-BH (containing the B-chains of mabinlin II downstream fused with His-tag) and MBL-ABH (containing the A- and B-chains of mabinlin II downstream fused with His-tag) were expressed in E. coli in the form of inclusion bodies. They were then purified and renatured. The refolded MBL-BH was found to be 100 times sweeter than sucrose by weight, but it was not heat-stable. Refolded MBL-ABH was neither sweet nor heat-stable. Recombinant mabinlin II proteins were secreted and expressed intracellularly in food-grade L. lactis, in which the concentrated cell samples and culture medium samples were detected using enzyme-linked immunosorbent assay and Western blotting analysis with anti-mabinlin II polyclonal antibody. This study demonstrated that the single B chain of mabinlin II has a sweet taste. The recombinant mabinlin II proteins have been successfully expressed in food-grade L. lactis, which is a crucial step in the production of mabinlin II through microorganism expression systems.

  20. PROTEINS IN NUCLEOCYTOPLASMIC INTERACTIONS : II. Turnover and Changes in Nuclear Protein Distribution with Time and Growth.

    PubMed

    Goldstein, L; Prescott, D M

    1968-01-01

    In previous studies, we showed that essentially all the proteins of the Amoeba proteus nucleus could be classified either as Rapidly Migrating Proteins (RMP), which shuttle between nucleus and cytoplasm continuously at a relatively rapid rate during interphase, or as Slow Turnover Proteins (STP), which seem to move hardly at all during interphase. In this paper, we report on the kinetics and direction of the movement of both classes of protein, as well as on aspects of their localization, with and without growth. The effects of growth were observed with and without cell division. These nuclear proteins have been studied in several ways: by transplantation of labeled nuclei into unlabeled cells and noting the rate of distribution to cytoplasm and host cell nuclei; by repeated amputation of cytoplasm from labeled cells-with and without initially labeled cytoplasm-each amputation being followed by refeeding on unlabeled food; by noting the redistribution of the various protein classes following growth and cell division. The data show (a) labeled RMP equilibrate between a grafted labeled nucleus and an unlabeled host nucleus in ca. 3 hr, but are detectable in the latter less than 30 min after the operation; (b) STP label does, indeed, leave the nucleus and does so at a rate of ca. 25% of the nuclear total per cell generation (ca. 36-40 hr at 23 degrees C); (c) the cytoplasm appears to have a reserve of material that is converted to RMP; (d) when labeled cells are amputated just before they would have divided and are refed unlabeled food after each amputation, there is a loss of 20-25% of the nuclear protein label with each amputation; (e) under the latter circumstances, an essentially complete turnover of all nuclear protein can be demonstrated.

  1. Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

    PubMed

    Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

    2010-06-01

    Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

  2. Paracrine Secreted Frizzled-Related Protein 4 Inhibits Melanocytes Differentiation in Hair Follicle

    PubMed Central

    Guo, Haiying; Lei, Mingxing; Li, Yuhong; Liu, Yingxin; Tang, Yinhong; Xing, Yizhan; Deng, Fang

    2017-01-01

    Wnt signaling plays crucial role in regulating melanocyte stem cells/melanocyte differentiation in the hair follicle. However, how the Wnt signaling is balanced to be overactivated to control follicular melanocytes behavior remains unknown. Here, by using immunofluorescence staining, we showed that secreted frizzled-related protein 4 (sFRP4) is preferentially expressed in the skin epidermal cells rather than in melanocytes. By overexpression of sFRP4 in skin cells in vivo and in vitro, we found that sFRP4 attenuates activation of Wnt signaling, resulting in decrease of melanocytes differentiation in the regenerating hair follicle. Our findings unveiled a new regulator that involves modulating melanocytes differentiation through a paracrine mechanism in hair follicle, supplying a hope for potential therapeutic application to treat skin pigmentation disorders. PMID:28337220

  3. Matrix Gla protein regulates differentiation of endothelial cells derived from mouse embryonic stem cells.

    PubMed

    Yao, Jiayi; Guihard, Pierre J; Blazquez-Medela, Ana M; Guo, Yina; Liu, Ting; Boström, Kristina I; Yao, Yucheng

    2016-01-01

    Matrix Gla protein (MGP) is an antagonist of bone morphogenetic proteins and expressed in vascular endothelial cells. Lack of MGP causes vascular abnormalities in multiple organs in mice. The objective of this study is to define the role of MGP in early endothelial differentiation. We find that expression of endothelial markers is highly induced in Mgp null organs, which, in wild type, contain high MGP expression. Furthermore, Mgp null embryonic stem cells express higher levels of endothelial markers than wild-type controls and an abnormal temporal pattern of expression. We also find that the Mgp-deficient endothelial cells adopt characteristics of mesenchymal stem cells. We conclude that loss of MGP causes dysregulation of early endothelial differentiation.

  4. Effects of ECM protein micropatterns on the migration and differentiation of adult neural stem cells.

    PubMed

    Joo, Sunghoon; Kim, Joo Yeon; Lee, Eunsoo; Hong, Nari; Sun, Woong; Nam, Yoonkey

    2015-08-12

    The migration and differentiation of adult neural stem cells (aNSCs) are believed to be strongly influenced by the spatial distribution of extracellular matrix (ECM) proteins in the stem cell niche. In vitro culture platform, which involves the specific spatial distribution of ECM protein, could offer novel tools for better understanding of aNSC behavior in the spatial pattern of ECM proteins. In this work, we applied soft-lithographic technique to design simple and reproducible laminin (LN)-polylysine cell culture substrates and investigated how aNSCs respond to the various spatial distribution of laminin, one of ECM proteins enriched in the aNSC niche. We found that aNSC preferred to migrate and attach to LN stripes, and aNSC-derived neurons and astrocytes showed significant difference in motility towards LN stripes. By changing the spacing of LN stripes, we were able to control the alignment of neurons and astrocytes. To the best of our knowledge, this is the first time to investigate the differential cellular responses of aNSCs on ECM protein (LN) and cell adhesive synthetic polymer (PDL) using surface micropatterns. Our findings would provide a deeper understanding in astrocyte-neuron interactions as well as ECM-stem cell interactions.

  5. Characterization of unique and differentially expressed proteins in anthracnose-tolerant Florida hybrid bunch grapes.

    PubMed

    Vasanthaiah, Hemanth K N; Katam, Ramesh; Basha, Sheikh M

    2009-06-01

    Anthracnose is a major disease in Florida hybrid bunch grapes, caused by a fungus viz. Elsinoe ampelina. Florida hybrid bunch grapes are grown in southeastern USA for their superior wine characteristics. However, the effect of anthracnose on grape productivity and wine quality is a major concern to grape growers. Our research is aimed at determining biochemical basis of anthracnose tolerance in Florida hybrid bunch grape. Leaf samples were collected from the plants infected with E. ampelina at different periods and analyzed for differential protein expression using high throughput two-dimensional gel electrophoresis. Among the 32 differentially expressed leaf proteins, two were uniquely expressed in tolerant genotypes in response to E. ampelina infection. These proteins were identified as mitochondrial adenosine triphosphate synthase and glutamine synthetase, which are known to play a major role in carbohydrate metabolism and defense. Several proteins including ribulose 1-5 bisphosphate-carboxylase involved in photosynthesis were found to be suppressed in susceptible genotypes compared to tolerant genotypes following E. ampelina infection. The results indicate that the anthracnose-tolerant genotypes have the ability to up-regulate and induce new proteins upon infection to defend the invasion of the pathogen as well as maintain the normal regulatory processes.

  6. Comparative proteomic analysis of differentially expressed proteins between peripheral sensory and motor nerves.

    PubMed

    He, Qianru; Man, Lili; Ji, Yuhua; Zhang, Shuqiang; Jiang, Maorong; Ding, Fei; Gu, Xiaosong

    2012-06-01

    Peripheral sensory and motor nerves have different functions and different approaches to regeneration, especially their distinct ability to accurately reinervate terminal nerve pathways. To understand the molecular aspects underlying these differences, the proteomics technique by coupling isobaric tags for relative and absolute quantitation (iTRAQ) with online two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) was used to investigate the protein profile of sensory and motor nerve samples from rats. A total of 1472 proteins were identified in either sensory or motor nerve. Of them, 100 proteins showed differential expressions between both nerves, and some of them were validated by quantitative real time RT-PCR, Western blot analysis, and immunohistochemistry. In the light of functional categorization, the differentially expressed proteins in sensory and motor nerves, belonging to a broad range of classes, were related to a diverse array of biological functions, which included cell adhesion, cytoskeleton, neuronal plasticity, neurotrophic activity, calcium-binding, signal transduction, transport, enzyme catalysis, lipid metabolism, DNA-binding, synaptosome function, actin-binding, ATP-binding, extracellular matrix, and commitment to other lineages. The relatively higher expressed proteins in either sensory or motor nerve were tentatively discussed in combination with their specific molecular characteristics. It is anticipated that the database generated in this study will provide a solid foundation for further comprehensive investigation of functional differences between sensory and motor nerves, including the specificity of their regeneration.

  7. Translational attenuation differentially alters the fate of disease-associated fibulin proteins

    PubMed Central

    Hulleman, John D.; Balch, William E.; Kelly, Jeffery W.

    2012-01-01

    Mutations in fibulin proteins that cause cellular secretion deficiencies are linked to a variety of diseases, ranging from retinopathies to cutis laxa (CL). One secretion-deficient fibulin mutant, R345W fibulin-3, causes the macular dystrophy malattia leventinese by increased endoplasmic reticulum retention and/or extracellular misfolding. Herein, we report that small-molecule activation of the PERK arm of the unfolded protein response partially rescues R345W secretion deficiencies through translational attenuation mediated by eIF2α phosphorylation. Enhanced mutant fibulin-3 secretion can also be achieved by activation of a PERK-independent eIF2α kinase through arsenite treatment and is independent of activating transcription factor 4 signaling and protein translation. However, this translational attenuation strategy was unsuccessful for enhancing the secretion deficiencies of fibulin-5 mutants associated with age-related macular degeneration or CL. While lowered growth temperature enhanced the secretion of mutants associated with CL (C217R and S227P), these effects were not mediated through translational attenuation. In stark contrast to the situation with fibulin-3, protein translation was required for efficient wild-type and mutant fibulin-5 secretion. These data suggest that alteration of specific cellular signaling pathways and proteostasis network components can differentially influence fibulin fate, a hypothesis that could be exploited as a therapy for fibulin-related diseases.—Hulleman, J. D., Balch, W. E., Kelly, J. W. Translational attenuation differentially alters the fate of disease-associated fibulin proteins. PMID:22872678

  8. Renoprotective and blood pressure-lowering effect of dietary soy protein via protein kinase C beta II inhibition in a rat model of metabolic syndrome.

    PubMed

    Palanisamy, Nallasamy; Viswanathan, Periyasamy; Ravichandran, Mambakkam Katchapeswaran; Anuradha, Carani Venkataraman

    2010-01-01

    We studied whether substitution of soy protein for casein can improve insulin sensitivity, lower blood pressure (BP), and inhibit protein kinase C betaII (PKCbetaII) activation in kidney in an acquired model of metabolic syndrome. Adult male rats were fed 4 different diets: (i) starch (60%) and casein (20%) (CCD), (ii) fructose (60%) and casein (20%) (FCD), (iii) fructose (60%) and soy protein (20%) (FSD), and (iv) starch (60%) and soy protein (20%) (CSD). Renal function parameters, BP, pressor mechanisms, PKCbetaII expression, oxidative stress, and renal histology were evaluated after 60 days. FCD rats displayed insulin resistance and significant changes in body weight, kidney weight, urine volume, plasma and urine electrolytes accompanied by significant changes in renal function parameters compared with CCD rats. Elevated BP, plasma angiotensin-converting enzyme (ACE) activity, renal oxidative stress, and reduced nitrite (NO) and kallikrein activity were observed. Western blot analysis revealed enhanced renal expression of membrane-associated PKCbetaII in the FCD group. Histology showed fatty infiltration and thickening of glomeruli while urinary protein profile revealed a 5-fold increase in albumin. Substitution of soy protein for casein improved insulin sensitivity, lowered BP and PKCbetaII activation and restored renal function. Antioxidant action, inhibitory effect on ACE and PKCbetaII activation, and increased availability of kinins and NO could be contributing mechanisms for the benefits of dietary soy protein.

  9. Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders

    DOEpatents

    Lelievre, Sophie; Bissell, Mina

    2001-01-01

    The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

  10. The RNA Binding Protein Csx1 Promotes Sexual Differentiation in Schizosaccharomyces pombe

    PubMed Central

    Matia-Gonzalez, Ana M.; Sotelo, Jael; Rodriguez-Gabriel, Miguel A.

    2012-01-01

    Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source. One of the key regulatory proteins in fission yeast sexual differentiation is the transcription factor Ste11. Ste11 regulates the transcription of many genes required for the initial steps of conjugation and meiosis, and its deficiency leads to sterility. Ste11 activity is mainly regulated at two levels: phosphorylation and abundance of its mRNA. Csx1 is an RNA binding protein that we have previously described to bind and regulate the turnover rate of the mRNA encoding the transcription factor Atf1 in the presence of oxidative stress. We have observed that Csx1-deficient cells have defects in sexual differentiation and are partially sterile. We investigated how Csx1 is regulating this process in S. pombe. Csx1 associates with ste11+ mRNA and cells lacking Csx1 are sterile with a reduced amount of ste11+ mRNA. Overexpression of ste11+ mRNA completely rescues the mating deficiencies of csx1Δ cells. Here, we present a novel mechanism of ste11+ mRNA positive regulation through the activity of Csx1, an RNA binding protein that also have key functions in the response to oxidative stress in fission yeast. This finding opens interesting question about the possible coordination of sexual differentiation and oxidative stress response in eukaryotes and the role of RNA binding proteins in the adaptation to environmental signals. PMID:22253882

  11. Relationships between milks differentiated on native milk fat globule characteristics and fat, protein and calcium compositions.

    PubMed

    Couvreur, S; Hurtaud, C

    2017-03-01

    Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a

  12. RNA-binding proteins related to stress response and differentiation in protozoa.

    PubMed

    Alves, Lysangela Ronalte; Goldenberg, Samuel

    2016-02-26

    RNA-binding proteins (RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein (RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress (nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

  13. Unique roles of the unfolded protein response pathway in fungal development and differentiation

    PubMed Central

    Jung, Kwang-Woo; So, Yee-Seul; Bahn, Yong-Sun

    2016-01-01

    Cryptococcus neoformans, a global fungal meningitis pathogen, employs the unfolded protein response pathway. This pathway, which consists of an evolutionarily conserved Ire1 kinase/endoribonuclease and a unique transcription factor (Hxl1), modulates the endoplasmic reticulum stress response and pathogenicity. Here, we report that the unfolded protein response pathway governs sexual and unisexual differentiation of C. neoformans in an Ire1-dependent but Hxl1-independent manner. The ire1∆ mutants showed defects in sexual mating, with reduced cell fusion and pheromone-mediated formation of the conjugation tube. Unexpectedly, these mating defects did not result from defective pheromone production because expression of the mating pheromone gene (MFα1) was strongly induced in the ire1∆ mutant. Ire1 controls sexual differentiation by modulating the function of the molecular chaperone Kar2 and by regulating mating-induced localisation of mating pheromone transporter (Ste6) and receptor (Ste3/Cprα). Deletion of IRE1, but not HXL1, also caused significant defects in unisexual differentiation in a Kar2-independent manner. Moreover, we showed that Rim101 is a novel downstream factor of Ire1 for production of the capsule, which is a unique structural determinant of C. neoformans virulence. Therefore, Ire1 uniquely regulates fungal development and differentiation in an Hxl1-independent manner. PMID:27629591

  14. Modification of proteins by cyclopentenone prostaglandins is differentially modulated by GSH in vitro.

    PubMed

    Gayarre, Javier; Avellano, M Isabel; Sánchez-Gómez, Francisco J; Carrasco, M Jesús; Cañada, F Javier; Pérez-Sala, Dolores

    2007-01-01

    Prostanoids with cyclopentenone structure (cyP) display a potent anti-inflammatory and antiproliferative activity. CyP are reactive compounds, which may modulate cellular functions by multiple mechanisms, including the direct covalent modification of cysteine residues by Michael addition. This interaction displays selectivity since only a subset of cellular proteins is modified by cyP. Several factors have been proposed to influence the selectivity and/or extent of cyP addition to proteins, including determinants related to protein and cyP structure, and levels of cellular thiols, such as glutathione (GSH). Here we have explored the ability of biotinylated cyP analogs to modify several recombinant proteins in vitro, and the influence of GSH in these effects. We have observed that protein modification by cyP is protein- and cyP-selective. Under our conditions, biotinylated 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) was more efficient than biotinylated PGA(1) (PGA(1)-B) at forming adducts with components of the transcription factors NF-kappaB and activator protein-1 (AP-1). However, both biotinylated cyP were nearly equipotent at modifying human GSTP1-1. Interestingly, the presence of GSH differentially modulated the formation of protein-cyP adducts. Under our conditions, GSH reduced the incorporation of cyP into GST, but improved their binding to p50, more intensely in the case of PGA(1)-B. These results evidence the importance of GSH-cyP and/or GSH-protein interactions for the selectivity of protein modification by cyP and suggest a complex role for GSH that may be related to its ability to prevent protein oxidation or induce conformational alterations. This may shed light on the factors involved in the pleiotropic effects of electrophiles with therapeutic potential.

  15. Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

    PubMed Central

    Mundy, John; Hejgaard, Jørn; Hansen, Annette; Hallgren, Lars; Jorgensen, Kim G.; Munck, Lars

    1986-01-01

    To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA3. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16664868

  16. Genome-wide Analysis of RNA Polymerase II Termination at Protein-Coding Genes.

    PubMed

    Baejen, Carlo; Andreani, Jessica; Torkler, Phillipp; Battaglia, Sofia; Schwalb, Bjoern; Lidschreiber, Michael; Maier, Kerstin C; Boltendahl, Andrea; Rus, Petra; Esslinger, Stephanie; Söding, Johannes; Cramer, Patrick

    2017-03-06

    At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.

  17. Papain digestion of crude Trichoderma reesei cellulase: Purification and properties of cellobiohydrolase I and II core proteins

    SciTech Connect

    Woodward, J.; Brown, J.P.; Evans, B.R.; Affholter, K.A.

    1992-12-01

    Papain digestion of a crude Trichoderma reesei cellulose preparation followed by gel filtration on a Superdex column resulted in the separation of cellobiohydrolase (CBH) I and II core proteins (cp). They were further purified to apparent homogeneity by chromatofocusing. N-terminal protein sequencing of the CBH II cp preparation confirmed its identity. A comparison of the catalytic activity and cellulose-binding ability of these core proteins was made. The major differences between them were the findings that CBH II cp possessed a sixfold higher specific activity toward p-nitrophenylcellobioside than the native CBH II preparation and still bound to microcrystalline cellulose, unlike CBH I cp. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyze barley b-glucan. These data suggest that removal of the cellulose-binding domain and hinge region from CBH I and II have different effects on their properties.

  18. Papain digestion of crude Trichoderma reesei cellulase: Purification and properties of cellobiohydrolase I and II core proteins

    SciTech Connect

    Woodward, J.; Brown, J.P.; Evans, B.R.; Affholter, K.A.

    1992-01-01

    Papain digestion of a crude Trichoderma reesei cellulose preparation followed by gel filtration on a Superdex column resulted in the separation of cellobiohydrolase (CBH) I and II core proteins (cp). They were further purified to apparent homogeneity by chromatofocusing. N-terminal protein sequencing of the CBH II cp preparation confirmed its identity. A comparison of the catalytic activity and cellulose-binding ability of these core proteins was made. The major differences between them were the findings that CBH II cp possessed a sixfold higher specific activity toward p-nitrophenylcellobioside than the native CBH II preparation and still bound to microcrystalline cellulose, unlike CBH I cp. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyze barley b-glucan. These data suggest that removal of the cellulose-binding domain and hinge region from CBH I and II have different effects on their properties.

  19. Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent.

    PubMed

    Samy, Ramar Perumal; Stiles, Bradley G; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman Ali; Franco, Octavio Luiz; Rowan, Edward G; Kumar, Alan Prem; Lim, Lina H K; Sethi, Gautam

    2015-01-01

    Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell's viper snake venom. Novel 15-kDa proteins called "Viperatoxin" (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of "Viperatoxin" showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100-3.1 μg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 μg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 μg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 μg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo.

  20. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-08-05

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

  1. Localization of a bacterial group II intron-encoded protein in human cells

    PubMed Central

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  2. Biochemical and Bioinformatic Characterization of Type II Metacaspase Protein (TaeMCAII) from Wheat.

    PubMed

    Piszczek, E; Dudkiewicz, M; Mielecki, M

    2012-01-01

    The biochemical analysis and homology modeling of a tertiary structure of a cereal type II metacaspase protein from wheat (Triticum aestivum), TaeMCAII, are presented. The biochemical characterization of synthetic oligopeptides and protease inhibitors of Escherichia coli-produced and purified recombinant TaeMCAII revealed that this metacaspase protein, similar to other known plant metacaspases, is an arginine/lysine-specific cysteine protease. Thus, a model of a plant type II metacaspase structure based on newly identified putative metacaspase-like template was proposed. Homology modeling of the TaeMCAII active site tertiary structure showed two cysteine residues, Cys140 and 23, in close proximity to the catalytic histidine, most likely participating in proton exchange during the catalytic process. The autoprocessing that leads to activation of TaeMCAII was highly dependent on Cys140. TaeMCAII required high levels of calcium ions for activity, which could indicate its involvement in stress signaling pathways connected to programmed cell death.

  3. Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

    PubMed

    Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

    2013-04-05

    To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

  4. Comparative proteomic changes of differentially expressed whey proteins in clinical mastitis and healthy yak cows.

    PubMed

    Li, X; Ding, X Z; Wan, Y L; Liu, Y M; Du, G Z

    2014-08-28

    Under the traditional grazing system on the Qinghai-Tibetan Plateau, the amount of milk in domesticated yak (Bos grunniens) with clinical mastitis decreases and the milk composition is altered. To understand the mechanisms of mammary gland secreted milk and disease infection, changes in the protein composition of milk during clinical mastitis were investigated using a proteomic approach. Milk whey from yak with clinical mastitis was compared to whey from healthy animals with two-dimensional gel electrophoresis using a mass spectrometer. Thirteen protein spots were identified to be four differentially expressed proteins. Increases in the concentrations of proteins of blood serum origin, including lactoferrin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins, casocidin-I, a-lactalbumin, and b-lactoglobulin, were downregulated in mastitic whey. These results indicated significant differences in protein expression between healthy yaks and those with clinical mastitis, and they may provide valuable information for finding new regulation markers and potential protein targets for the treatment of mastitis.

  5. Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae.

    PubMed

    Zhao, Fu'an; Fang, Weiping; Xie, Deyi; Zhao, Yuanming; Tang, Zhongjie; Li, Wu; Nie, Lihong; Lv, Shuping

    2012-04-01

    Thurber's cotton (Gossypium thurberi) is the wild relative of cultivated cotton. It is highly resistant to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. To reveal the mechanism of disease resistance in G. thurberi and to clone resistance-related genes, we used two-dimensional electrophoresis (2-DE) and tandem time-of-flight mass spectrometry (MALDI-TOF-MS) to identify differentially expressed proteins in Thurber's cotton after inoculation with Verticillium dahliae. A total of 57 different protein spots were upregulated, including 52 known proteins representing 11% of the total protein spots. These proteins are involved in resistance to stress and disease, transcriptional regulation, signal transduction, protein processing and degradation, photosynthesis, production capacity, basic metabolism, and other processes. In addition, five disease resistance proteins showed intense upregulation, indicating that resistance genes (R genes) may play a critical role in resistance to Verticillium wilt in Thurber's cotton. Our results suggest that disease and stress resistance are the combined effects of multiple co-expressed genes. This provides a basis for further, detailed investigation into the mechanisms underlying Verticillium wilt resistance of G. thurberi and for cloning essential genes into cotton cultivars to produce Verticillium wilt resistant plants.

  6. dbDEPC 2.0: updated database of differentially expressed proteins in human cancers

    PubMed Central

    He, Ying; Zhang, Menghuan; Ju, Yuanhu; Yu, Zhonghao; Lv, Daqing; Sun, Han; Yuan, Weilan; He, Fei; Zhang, Jianshe; Li, Hong; Li, Jing; Wang-Sattler, Rui; Li, Yixue; Zhang, Guoqing; Xie, Lu

    2012-01-01

    A large amount of differentially expressed proteins (DEPs) have been identified in various cancer proteomics experiments, curation and annotation of these proteins are important in deciphering their roles in oncogenesis and tumor progression, and may further help to discover potential protein biomarkers for clinical applications. In 2009, we published the first database of DEPs in human cancers (dbDEPCs). In this updated version of 2011, dbDEPC 2.0 has more than doubly expanded to over 4000 protein entries, curated from 331 experiments across 20 types of human cancers. This resource allows researchers to search whether their interested proteins have been reported changing in certain cancers, to compare their own proteomic discovery with previous studies, to picture selected protein expression heatmap across multiple cancers and to relate protein expression changes with aberrance in other genetic level. New important developments include addition of experiment design information, advanced filter tools for customer-specified analysis and a network analysis tool. We expect dbDEPC 2.0 to be a much more powerful tool than it was in its first release and can serve as reference to both proteomics and cancer researchers. dbDEPC 2.0 is available at http://lifecenter.sgst.cn/dbdepc/index.do. PMID:22096234

  7. Effects of enamel matrix proteins on multi-lineage differentiation of periodontal ligament cells in vitro.

    PubMed

    Amin, Harsh D; Olsen, Irwin; Knowles, Jonathan C; Dard, Michel; Donos, Nikolaos

    2013-01-01

    The adult periodontal ligament (PDL) is considered to contain progenitor cells that are involved in the healing of periodontal wounds. Treatment with enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs), has been shown to be of some clinical benefit in eliciting periodontal regeneration in vivo. Although there is extensive information available about the effects of EMD on periodontal regeneration, the precise influence of this material on alveolar bone and the formation of blood vessels and proprioceptive sensory nerves, prominent features of functionally active periodontal tissue, remain unclear. The aim of the present study was therefore to examine the effects of EMD on the ability of human periodontal ligament cells (HPCs) to undergo multi-lineage differentiation in vitro. Our results showed that HPCs treated with EMD under non-selective growth conditions did not show any evidence of osteogenic, adipogenic, chondrogenic, neovasculogenic, neurogenic and gliogenic "terminal" differentiation. In contrast, under selective lineage-specific culture conditions, EMD up-regulated osteogenic, chondrogenic and neovasculogenic genes and "terminal" differentiation, but suppressed adipogenesis, neurogenesis and gliogenesis. These findings thus demonstrate for the first time that EMD can differentially modulate the multi-lineage differentiation of HPCs in vitro.

  8. Paired and LIM class homeodomain proteins coordinate differentiation of the C. elegans ALA neuron.

    PubMed

    Van Buskirk, Cheryl; Sternberg, Paul W

    2010-06-01

    The ancient origin of sleep is evidenced by deeply conserved signaling pathways regulating sleep-like behavior, such as signaling through the Epidermal growth factor receptor (EGFR). In Caenorhabditis elegans, a sleep-like state can be induced at any time during development or adulthood through conditional expression of LIN-3/EGF. The behavioral response to EGF is mediated by EGFR activity within a single cell, the ALA neuron, and mutations that impair ALA differentiation are expected to confer EGF-resistance. Here we describe three such EGF-resistant mutants. One of these corresponds to the LIM class homeodomain (HD) protein CEH-14/Lhx3, and the other two correspond to Paired-like HD proteins CEH-10/Chx10 and CEH-17/Phox2. Whereas CEH-14 is required for ALA-specific gene expression throughout development, the Prd-like proteins display complementary temporal contributions to gene expression, with the requirement for CEH-10 decreasing as that of CEH-17 increases. We present evidence that CEH-17 participates in a positive autoregulatory loop with CEH-14 in ALA, and that CEH-10, in addition to its role in ALA differentiation, functions in the generation of the ALA neuron. Similarly to CEH-17, CEH-10 is required for the posterior migration of the ALA axons, but CEH-14 appears to regulate an aspect of ALA axon outgrowth that is distinct from that of the Prd-like proteins. Our findings reveal partial modularity among the features of a neuronal differentiation program and their coordination by Prd and LIM class HD proteins.

  9. Comparative modeling of the three-dimensional structure of type II antifreeze protein.

    PubMed Central

    Sönnichsen, F. D.; Sykes, B. D.; Davies, P. L.

    1995-01-01

    Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7540906

  10. Protein isoprenylation regulates osteogenic differentiation of mesenchymal stem cells: effect of alendronate, and farnesyl and geranylgeranyl transferase inhibitors

    PubMed Central

    Duque, G; Vidal, C; Rivas, D

    2011-01-01

    BACKGROUND AND PURPOSE Protein isoprenylation is an important step in the intracellular signalling pathway conducting cell growth and differentiation. In bone, protein isoprenylation is required for osteoclast differentiation and activation. However, its role in osteoblast differentiation and function remains unknown. In this study, we assessed the role of protein isoprenylation in osteoblastogenesis in a model of mesenchymal stem cells (MSC) differentiation. EXPERIMENTAL APPROACH We tested the effect of an inhibitor of farnesylation [farnesyl transferase inhibitor-277 (FTI-277)] and one of geranylgeranylation [geranylgeranyltransferase inhibitor-298 (GGTI-298)] on osteoblast differentiating MSC. In addition, we tested the effect of alendronate on protein isoprenylation in this model either alone or in combination with other inhibitors of isoprenylation. KEY RESULTS Initially, we found that levels of unfarnesylated proteins (prelamin A and HDJ-2) increased after treatment with FTI-277 concomitantly affecting osteoblastogenesis and increasing nuclear morphological changes without affecting cell survival. Furthermore, inhibition of geranylgeranylation by GGTI-298 alone increased osteoblastogenesis. This effect was enhanced by the combination of GGTI-298 and alendronate in the osteogenic media. CONCLUSIONS AND IMPLICATIONS Our data indicate that both farnesylation and geranylgeranylation play a role in osteoblastogenesis. In addition, a new mechanism of action for alendronate on protein isoprenylation in osteogenic differentiating MSC in vitro was found. In conclusion, protein isoprenylation is an important component of the osteoblast differentiation process that could constitute a new therapeutic target for osteoporosis in the future. PMID:21077849

  11. Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasis.

    PubMed

    Luque-García, Jose Luis; Martínez-Torrecuadrada, Jorge Luis; Epifano, Carolina; Cañamero, Marta; Babel, Ingrid; Casal, J Ignacio

    2010-03-01

    Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS-PAGE and analyzed using nanoflow LC-ESI-LTQ. A total of 291 membrane and membrane-associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5-fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17-beta-hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis.

  12. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  13. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    PubMed

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

  14. Liver-enriched inhibitory protein (LIP) actively inhibits preadipocyte differentiation through histone deacetylase 1 (HDAC1).

    PubMed

    Abdou, Houssein-Salem; Atlas, Ella; Haché, Robert J G

    2011-06-17

    The CCAAT/enhancer-binding protein β (C/EBPβ) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBPβ to stimulate preadipocyte differentiation through activation of C/ebpα transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebpα promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebpα transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebpα promoter, mutations in C/ebpβ that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBPα transcription and preadipocyte differentiation are discussed.

  15. Special AT-rich Binding Protein-2 (SATB2) Differentially Affects Disease-causing p63 Mutant Proteins*

    PubMed Central

    Chung, Jacky; Grant, R. Ian; Kaplan, David R.; Irwin, Meredith S.

    2011-01-01

    p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins. PMID:21965674

  16. Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy; Savas, Christopher J.; Kartje, Zachary; Hoops, Geoffrey C.

    2014-01-01

    Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal…

  17. Sex differences in angiotensin II responses contribute to a differential regulation of cox-mediated vascular dysfunction during aging.

    PubMed

    Costa, Gustavo; Garabito, Manel; Jiménez-Altayó, Francesc; Onetti, Yara; Sabate, Manel; Vila, Elisabet; Dantas, Ana Paula

    2016-12-01

    Aging is a cardiovascular risk factor partially related to activation of the Renin-Angiotensin System (RAS). RAS activation is also influenced by sex. In this regard, our study aims to determine whether sex-associated differences in RAS contribute to a differential regulation of vascular aging and associated dysfunction. Male and female outbreed CD-1 mice were studied at 3 and 12months of age (M). Contribution of RAS was determined by treating mice from 3M to 12M with the AngII type 1 receptor blocker losartan (0.6g/L in the drinking water). At 12M, contractions to AngII were higher in males compared to females (P<0.05). This effect was paralleled by a decrease in AngII type 2 receptors in 12M males. Aging also diminished ACh relaxation in males, but did not modify female responses. Treatment of aortas with indomethacin (10μM) restored the impaired endothelium-dependent relaxation in 12M males, suggesting an increase of cyclooxygenase (COX)-derived vasoconstrictors in aged males. Chronic treatment of mice with losartan also improved endothelium-dependent relaxation. Besides, losartan significantly decreased COX-2 expression and activity in 12M male, with a minor effect in aged females. Aging increases AngII contraction and induces endothelial dysfunction differently in males and females. In aged males, RAS contributed to increased COX-2 expression and activity, which in turn may lead to vascular dysfunction.

  18. Apo-neocarzinostatin: a protein carrier for Cu(II) glycocomplexes and Cu(II) into U937 and HT29 cell lines.

    PubMed

    Garcia, Ludivine; Franzoni, Susanna; Mussi, Francesca; Aumont-Niçaise, Magali; Bertrand, Hélène; Desmadril, Michel; Pelosi, Giorgio; Buschini, Annamaria; Policar, Clotilde

    2014-06-01

    In the field of pharmaceuticals there is an increasing need for new delivery systems to overcome the issues of solubility, penetration, toxicity and drug resistance. One of the possible strategies is to use biocarriers such as proteins to encourage the cell-penetration of drugs. In this paper, the use of the apo-protein neocarzinostatin (apo-NCS) as a carrier-protein for two Cu(II) glycocomplexes, previously characterized, and Cu(II) ions was investigated. Its interaction with the metallic compounds was analyzed using microcalorimetry. The dissociation constants were shown to be in the micromolar range. The Cu(II) glycocomplexes, in absence of apo-NCS, were found to be cytotoxic in the U937 and HT29 cell lines whereas the corresponding glycoligands showed no toxicity. The leukemic cell line (U937) seems to be more sensitive to glycocomplexes than the colon cancer cell line (HT29). Interestingly, apo-NCS was shown to increase systematically the antiproliferative activity by a factor of 2 and 3 for Cu(II) glycocomplexes and Cu(II) respectively. The antiproliferative activity detected was not related to proteasome inhibition. This result stresses the importance of new molecular tools for the delivery of Cu(II) to tumor cells using non-covalent association with carriers proteins.

  19. Class II G Protein-Coupled Receptors and Their Ligands in Neuronal Function and Protection

    PubMed Central

    Martin, Bronwen; de Maturana, Rakel Lopez; Brenneman, Randall; Walent, Tom; Mattson, Mark P.; Maudsley, Stuart

    2008-01-01

    G protein-coupled receptors (GPCRs) play pivotal roles in regulating the function and plasticity of neuronal circuits in the nervous system. Among the myriad of GPCRs expressed in neural cells, class II GPCRs which couples predominantly to the Gs–adenylate cyclase–cAMP signaling pathway, have recently received considerable attention for their involvement in regulating neuronal survival. Neuropeptides that activate class II GPCRs include secretin, glucagon-like peptides (GLP-1 and GLP-2), growth hormone-releasing hormone (GHRH), pituitary adenylate cyclase activating peptide (PACAP), corticotropin-releasing hormone (CRH), vasoactive intestinal peptide (VIP), parathyroid hormone (PTH), and calcitonin-related peptides. Studies of patients and animal and cell culture models, have revealed possible roles for class II GPCRs signaling in the pathogenesis of several prominent neurodegenerative conditions including stroke, Alzheimer's, Parkinson's, and Huntington's diseases. Many of the peptides that activate class II GPCRs promote neuron survival by increasing the resistance of the cells to oxidative, metabolic, and excitotoxic injury. A better understanding of the cellular and molecular mechanisms by which class II GPCRs signaling modulates neuronal survival and plasticity will likely lead to novel therapeutic interventions for neurodegenerative disorders. PMID:16052036

  20. Differential expression of genes and proteins associated with wool follicle cycling.

    PubMed

    Liu, Nan; Li, Hegang; Liu, Kaidong; Yu, Juanjuan; Cheng, Ming; De, Wei; Liu, Jifeng; Shi, Shuyan; He, Yanghua; Zhao, Jinshan

    2014-08-01

    Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.

  1. Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

    PubMed

    Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

    2016-05-17

    Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production.

  2. A Guide to Differential Scanning Calorimetry of Membrane and Soluble Proteins in Detergents.

    PubMed

    Yang, Zhengrong; Brouillette, Christie G

    2016-01-01

    Differential scanning calorimetry (DSC) detects protein thermal unfolding by directly measuring the heat absorbed. Simple DSC experiments that require relatively small amounts of pure material can provide a wealth of information related to structure, especially with respect to domain architecture, without the need for a complete thermodynamic analysis. Thus, DSC is an ideal additional tool for membrane protein characterization and also offers several advantages over indirect thermal unfolding methods. Integral membrane proteins (IMPs) that comprise both large multitopic transmembrane domains (TMDs) and extramembranous domains (EMDs) are differentially affected by detergent interactions with both domains. In fact, in some cases, destabilization of the EMD by detergent may dominate overall IMP stability. This chapter will (1) provide a perspective on the advantages of DSC for membrane protein characterization and stability measurements, including numerous examples spanning decades of research; (2) introduce models for the interaction and destabilization of IMPs by detergents; (3) discuss two case studies from the authors' lab; and (4) offer practical advice for performing DSC in the presence of detergents.

  3. Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

    PubMed Central

    Akanuma, Genki; Nanamiya, Hideaki; Natori, Yousuke; Yano, Koichi; Suzuki, Shota; Omata, Shuya; Ishizuka, Morio; Sekine, Yasuhiko

    2012-01-01

    Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation. PMID:23002217

  4. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  5. Nitric oxide (N0) donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair c...

  6. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

    PubMed Central

    Wieczorek, Marek; Abualrous, Esam T.; Sticht, Jana; Álvaro-Benito, Miguel; Stolzenberg, Sebastian; Noé, Frank; Freund, Christian

    2017-01-01

    Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

  7. Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH.

    PubMed

    Zhang, Ying; Guo, Xiaoyu; Dong, Juan

    2016-11-07

    Cell polarization is commonly used for the regulation of stem cell asymmetric division in both animals and plants. Stomatal development in Arabidopsis, a process that produces breathing pores in the epidermis, requires asymmetric cell division to differentiate highly specialized guard cells while maintaining a stem cell population [1, 2]. The BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) protein exhibits a polarized localization pattern in the cell and is required for differential cell fates resulting from asymmetric cell division [3]. The polarization of BASL is made possible by a positive feedback loop with a canonical mitogen-activated protein kinase (MAPK) pathway that recruits the MAPKK kinase YODA (YDA) and MAPK 6 (MPK6) to the cortical polarity site [4]. Here, we study BASL intracellular dynamics and show that the membrane-associated BASL is slowly replenished at the cortical polarity site and that the mobility is tightly linked to its phosphorylation status. Because BASL polarity is only exhibited by one daughter cell after an asymmetric cell division, we study how BASL differentially functions in the two daughter cells. The YDA MAPK cascade transduces upstream ligand-receptor signaling [5-13] to the transcription factor SPEECHLESS (SPCH), which controls stomatal initiation and is directly suppressed by MPK3/6-mediated phosphorylation [14, 15]. We show that BASL polarization leads to elevated nuclear MPK6 signaling and lowered SPCH abundance in one of the two daughter cells. Therefore, two daughter cells are differentiated by BASL polarity-mediated differential suppression of SPCH, which may provide developmental plasticity in plant stem cell asymmetric cell division (ACD).

  8. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  9. [In vitro encystation of Giardia lamblia: analysis with two-dimensional electrophoresis of differentially expressed proteins].

    PubMed

    Hernández, Paula C; Caldas, María Leonor; Wasserman, Moisés

    2002-09-01

    The reconstruction of Giardia lamblia life cycle in vitro is an excellent tool for the study of the parasite's molecular biology. The present work describes techniques developed that better define parasite differentiation. An encystation protocol is presented along with a method for isolation and purification of the produced cysts. The cyst morphology at the light microscopy level is identical to that of in vivo cysts. A two-dimension protein map obtained by high-resolution electrophoresis indicated that most of the parasite's proteins are acid. Based on this result, the two dimension gel electrophoresis used a pH 4-7 gradient in the first, isoelectric focusing dimension. Differences in protein expression during the stages of encystation were clearly discerned, as well as images of the parasite obtained by light and by transmission electron microscopy that describe the morphological and the ultrastructural changes that occur as the cysts are produced in vitro.

  10. Subtype-specific roles of phospholipase C-β via differential interactions with PDZ domain proteins.

    PubMed

    Kim, Jung Kuk; Lim, Seyoung; Kim, Jinho; Kim, Sanguk; Kim, Jae Ho; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-01-01

    Since we first identified the PLC-β isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-β subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-β subtype exist. Most cells express multiple subtypes of PLC-β in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-β subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-β interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-β subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-β subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-β subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-β subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-β in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-β subtypes. It has been

  11. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.

    NASA Astrophysics Data System (ADS)

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-07-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted `silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers.

  12. Translational diffusion of individual class II MHC membrane proteins in cells.

    PubMed Central

    Vrljic, Marija; Nishimura, Stefanie Y; Brasselet, Sophie; Moerner, W E; McConnell, Harden M

    2002-01-01

    Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells. PMID:12414700

  13. Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate.

    PubMed

    Zhang, Ying-Ying; Tang, Lei-Lei; Zheng, Bei; Ge, Ren-Shan; Zhu, Dan-Yan

    2016-05-01

    Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α-actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L-threonine dehydrogenase, X-ray repair cross-complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system.

  14. Differential surface deposition of complement proteins on logarithmic and stationary phase Leishmania chagasi promastigotes.

    PubMed

    Ramer-Tait, Amanda E; Lei, Soi Meng; Bellaire, Bryan H; Beetham, Jeffrey K

    2012-12-01

    Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHS-exposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of

  15. Cell surface differentiation of Mycoplasma mobile visualized by surface protein localization.

    PubMed

    Kusumoto, Akiko; Seto, Shintaro; Jaffe, Jacob D; Miyata, Makoto

    2004-12-01

    Mycoplasma mobile has a flask-shaped cell morphology and glides toward its tapered end at a rate of 3-7 cell lengths per s (2.0-4.5 microm s(-1)) by an unknown mechanism. Gliding requires that the surface of the cell is in contact with a solid substrate, such as glass or plastic. In order to characterize the nature of the outer surface of M. mobile, monoclonal antibodies were raised against intact cells and screened for their ability to recognize surface proteins. Four antibodies were identified and their protein targets were determined. One antibody recognized the Gli349 protein, which is known to be involved in glass binding and gliding. This antibody was also able to displace attached M. mobile cells from glass, suggesting that Gli349 is the major adhesion protein in M. mobile. The other three antibodies recognized members of the Mvsp family of proteins, which are presumably the major surface antigens of M. mobile. Immunofluorescence studies were performed to localize these proteins on the surface of M. mobile cells. Gli349 localized to the proximal region of the tapered part of the cell (the 'neck'), while the various Mvsp family members showed several distinct patterns of subcellular localization. MvspN and MvspO localized to the distal end of the tapered part of the cell (the 'head'), MvspK localized to the main part of the cell (the 'body'), and MvspI localized to both the head and body but not the neck. This analysis shows that M. mobile surprisingly expresses multiple versions of its major surface antigen at once but differentiates its surface by differential localization of the various paralogues.

  16. Information theory in systems biology. Part II: protein-protein interaction and signaling networks.

    PubMed

    Mousavian, Zaynab; Díaz, José; Masoudi-Nejad, Ali

    2016-03-01

    By the development of information theory in 1948 by Claude Shannon to address the problems in the field of data storage and data communication over (noisy) communication channel, it has been successfully applied in many other research areas such as bioinformatics and systems biology. In this manuscript, we attempt to review some of the existing literatures in systems biology, which are using the information theory measures in their calculations. As we have reviewed most of the existing information-theoretic methods in gene regulatory and metabolic networks in the first part of the review, so in the second part of our study, the application of information theory in other types of biological networks including protein-protein interaction and signaling networks will be surveyed.

  17. Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors.

    PubMed

    Thomsen, Morten S; Mikkelsen, Jens D

    2012-10-01

    Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL-3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine-induced receptor up-regulation, suggesting that nicotine and A-582941 induce up-regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU-120596 inhibits up-regulation of the α7 nAChR induced by 10 mg/kg A-582941, as measured by [(125)I]-bungarotoxin autoradiography, whereas 1 mg/kg AVL-3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A-582941-induced up-regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist-dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.

  18. Applications of differential scanning calorimetry for thermal stability analysis of proteins: qualification of DSC.

    PubMed

    Wen, Jie; Arthur, Kelly; Chemmalil, Letha; Muzammil, Salman; Gabrielson, John; Jiang, Yijia

    2012-03-01

    Differential scanning calorimetry (DSC) has been used to characterize protein thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical industry. Recently, there have been increased requests from regulatory agencies for the qualification of characterization methods including DSC. Understanding the method precision can help determine what differences between samples are significant and also establish the acceptance criteria for comparability and other characterization studies. In this study, we identify the parameters for the qualification of DSC for thermal stability analysis of proteins. We use these parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable for protein thermal stability analysis for these purposes. Several molecules from different structural families were studied. The experiments and data analyses were performed by different analysts using different instruments at different sites. The results show that the (apparent) thermal transition midpoint (T(m)) values obtained for the same protein by same and different instruments and/or analysts are quite reproducible, and the profile similarity values obtained for the same protein from the same instrument are also high. DSC is an appropriate method for assessing protein thermal stability and conformational changes.

  19. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    PubMed

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  20. Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing.

    PubMed

    Lee, Ho Young; Zhou, Kaihong; Smith, Alison Marie; Noland, Cameron L; Doudna, Jennifer A

    2013-07-01

    During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer-TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer-dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.

  1. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

    PubMed Central

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

  2. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARgamma Expression and Activation in Differentiating Mesenchymal Stem Cells.

    PubMed

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARgamma2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARgamma, and SREBP-1 were determined by western blot. Finally, DNA binding PPARgamma activity was determined using an ELISA-based PPARgamma activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARgamma expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARgamma activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARgamma expression and activity.

  3. Angiotensin II induces the expression of c-reactive protein via MAPK-dependent signal pathway in U937 macrophages.

    PubMed

    Li, Ming; Liu, Juntian; Han, Chunjie; Wang, Bin; Pang, Xiaoming; Mao, Junjun

    2011-01-01

    Atherosclerosis is an inflammatory disease in the vessel wall. As an inflammatory molecule, C-reactive protein (CRP) participates in all stages of atherosclerotic process. Although angiotensin II (Ang II) can stimulate the vascular cells to produce CRP, it is unknown whether Ang II induces CRP expression in macrophages. The present study was to observe effect of Ang II on CRP production and the related signal pathway in U937 macrophages so as to provide more evidence for the proinflammatory action of Ang II. The results showed that Ang II significantly increased mRNA and protein expression of CRP in U937 macrophages in time- and concentration-dependent manners. AT(1) receptor blocker losartan blocked Ang II -induced CRP expression in mRNA and protein levels in U937 macrophages. Losartan and complex II inhibitor TIFA decreased Ang II -stimulated reactive oxygen species (ROS) generation, and antioxidant NAC completely abolished Ang II -induced CRP expression in U937 macrophages. The further study indicated that losartan, NAC, MEK1/2 inhibitor PD98059, p38MAPK inhibitor SB203580 obviously inhibited ERK1/2 and p38MAPK phosphorylation, and PD98059, SB203580 and NF-κB inhibitor PDTC reduced Ang II -induced mRNA and protein expression of CRP in U937 macrophages. These demonstrate that Ang II is capable of inducing CRP generation in macrophages via AT(1)-ROS-ERK1/2/p38MAPK-NF-κB signal pathway, which contributes to better understanding of the proinflammatory and proatherosclerotic actions of Ang II.

  4. Cross-reconstitution of the extrinsic proteins and photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea.

    PubMed

    Suzuki, T; Ohta, H; Enami, I

    2005-06-01

    Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.

  5. Rictor regulates phosphorylation of the novel protein kinase C Apl II in Aplysia sensory neurons.

    PubMed

    Labban, Margaret; Dyer, John R; Sossin, Wayne S

    2012-09-01

    Rapamycin-insensitive companion of TOR (Rictor) is a conserved component of target of rapamycin complex 2 (TORC2), a complex implicated in phosphorylation of a number of signal transduction-related kinases, including protein kinase Cs (PKCs) at their 'hydrophobic' site in the carboxy-terminal extension domain. In the marine mollusk, Aplysia californica, an increase in phosphorylation of the novel PKC, Apl II, at the hydrophobic site is associated with a protein synthesis-dependent increase in synaptic strength seen after continuous application of serotonin. To determine if Rictor plays a role in this increase, we cloned the Aplysia ortholog of Rictor (ApRictor). An siRNA-mediated decrease in ApRictor levels in Aplysia sensory neurons led to a decrease in the phosphorylation of PKC Apl II at the hydrophobic site suggesting a role for ApRictor in hydrophobic site phosphorylation. However, over-expression of ApRictor was not sufficient to increase phosphorylation of PKC Apl II. Continuous application of serotonin increased phosphorylation of PKC Apl II at the hydrophobic site in cultured sensory neurons, and this was blocked by Torin, which inhibits both TORC1 and TORC2. Over-expression of ApRictor did not lead to change in the magnitude of serotonin-mediated phosphorylation, but did lead to a small increase in the membrane localization of phosphorylated PKC Apl II. In conclusion, these studies implicate Rictor in phosphorylation of a novel PKC during synaptic plasticity and suggest an additional role for Rictor in regulating the localization of PKCs.

  6. Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

    PubMed Central

    Linares, Gabriel R.; Xing, Weirong; Burghardt, Hans; Baumgartner, Bernhard; Chen, Shin-Tai; Ricart, Wifredo; Fernández-Real, José Manuel; Zorzano, Antonio

    2011-01-01

    Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men. PMID:21467300

  7. Protein serine/threonine Phosphotase-2A is differentially expressed and regulates eye development in vertebrates.

    PubMed

    Liu, W-B; Hu, X-H; Zhang, X-W; Deng, M-X; Nie, L; Hui, S-S; Duan, W; Tao, M; Zhang, C; Liu, J; Hu, W-F; Huang, Z-X; Li, L; Yi, M; Li, T-T; Wang, L; Liu, Y; Liu, S-J; Li, D W-C

    2013-09-01

    Protein serine/threonine phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that protein phosphatase-2A is important for normal eye development in goldfish.

  8. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    PubMed

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses.

  9. The effects of simultaneous RNAi suppression of PsbO and PsbP protein expression in photosystem II of Arabidopsis.

    PubMed

    Yi, Xiaoping; Hargett, Stefan R; Frankel, Laurie K; Bricker, Terry M

    2008-01-01

    Interfering RNA was used to suppress simultaneously the expression of the four genes which encode the PsbO and PsbP proteins of Photosystem II in Arabidopsis (PsbO: At5g66570, At3g50820 and PsbP: At1g06680, At2g30790). A phenotypic series of transgenic plants was obtained that expressed variable amounts of the PsbO proteins and undetectable amounts of the PsbP proteins. Immunological studies indicated that the loss of PsbP expression was correlated with the loss of expression of the PsbQ, D2, and CP47 proteins, while the loss of PsbO expression was correlated with the loss of expression of the D1 and CP43 proteins. Q(A)(-) reoxidation kinetics in the absence of DCMU indicated that the slowing of electron transfer from Q(A)(-) to Q(B) was correlated with the loss of the PsbP protein. Q(A)(-) reoxidation kinetics in the presence of DCMU indicated that charge recombination between Q(A)(-) and donor side components of the photosystem was retarded in all of the mutants. Decreasing amounts of the PsbO protein in the absence of the PsbP component also led to a progressive loss of variable fluorescence yield (F(V)/F(M)). During fluorescence induction, the loss of PsbP was correlated with a more rapid O to J transition and a loss of the J to I transition. These results indicate that the losses of the PsbO and PsbP proteins differentially affect separate protein components and different PS II functions and can do so, apparently, in the same plant.

  10. The stability of differentially rotating self-gravitating gas clouds. II - Polytropic configurations

    NASA Astrophysics Data System (ADS)

    Schmitz, F.; Ebert, R.

    1987-07-01

    The stability of differentially rotating gas clouds with polytropic equations of state is investigated. The equilibrium states are self-consistent. The structure of the infinitely extended configurations is very simple. Perpendicular to the rotation axis, the density distribution and the differential rotational velocity are described by power laws. In the limit of vanishing rotation the authors obtain the familiar polytropic gas spheres. Only marginal axisymmetric perturbations are considered. The structure of the equilibrium states and the form of the perturbations are both calculated with the approximation method presented in paper I.

  11. Differential effect of cholesterol on type I and II feline coronavirus infection.

    PubMed

    Takano, Tomomi; Satomi, Yui; Oyama, Yuu; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal disease of domestic and wild felidae that is caused by feline coronavirus (FCoV). FCoV has been classified into types I and II. Since type I FCoV infection is dominant in the field, it is necessary to develop antiviral agents and vaccines against type I FCoV infection. However, few studies have been conducted on type I FCoV. Here, we compare the effects of cholesterol on types I and II FCoV infections. When cells were treated methyl-β-cyclodextrin (MβCD) and inoculated with type I FCoV, the infection rate decreased significantly, and the addition of exogenous cholesterol to MβCD-treated cells resulted in the recovery of the infectivity of type I FCoV. Furthermore, exogenous cholesterol increased the infectivity of type I FCoV. In contrast, the addition of MβCD and exogenous cholesterol had little effect on the efficiency of type II FCoV infection. These results strongly suggest that the dependence of infection by types I and II FCoV on cholesterol differs.

  12. The CCN proteins: important signaling mediators in stem cell differentiation and tumorigenesis

    PubMed Central

    Zuo, Guo-Wei; Kohls, Christopher D.; He, Bai-Cheng; Chen, Liang; Zhang, Wenli; Shi, Qiong; Zhang, Bing- Qiang; Kang, Quan; Luo, Jinyong; Luo, Xiaoji; Wagner, Eric R.; Kim, Stephanie H.; Restegar, Farbod; Haydon, Rex C.; Deng, Zhong-Liang; Luu, Hue H.; He, Tong-Chuan; Luo, Qing

    2010-01-01

    Summary The CCN proteins contain six members, namely CCN1 to CCN6, which are small secreted cysteine-rich proteins. The CCN proteins are modular proteins, containing up to four functional domains. Many of the CCN members are induced by growth factors, cytokines, or cellular stress. The CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues. The CCN proteins can integrate and modulate the signals of integrins, BMPs, VEGF, Wnts, and Notch. The involvement of integrins in mediating CCN signaling may provide diverse context-dependent responses in distinct cell types. CCN1 and CCN2 play an important role in development, angiogenesis and cell adhesion, whereas CCN3 is critical to skeletal and cardiac development. CCN4, CCN5 and CCN6 usually inhibit cell growth. Mutations of Ccn6 are associated with the progressive pseudorheumatoid dysplasia and spondyloepiphyseal dysplasia tarda. In stem cell differentiation, CCN1, CCN2, and CCN3 play a principal role in osteogenesis, chondrogenesis, and angiogenesis. Elevated expression of CCN1 is associated with more aggressive phenotypes of human cancer, while the roles of CCN2 and CCN3 in tumorigenesis are tumor type-dependent. CCN4, CCN5 and CCN6 function as tumor suppressors. Although CCN proteins may play important roles in fine-tuning other major signaling pathways, the precise function and mechanism of action of these proteins remain undefined. Understanding of the biological functions of the CCN proteins would not only provide insight into their roles in numerous cellular processes but also offer opportunities for developing therapeutics by targeting CCN functions. PMID:20376786

  13. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

    PubMed

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  14. The CCN proteins: important signaling mediators in stem cell differentiation and tumorigenesis.

    PubMed

    Zuo, Guo-Wei; Kohls, Christopher D; He, Bai-Cheng; Chen, Liang; Zhang, Wenli; Shi, Qiong; Zhang, Bing-Qiang; Kang, Quan; Luo, Jinyong; Luo, Xiaoji; Wagner, Eric R; Kim, Stephanie H; Restegar, Farbod; Haydon, Rex C; Deng, Zhong-Liang; Luu, Hue H; He, Tong-Chuan; Luo, Qing

    2010-06-01

    The CCN proteins contain six members, namely CCN1 to CCN6, which are small secreted cysteine-rich proteins. The CCN proteins are modular proteins, containing up to four functional domains. Many of the CCN members are induced by growth factors, cytokines, or cellular stress. The CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues. The CCN proteins can integrate and modulate the signals of integrins, BMPs, VEGF, Wnts, and Notch. The involvement of integrins in mediating CCN signaling may provide diverse context-dependent responses in distinct cell types. CCN1 and CCN2 play an important role in development, angiogenesis and cell adhesion, whereas CCN3 is critical to skeletal and cardiac development. CCN4, CCN5 and CCN6 usually inhibit cell growth. Mutations of Ccn6 are associated with the progressive pseudorheumatoid dysplasia and spondyloepiphyseal dysplasia tarda. In stem cell differentiation, CCN1, CCN2, and CCN3 play a principal role in osteogenesis, chondrogenesis, and angiogenesis. Elevated expression of CCN1 is associated with more aggressive phenotypes of human cancer, while the roles of CCN2 and CCN3 in tumorigenesis are tumor type-dependent. CCN4, CCN5 and CCN6 function as tumor suppressors. Although CCN proteins may play important roles in fine-tuning other major signaling pathways, the precise function and mechanism of action of these proteins remain undefined. Understanding of the biological functions of the CCN proteins would not only provide insight into their roles in numerous cellular processes but also offer opportunities for developing therapeutics by targeting CCN functions.

  15. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development

    PubMed Central

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  16. Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation.

    PubMed Central

    Reith, W; Barras, E; Satola, S; Kobr, M; Reinhart, D; Sanchez, C H; Mach, B

    1989-01-01

    The regulation of major histocompatibility complex class II gene expression is directly involved in the control of normal and abnormal immune responses. In humans, HLA-DR, -DQ, and -DP class II heterodimers are encoded by a family of alpha- and beta-chain genes clustered in the major histocompatibility complex. Their expression is developmentally controlled and normally restricted to certain cell types. This control is mediated by cis-acting sequences in class II promoters and by trans-acting regulatory factors. Several nuclear proteins bind to class II promoter sequences. In a form of hereditary immunodeficiency characterized by a defect in a trans-acting regulatory factor controlling class II gene transcription, we have observed that one of these nuclear factors (RF-X) does not bind to its target sequence (the class II X box). A cDNA encoding RF-X was isolated by screening a phage expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in the regulatory mutants, indicating a defect in the synthesis of a functional form of the RF-X protein. Images PMID:2498880

  17. Thermal bleaching induced changes in photosystem II function not reflected by changes in photosystem II protein content of Stylophora pistillata

    NASA Astrophysics Data System (ADS)

    Jeans, J.; Szabó, M.; Campbell, D. A.; Larkum, A. W. D.; Ralph, P. J.; Hill, R.

    2014-03-01

    Scleractinian corals exist in a symbiosis with marine dinoflagellates of the genus Symbiodinium that is easily disrupted by changes in the external environment. Increasing seawater temperatures cause loss of pigments and expulsion of the symbionts from the host in a process known as coral bleaching; though, the exact mechanism and trigger of this process has yet to be elucidated. We exposed nubbins of the coral Stylophora pistillata to bleaching temperatures over a period of 14 daylight hours. Fifty-nine percent of the symbiont population was expelled over the course of this short-term treatment. Maximum quantum yield ( F V/ F M) of photosystem (PS) II for the in hospite symbiont population did not change significantly over the treatment period, but there was a significant decline in the quantity of PSII core proteins (PsbA and PsbD) at the onset of the experimental increase in temperature. F V/ F M from populations of expelled symbionts dropped sharply over the first 6 h of temperature treatment, and then toward the end of the experiment, it increased to an F V/ F M value similar to that of the in hospite population. This suggests that the symbionts were likely damaged prior to expulsion from the host, and the most damaged symbionts were expelled earlier in the bleaching. The quantity of PSII core proteins, PsbA and PsbD, per cell was significantly higher in the expelled symbionts than in the remaining in hospite population over 6-10 h of temperature treatment. We attribute this to a buildup of inactive PSII reaction centers, likely caused by a breakdown in the PSII repair cycle. Thus, thermal bleaching of the coral S. pistillata induces changes in PSII content that do not follow the pattern that would be expected based on the results of PSII function.

  18. Comparative proteomic study for profiling differentially expressed proteins between Chinese left- and right-sided colon cancers.

    PubMed

    Shen, Hong; Huang, Jinlin; Pei, Haiping; Zeng, Shan; Tao, Yiming; Shen, Liangfang; Zeng, Liang; Zhu, Hong

    2013-01-01

    The aim of the present study is to profile differentially expressed protein markers between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). Fresh tumor tissue samples from LSCC (n = 7) and RSCC (n = 7) groups were analyzed by two-dimensional electrophoresis coupled with MALDI-TOF-MS, followed by Western blotting. In 50 paraffin embedded samples from each group, levels of four differentially expressed proteins (identified by proteomics analysis) were measured by tissue microarray with immunohistochemistry staining to compare the different protein markers between LSCC and RSCC. Sixteen proteins were found to be differentially expressed between LSCC and RSCC. Ten proteins including HSP-60 and PDIA1 were identified to be highly expressed in LSCC (P < 0.01 or P < 0.05), while the expression of six proteins including EEF1D and HSP-27 were higher in RSCC (P < 0.01 or P < 0.05). Virtually all of the indentified proteins were involved in cellular energy metabolism, protein folding/unfolding, and/or oxidative stress. Human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognosis between left- and right-sided colon cancer.

  19. Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein

    SciTech Connect

    Allen, C. Leigh; Gulick, Andrew M.

    2014-06-01

    The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA-003406–ABBFA-003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented. Microorganisms produce a variety of natural products via secondary metabolic biosynthetic pathways. Two of these types of synthetic systems, the nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), use large modular enzymes containing multiple catalytic domains in a single protein. These multidomain enzymes use an integrated carrier protein domain to transport the growing, covalently bound natural product to the neighboring catalytic domains for each step in the synthesis. Interestingly, some PKS and NRPS clusters contain free-standing domains that interact intermolecularly with other proteins. Being expressed outside the architecture of a multi-domain protein, these so-called type II proteins present challenges to understand the precise role they play. Additional structures of individual and multi-domain components of the NRPS enzymes will therefore provide a better understanding of the features that govern the domain interactions in these interesting enzyme systems. The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA-003406–ABBFA-003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented here. Comparison with the closest structural homologs of other carrier proteins identifies the requirements for a conserved glycine residue and additional important sequence and structural requirements within the regions that interact with partner proteins.

  20. Activation of RNA polymerase II by topologically linked DNA-tracking proteins

    PubMed Central

    Ouhammouch, Mohamed; Sayre, Michael H.; Kadonaga, James T.; Geiduschek, E. Peter

    1997-01-01

    Almost all proteins mediating transcriptional activation from promoter-distal sites attach themselves, directly or indirectly, to specific DNA sequence elements. Nevertheless, a single instance of activation by a prokaryotic topologically linked DNA-tracking protein has also been demonstrated. The scope of the latter class of transcriptional activators is broadened in this work. Heterologous fusion proteins linking the transcriptional activation domain of herpes simplex virus VP16 protein to the sliding clamp protein β of the Escherichia coli DNA polymerase III holoenzyme are shown to function as topologically DNA-linked activators of yeast and Drosophila RNA polymerase II. The β:VP16 fusion proteins must be loaded onto DNA by the clamp-loading E. coli γ complex to be transcriptionally active, but they do not occupy fixed sites on the DNA. The DNA-loading sites of these activators have all the properties of enhancers: they can be inverted and their locations relative to the transcriptional start site are freely adjustable. PMID:9192631

  1. Interaction of polyamines with proteins of photosystem II: Cation binding and photosynthetic oxygen evolution

    NASA Astrophysics Data System (ADS)

    Beauchemin, R.; Harnois, J.; Rouillon, R.; Tajmir-Riahi, H. A.; Carpentier, R.

    2007-05-01

    Polyamines are organic cations that function in diverse physiological processes that share as a common thread a close relationship to cell proliferation and growth. Polyamines also affect photosynthetic oxygen evolution and therefore, this study was designed to investigate the interaction of 1,3-diaminopropane, 1,4-diaminobutane (putrescine), and 1,5-diaminopentane (cadaverine) cations with proteins of photosystem II (PSII) using PSII-enriched submembrane fractions with diamine concentrations between 0.01 and 20 mM. Fourier transformed infrared (FTIR) difference spectroscopy with its self-deconvolution and second derivative resolution enhancement, as well as curve-fitting procedures were applied in order to determine the diamine binding mode, the protein conformational changes, and the structural properties of diamine-protein complexes. Spectroscopic evidence showed that diamines interact with proteins (H-bonding) through polypeptide C dbnd O groups with no major perturbations of protein secondary structure. At very low diamine concentration (0.01 mM), no inhibition of oxygen-evolution occurred, while at higher diamine content (5-10 mM), 100% inhibition was observed. Chorophyll fluorescence measurements demonstrated that the inhibition mainly affects the oxygen evolving complex of PSII. Comparisons of the effects of these dipositive organic cations with divalent metal cations on one hand and with polyvalent spermine and spermidine on the other hand, show major alterations of the protein secondary structure as positive charge increases.

  2. Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.

    PubMed

    Seebald, Leah M; DeMott, Christopher M; Ranganathan, Srivathsan; Asare Okai, Papa Nii; Glazunova, Anastasia; Chen, Alan; Shekhtman, Alexander; Royzen, Maksim

    2017-04-03

    Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.

  3. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular {beta}-catenin protein

    SciTech Connect

    Cho, Munju; Park, Seoyoung; Gwak, Jungsug; Kim, Dong-Eun; Yea, Sung Su; Shin, Jae-Gook; Oh, Sangtaek

    2008-02-29

    The Wnt/{beta}-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/{beta}-catenin signaling pathway. BIM increased {beta}-catenin responsive transcription (CRT) and up-regulated intracellular {beta}-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}) and CAATT enhancer-binding protein {alpha} (C/EBP{alpha}) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of {beta}-catenin protein in 3T3-L1 preadipocyte cells.

  4. Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity

    PubMed Central

    Hao, Yuhui; Huang, Jiawei; Liu, Cong; Li, Hong; Liu, Jing; Zeng, Yiping; Yang, Zhangyou; Li, Rong

    2016-01-01

    The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT−/− and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT−/− mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis. PMID:27966587

  5. STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post-Translational Modifications

    PubMed Central

    Spencer, Jean L.; Bhatia, Vivek N.; Whelan, Stephen A.; Costello, Catherine E.

    2014-01-01

    The identification of protein post-translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post-Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi-sample comparison by collating, scoring, and ranking PTMs and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting. PMID:25422678

  6. Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis.

    PubMed

    Hayashi, Makoto; Yagi, Mina; Nito, Kazumasa; Kamada, Tomoe; Nishimura, Mikio

    2005-04-15

    Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.

  7. Muscle Satellite Cell Protein Teneurin-4 Regulates Differentiation During Muscle Regeneration.

    PubMed

    Ishii, Kana; Suzuki, Nobuharu; Mabuchi, Yo; Ito, Naoki; Kikura, Naomi; Fukada, So-Ichiro; Okano, Hideyuki; Takeda, Shin'ichi; Akazawa, Chihiro

    2015-10-01

    Satellite cells are maintained in an undifferentiated quiescent state, but during muscle regeneration they acquire an activated stage, and initiate to proliferate and differentiate as myoblasts. The transmembrane protein teneurin-4 (Ten-4) is specifically expressed in the quiescent satellite cells; however, its cellular and molecular functions remain unknown. We therefore aimed to elucidate the function of Ten-4 in muscle satellite cells. In the tibialis anterior (TA) muscle of Ten-4-deficient mice, the number and the size of myofibers, as well as the population of satellite cells, were reduced with/without induction of muscle regeneration. Furthermore, we found an accelerated activation of satellite cells in the regenerated Ten-4-deficient TA muscle. The cell culture analysis using primary satellite cells showed that Ten-4 suppressed the progression of myogenic differentiation. Together, our findings revealed that Ten-4 functions as a crucial player in maintaining the quiescence of muscle satellite cells.

  8. Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation

    PubMed Central

    Fritz-Laylin, Lillian K.; Cande, W. Zacheus

    2010-01-01

    Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism, signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55 genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late). The early set includes genes encoding nearly every known conserved centriole component, as well as eight previously uncharacterized, highly conserved genes. The human orthologs of at least five genes localize to the centrosomes of human cells, one of which (here named Friggin) localizes specifically to mother centrioles. PMID:21045110

  9. The effect of hydration on protein flexibility in photosystem II of green plants studied by quasielastic neutron scattering.

    PubMed

    Pieper, J; Hauss, T; Buchsteiner, A; Renger, G

    2008-06-01

    The effect of hydration on protein dynamics in photosystem II (PS II) membrane fragments from spinach has been investigated by using the method of quasielastic neutron scattering (QENS) at room temperature. The QENS data obtained indicate that the protein dynamics is strongly dependent on the extent of hydration. In particular, the hydration-induced activation of localized diffusive protein motions and QA- reoxidation by QB in PS II appear to be correlated in their onset at a hydration value of about 45% relative humidity (r.h.). These findings underline the crucial functional relevance of localized diffusive protein motions on the picosecond-timescale for the reactions of light-induced photosynthetic water splitting under formation of plastoquinol and molecular oxygen in PS II of green plants.

  10. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  11. cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolor A3(2)

    PubMed Central

    2009-01-01

    Background Streptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying differentiation and antibiotic production. To date, many genes have been identified to be required for its differentiation (e.g. bld genes for aerial growth and whi genes for sporulation) and antibiotics production (including actII-orf4, redD, cdaR as pathway-specific regulatory genes and afsR, absA1/A2 as pleiotropic regulatory genes). Results A gene cluster containing six genes (SCO4126-4131) was proved to be co-transcribed in S. coelicolor. Deletions of cmdABCDEF (SCO4126-4131) displayed defective sporulation including formation of aberrant branches, and abnormalities in chromosome segregation and spore septation. Disruption mutants of apparently orthologous genes of S. lividans and S. avermitilis also showed defective sporulation, implying that the role of these genes is similar among Streptomyces. Transcription of cmdB, and therefore presumably of the whole operon, was regulated developmentally. Five of the encoded proteins (CmdA, C, D, E, F) were predicted membrane proteins. The other, CmdB, a predicted ATP/GTP-binding protein with an ABC-transporter-ATPase domain shown here to be essential for its function, was also located on the cell membrane. These results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation at an early stage of aerial hyphae formation, and suggest that these proteins may form a complex on cell membrane for proper segregation of chromosomes. In addition, deletions of cmdABCDEF also revealed over-production of blue-pigmented actinorhodin (Act) via activation of transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis. Conclusion In this study, six co-transcribed genes cmdABCDEF were identified by their effects on differentiation and antibiotic production in Streptomyces coelicolor A3(2). These six membrane-located proteins are possibly assembled into a complex to

  12. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5′ splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5′ exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  13. Functional variation of the antigen I/II surface protein in Streptococcus mutans and Streptococcus intermedius.

    PubMed

    Petersen, F C; Assev, S; van der Mei, H C; Busscher, H J; Scheie, A A

    2002-01-01

    Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

  14. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  15. Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC).

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Sykes, Donald E; Schwartz, Stanley A; Nair, Madhavan P N

    2007-04-01

    In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

  16. Differential localization of Mox-1 and Mox-2 proteins indicates distinct roles during development.

    PubMed

    Candia, A F; Wright, C V

    1996-12-01

    Transcript localizations for Mox genes have implicated this homeobox gene subfamily in the early steps of mesoderm formation. We have extended these studies by determining the protein expression profile of Mox-1 and Mox-2 during mouse development. The time of onset of Mox protein expression has been accurately obtained to provide clues as to their roles during gastrulation. Expression of Mox-1 protein is first detected in the newly formed mesoderm of primitive streak stage mouse embryos (7.5 days post-coitum, d.p.c.). In contrast, Mox-2 protein is first detected at 9.0 d.p.c. in thr already formed somites. Additionally, immunostaining reveals new and distinct areas of Mox expression in the branchial arches and limbs that were not reported in our previous mRNA localization analysis. Mouse Mox-2 antibodies cross-react specifically in similar embryonic tissues in chick indicating the conservation of function of Mox genes in vertebrates. These expression data suggest that the Mox genes function transiently in the formation of mesodermal and mesenchymal derivatives, after their initial specification, but before their overt differentiation. Furthermore, while there appears to be some overlap in protein expression between Mox-1 and Mox-2 during somitogenesis, unique areas of expression indicate several distinct roles for the Mox genes during development.

  17. STIM proteins: integrators of signalling pathways in development, differentiation and disease

    PubMed Central

    Johnstone, Lorna S; Graham, Sarah J L; Dziadek, Marie A

    2010-01-01

    Abstract The stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum Ca2+ sensors, serving to detect changes in receptor-mediated ER Ca2+ store depletion and to relay this information to plasma membrane localized proteins, including the store-operated Ca2+ channels of the ORAI family. The resulting Ca2+ influx sustains the high cytosolic Ca2+ levels required for activation of many intracellular signal transducers such as the NFAT family of transcription factors. Models of STIM protein deficiency in mice, Drosophila melanogaster and Caenorhabditis elegans, in addition to the phenotype of patients bearing mutations in STIM1 have provided great insight into the role of these proteins in cell physiology and pathology. It is now becoming clear that STIM1 and STIM2 are critical for the development and functioning of many cell types, including lymphocytes, skeletal and smooth muscle myoblasts, adipocytes and neurons, and can interact with a variety of signalling proteins and pathways in a cell- and tissue-type specific manner. This review focuses on the role of STIM proteins in development, differentiation and disease, in particular highlighting the functional differences between STIM1 and STIM2. PMID:20561111

  18. Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

    PubMed

    Sözgen, Kevser; Cekic, Sema Demirci; Tütem, Esma; Apak, Resat

    2006-02-28

    Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat

  19. Genetic differentiation and phylogeny relationships of functional ApoVLDL-II gene in red jungle fowl and domestic chicken populations.

    PubMed

    Musa, Hassan H; Cheng, Jin H; Bao, Wen B; Li, Bi C; Mekki, Dafaalla M; Chen, Guo H

    2007-08-01

    A total of 243 individuals from Red Jungle Fowl (Gallus gallus spadiceus), Rugao, Anka, Wenchang and Silikes chicken populations were used for polymorphism analysis in functional apoVLDL-II gene by Restriction fragment length polymorphism and single strand conformation polymorphism markers. The results show that Anka population has highest gene diversity and Shannon information index, while Red jungle fowl shows highest effective number of allele. In addition, the higher coefficient of genetic differentiation (Gst) across all loci in apoVLDL-II was indicating that high variation is proportioned among populations. As expected total gene diversity (Ht) has upper estimate compared with within population genetic diversity (Hs) across all loci. The mean Gst value across all loci was (0.194) indicating about 19.4% of total genetic variation could be explained by breeds differences, while the remaining 80.6% was accounted for differences among individuals. The average apoVLDL-II gene flow across all loci in five chicken populations was 1.189. The estimates of genetic identity and distance confirm that these genes are significantly different between genetically fat and lean population, because fat type breed Anka shows highest distance with the other Silikes and Rugao whish are genetically lean. In addition, Wenchang and Red jungle fowl were found more closely and genetically related than the other breeds with 49.4% bootstrapping percentages, then they were related to Silikes by 100% bootstrapping percentages followed by Rugao and finally all of them are related with exotic fat breed Anka.

  20. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

    PubMed

    James, P L; Stewart, C E; Rotwein, P

    1996-05-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

  1. Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress.

    PubMed

    Ebrahimian, Talin; Li, Melissa Wei; Lemarié, Catherine A; Simeone, Stefania M C; Pagano, Patrick J; Gaestel, Matthias; Paradis, Pierre; Wassmann, Sven; Schiffrin, Ernesto L

    2011-02-01

    Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation

  2. LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions.

    PubMed

    Jepson, Scott; Vought, Bryan; Gross, Christian H; Gan, Lu; Austen, Douglas; Frantz, J Daniel; Zwahlen, Jacque; Lowe, Derek; Markland, William; Krauss, Raul

    2012-06-22

    Overcoming remyelination failure is a major goal of new therapies for demyelinating diseases like multiple sclerosis. LINGO-1, a key negative regulator of myelination, is a transmembrane signaling protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 is an integral component of the Nogo receptor complex, which inhibits axonal growth via RhoA. Because the only ligand-binding subunit of this complex, the Nogo receptor, is absent in oligodendrocytes, the extracellular signals that inhibit myelination through a LINGO-1-mediated mechanism are unknown. Here we show that LINGO-1 inhibits oligodendrocyte terminal differentiation through intercellular interactions and is capable of a self-association in trans. Consistent with previous reports, overexpression of full-length LINGO-1 inhibited differentiation of oligodendrocyte precursor cells (OPCs). Unexpectedly, treatment with a soluble recombinant LINGO-1 ectodomain also had an inhibitory effect on OPCs and decreased myelinated axonal segments in cocultures with neurons from dorsal root ganglia. We demonstrated LINGO-1-mediated inhibition of OPCs through intercellular signaling by using a surface-bound LINGO-1 construct expressed ectopically in astrocytes. Further investigation showed that the soluble LINGO-1 ectodomain can interact with itself in trans by binding to CHO cells expressing full-length LINGO-1. Finally, we observed that soluble LINGO-1 could activate RhoA in OPCs. We propose that LINGO-1 acts as both a ligand and a receptor and that the mechanism by which it negatively regulates OPC differentiation and myelination is mediated by a homophilic intercellular interaction. Disruption of this protein-protein interaction could lead to a decrease of LINGO-1 inhibition and an increase in myelination.

  3. MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.

    PubMed

    Buschow, Sonja I; van Balkom, Bas W M; Aalberts, Marian; Heck, Albert J R; Wauben, Marca; Stoorvogel, Willem

    2010-01-01

    Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II.

  4. The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis.

    PubMed

    Schneider, Anja; Steinberger, Iris; Strissel, Henning; Kunz, Hans-Henning; Manavski, Nikolay; Meurer, Jörg; Burkhard, Gabi; Jarzombski, Sabine; Schünemann, Danja; Geimer, Stefan; Flügge, Ulf-Ingo; Leister, Dario

    2014-04-01

    Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C-terminally to GFP in the terc-1 background, and the resulting terc-1TerC- GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc-1TerC- GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.

  5. Isolation and characterization of lung resident mesenchymal stem cells capable of differentiating into alveolar epithelial type II cells.

    PubMed

    Gong, Xuemin; Sun, Zhaorui; Cui, Di; Xu, Xiaomeng; Zhu, Huiming; Wang, Lihui; Qian, Weiping; Han, Xiaodong

    2014-04-01

    Controversies and risks continue to be reported about exogenous mesenchymal stem cell-based therapies. In contrast with employing exogenous stem cells, making use of lung resident mesenchymal stem cells (LR-MSCs) could be advantageous. Our study sought to isolate the LR-MSCs and explore their potential to differentiate into alveolar epithelial type II cells (ATII cells). Total lung cells were first precultured, from which the Sca-1(+) CD45(-) CD31(-) population was purified using fluorescence activated cell sorting (FACS). By these methods, it would seem that the Sca-1(+) CD45(-) CD31(-) cells were LR-MSCs. Similar to bone marrow derived mesenchymal stem cells (BM-MSCs), these cells express Sca-1, CD29, CD90, CD44 and CD106, but not CD31 or CD45. They share the same gene expression file with the BM-MSCs and have a similar DNA content during long-term culturing. Furthermore, they could be serially passaged with all these properties being sustained. Above all, LR-MSCs could differentiate into ATII cells when co-cultured with ATII cells in a trans-well system. These findings demonstrated that the Sca-1(+) CD45(-) CD31(-) cells appear to be LR-MSCs that can differentiate into ATII cells. This approach may hold promise for their use in the treatment of lung disease.

  6. p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells.

    PubMed

    Nakajima, Masahiro; Negishi, Yoichi; Tanaka, Hiroyasu; Kawashima, Kohtaro

    2004-08-06

    The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory ef