Real-time monitoring of cellular dynamics in living organisms is highly challenging. We developed a multi-imaging vector based on 2A peptides. Live imaging of subcellular compartments can be performed following the transfection of cells with another vector, the multi-labeling vector, which contains localization signals and various fluorescent protein variants.
Protein kinase C (PKC) induces mitochondrial dysfunction, which is an important pathological factor in cardiovascular diseases. The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) on PKC-induced mitochondrial dysfunction has not been variously investigated. In this study, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced mitochondrial hyperpolarization and reactive oxygen species generation and also increased mitochondrial translocation of APE1/Ref-1. APE1/Ref-1 overexpression suppressed PMA-induced mitochondrial dysfunction. In contrast, gene silencing of APE1/Ref-1 increased the sensitivity of mitochondrial dysfunction. Moreover, mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 more effectively suppressed PMA-induced mitochondrial dysfunctions. These results suggest that mitochondrial APE1/Ref-1 is contributed to the protective role to protein kinase C-induced mitochondrial dysfunction in endothelial cells.
The peripheral terminals of primary nociceptive neurons play an essential role in pain detection mediated by membrane receptors like TRPV1, a molecular sensor of heat and capsaicin. However, the contribution of central terminal TRPV1 in the dorsal horn to chronic pain has not been investigated directly. Combining primary sensory neuron-specific GCaMP3 imaging with a trigeminal neuropathic pain model, we detected robust neuronal hyperactivity in injured and uninjured nerves in the skin, soma in trigeminal ganglion, and central terminals in the spinal trigeminal nucleus. Extensive TRPV1 hyperactivity was observed in central terminals innervating all dorsal horn laminae. The central terminal TRPV1 sensitization was maintained by descending serotonergic (5-HT) input from the brainstem. Central blockade of TRPV1 or 5-HT/5-HT3A receptors attenuated central terminal sensitization, excitatory primary afferent inputs, and mechanical hyperalgesia in the territories of injured and uninjured nerves. Our results reveal central mechanisms facilitating central terminal sensitization underlying chronic pain.
A simple, rapid, and efficient live cell surface labeling method has been developed that uses a direct conjugation between Sortase A expressed transiently at the cell surface and Sortase A specific binding peptide.
Human dental mesenchymal stem cells (hDMSCs) have been isolated from extracted human teeth and proven to have different proliferation and differentiation abilities among the subtypes. Despite increasing interest in the clinical use of hDMSCs, a well-defined specific marker has been absent for these stem cells. In this study, a comparative analysis with known mesenchymal stem cell surface markers such as STRO-1, CD90, CD146, CD34 and TfR (CD71) was performed.
Pirt is a membrane protein that is specifically expressed in the peripheral nervous system, where it has been shown to increase the sensitivity of the transient receptor potential vanilloid 1 channel and modulate its role in heat pain. The broad expression of Pirt among dorsal root ganglion neurons suggests it may modulate other transient receptor potentials, such as the menthol and cooling sensor TRPM8. The discrepancies in the channel properties of TRPM8 in native neurons versus heterologous cells indicate the existence of endogenous modulators of the channel. Here we show that Pirt regulates the function of TRPM8 and its role in detecting cold. Pirt(-/-) mice exhibit decreased behavioural responses to cold and cool temperatures, and Pirt increases the sensitivity of TRPM8 to menthol and cool temperature. Our data suggest Pirt is an endogenous regulator of TRPM8.
Celastrol (CEL) is known as a potent inducer of heat shock protein (HSP) in non-muscle cells and exhibits cytoprotective function and inhibitory effects on proteasome and glucocorticoid receptor activities. To investigate an anti-atrophic effect of CEL on skeletal muscle cells, C2C12 myotubes were treated with 150 ?M dexamethasone (DEX) for 24h and 1.5 ?M CEL was added for the last 6h during the 24h DEX treatment. Compared to the control, the myotube diameter was reduced by a factor of 0.30 by DEX, but CEL treatment almost abrogated the DEX-induced atrophy. CEL treatment also increased expression of HSP72 and phosphorylation of heat shock transcription factor 1 (p-HSF1) 11-fold and 3.4-fold, respectively, as well as accumulation of p-HSF1 in the nucleus. Furthermore, CEL treatment elevated activities of Akt1, p70/S6K and ERK1/2 2.0- to 4.4-fold whereas DEX had no effect on these signaling activities. Inhibition of Akt1 and ERK1/2 pathways by specific inhibitors confirmed CEL-induced anti-atrophic effect. Moreover, DEX-mediated downregulation of FoxO3 phosphorylation and upregulation of MuRF1 expression and proteasome activity were abrogated by CEL treatment. These results demonstrate a novel anti-atrophic function of CEL in muscle cells via both activation of protein anabolic signals and suppression of catabolic signaling activities.
Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.
Despite significant medical benefits as in space exploration or emergency care, prolonged torpidity of non-hibernator mammals remains unexplored to date. Here, we report that male Institute of Cancer Research mice could sustain two separate 2-day torpor bouts and maintain body temperature of 28-33°C following repeated treatments of 3-iodothyronamine (T(1) AM), a natural derivative of thyroid hormone. A 1-day interbout arousal period, adopted to mimic the behavior of true hibernators, seemed critical for the subjects to restore physiological homeostasis. Molecular studies of neuron-specific enolase, S100 calcium binding protein B and heat shock protein 72 suggested that the brain maintains functional and cytoprotective activities during sustained torpidity. Together, the results of this study propose a practical protocol using a torpor-arousal cycle that can be applied to the extreme medical situations.
Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.
O-linked beta-N-acetylglucosamine (O-GlcNAc) plays an important role in the development of insulin resistance and glucose toxicity. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT), which attaches O-GlcNAc to serine and/or threonine residues of proteins and by O-GlcNAcase, which removes O-GlcNAc. We investigated the expression of these two enzymes in erythrocytes of human subjects with diabetes or pre-diabetes.
We assessed the abilities of wild p53 and mutant p53 proteins to interact with the consensus DNA-binding sequence using a MOSFET biosensor. This is the first report in which mutant p53 has been detected on the basis of DNA-protein interaction using a FET-type biosensor. In an effort to evaluate the performance of this protocol, we constructed the core domain of wild p53 and mutant p53 (R248W), which is DNA-binding-defective. After the immobilization of the cognate DNA to the sensing layer, wild p53 and mutant p53 were applied to the DNA-coated gate surface, and subsequently analyzed using a semiconductor analyzer. As a consequence, a significant up-shift in drain current was noted in response to wild p53, but not mutant p53, thereby indicating that sequence-specific DNA-protein interactions could be successfully monitored using a field-effect-based biosensor. These data also corresponded to the results obtained using surface plasmon resonance (SPR) measurements. Taken together, our results show that a FET-type biosensor might be promising for the monitoring of mutant p53 on the basis of its DNA-binding activity, providing us with very valuable insights into the monitoring for diseases, particularly those associated with DNA-protein binding events.
Promyelocytic leukemia zinc finger is a negative regulator of cell cycle progression. In this study, we showed that PLZF inhibits endothelial cell angiogenesis using a human umbilical vein endothelial cell system. We also focused on characterizing the specific function of the BTB domain of PLZF as a novel apoptotic and anti-angiogenic protein via deletion mapping analysis. The BTB domain directly inhibited tube formation, as well as the biological functions of angiostatic activity in vivo, and reduced the expression of p-Akt and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. These results strongly suggest that the BTB domain could potentially modulate the apoptotic and anti-angiogenic effects of PLZF.
The apoptotic caspases have been classified in accordance with their substrate specificities, as the optimal tetrapeptide recognition motifs for a variety of caspases have been determined via positional scanning substrate combinatorial library technology. Here, we focused on two proteolytic recognition motifs, DEVD and IETD, owing to their extensive use in cell death assay. Although DEVE and IETD have been generally considered to be selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, we attempted to monitor the cleavage preference for caspase-3, particularly using the recombinant protein substrates. For this aim, the chimeric GST:DEVD:EGFP and GST:IETD:EGFP proteins were genetically constructed by linking GST and EGFP with the linkers harboring DEVD and IETD. To our best knowledge, this work constitutes the first application for the monitoring of cleavage preference employing the recombinant protein substrates that simultaneously allow for mass and fluorescence analyses. Consequently, GST: IETD:EGFP was cleaved partially in response to caspase-3, whereas GST:DEVD:EGFP was completely proteolyzed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Collectively, using these chimeric protein substrates, we have successfully evaluated the feasibility of the recombinant protein substrate for applicability to the monitoring of cleavage preference for caspase-3.
The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)= approximately 0.11, Distance (D)= approximately 6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.
Since its discovery in the early 1980s, O-linked-beta-N-acetylglucosamine (O-GlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc, and beta-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of O-GlcNAcs functions has grown rapidly.
O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals.
During carcinogenesis, NF-gammaB mediates processes associated with deregulation of the normal control of proliferation, angiogenesis, and metastasis. Thus, suppression of NF-gammaB has been linked with chemoprevention of cancer. Accumulating findings reveal that heat shock protein 90 (HSP90) is a molecular chaperone and a component of the IgammaB kinase (IKK) complex that plays a central role in NF-gammaB activation. HSP90 also stabilizes key proteins involved in cell cycle control and apoptosis signaling. We have determined whether the exogenous administration of isoflavone-deprived soy peptide prevents 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced rat mammary tumorigenesis and investigated the mechanism of action. Dietary administration of soy peptide (3.3 g/rat/day) significantly reduced the incidence of ductal carcinomas (50%), the number of tumors per multiple tumor-bearing rats (49%; P<0.05), and extended the latency period of tumor development (8.07+/-0.92 weeks) compared to control diet animals (10.80+/-1.30; P<0.05). Our results have further demonstrated that soy peptide (1) dramatically inhibits the expression of HSP90, thereby suppressing signaling pathway leading to NF-gammaB activation; (2) induces expression of p21, p53, and caspase-3 proteins; and (3) inhibits expression of VEGF. In agreement with our in vivo data, soy peptide treatment inhibited the growth of human breast MCF-7 tumor cells in a dose-dependent manner and induced apoptosis. Taken together, our in vivo and in vitro results suggest chemopreventive and tumor suppressive functions of isoflavone-deprived soy peptide by inducing growth arrest and apoptosis.
Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.
Maspin is expressed aberrantly in pancreatic ductal adenocarcinoma (PDAC), and its function is still unknown. To explore the role of maspin in PDAC, we constructed wild-type maspin-expressing Panc-1 clones (Panc-1-maspin) by transfecting maspin cDNA into Panc-1, a PDAC-derived cell line that lacks maspin expression. As a control, mock-transfected clones (Panc-1-mock) were constructed using the same method. The invasive ability of the stable transfectants was evaluated with an in vitro invasion assay. The ability of Panc-1-maspin cells to migrate through a Matrigel-coated filter was significantly reduced compared to that of Panc-1-mock cells (p=0.012). In addition, we identified the c.1022A>G variant of maspin in human PDAC cells; however, this polymorphism was not involved in the clinical characteristics of PDAC nor did it alter the invasive ability of PDAC cells. The results of the present study indicate that maspin suppresses the invasive ability of PDAC cells.
The Wnt/beta-catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt-induced stabilization of the beta-catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real-time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated beta-catenin-responsive luciferase activity in a dose-dependent manner and decreased the beta-catenin levels. By utilizing a yeast two-hybrid screen, betaTrCP, a negative regulator of beta-catenin was identified as a novel Dkk3-interacting partner. Coexpression with betaTrCP synergistically enhanced the inhibitory function of Dkk3 on beta-catenin. The stable expression of Dkk3 blocks the nuclear translocation of beta-catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of beta-catenin and its downregulation contribute to an activation of the beta-catenin signaling pathway.
The real-time monitoring of the caspase cascade in cancer cells during apoptosis was performed using various caspase substrate-linked fluorescent proteins-conjugated gold nanoparticles as a new imaging probe.
We previously reported that among a series of artemisinin-derived monomers and dimers, dimer diphenyl phosphate (838) was the most potent inhibitor of human cytomegalovirus (CMV) replication. Our continued investigation of a prototypic artemisinin monomer (artesunate [AS]) and dimer (838) now reveals that both compounds have specific activity against CMV but do not inhibit lytic replication of human herpesvirus 1 or 2 or Epstein-Barr virus. AS and 838 inhibited CMV replication during the first 24 h of the virus replication cycle, earlier than the time of ganciclovir (GCV) activities and prior to DNA synthesis. Neither compound inhibited virus entry. Quantification of DNA replication and virus yield revealed a similar level of inhibition by GCV, but AS and 838 had a 10-fold-higher inhibition of virus yield than of DNA replication, suggesting that artemisinins could inhibit CMV through multiple steps: a predominant early inhibition and possibly an additional step following DNA replication. During the strong early CMV inhibition, the transcription of immediate-early genes was not significantly downregulated, and viral protein expression was reduced only after 48 h. AS and GCV were reversible CMV inhibitors, but the inhibition of CMV replication by 838 was irreversible. Combinations of GCV and 838 as well as GCV and AS were highly synergistic. Finally, treatment with 838, but not AS, prior to CMV infection demonstrated strong anti-CMV activity. These findings illustrate the unique activities of dimer 838, including early and irreversible CMV inhibition, possibly by tight binding to its target.
Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Ig? locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Ig? intron enhancer. However, the ?E3 enhancer must also be regulated as this enhancer plays a role in Ig? rearrangement. We show in this study that STAT5 can repress ?E3 enhancer activity. We find that STAT5 binds to a site that overlaps the ?E3 PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the ?E3 enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the ?E3 enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates ?E3 enhancer function.
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