32 results on '"Tang, D."'
Search Results
2. Identification of the alpha IIb beta 3 integrin in murine tumor cells.
- Author
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Chen, Y.Q., primary, Gao, X, additional, Timar, J, additional, Tang, D, additional, Grossi, I.M., additional, Chelladurai, M, additional, Kunicki, T.J., additional, Fligiel, S.E., additional, Taylor, J.D., additional, and Honn, K.V., additional
- Published
- 1992
- Full Text
- View/download PDF
3. Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.
- Author
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Nagalla, S.R., primary, Gibson, B.W., additional, Tang, D, additional, Reeve, J R, additional, and Spindel, E.R., additional
- Published
- 1992
- Full Text
- View/download PDF
4. Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis.
- Author
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Tang, D, Lahti, J M, and Kidd, V J
- Abstract
There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase-8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame.
- Published
- 2000
5. Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism.
- Author
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Tang, D, Lahti, J M, Grenet, J, and Kidd, V J
- Abstract
Cycloheximide (CHX) can contribute to apoptotic processes, either in conjunction with another agent (e.g. tumor necrosis factor-alpha) or on its own. However, the basis of this CHX-induced apoptosis has not been clearly established. In this study, the molecular mechanisms of CHX-induced cell death were examined in two different human T-cell lines. In T-cells undergoing CHX-induced apoptosis (Jurkat), but not in T-cells resistant to the effects of CHX (CEM C7), caspase-8 and caspase-3 were activated. However, the Fas ligand was not expressed in Jurkat cells either before or after treatment with CHX, suggesting that the activation of these caspases does not involve the Fas receptor. To determine whether CHX-induced apoptosis was mediated by a Fas-associated death domain (FADD)-dependent mechanism, a FADD-DN protein was expressed in cells prior to CHX treatment. Its expression effectively inhibited CHX-induced cell death, suggesting that CHX-mediated apoptosis primarily involves a FADD-dependent mechanism. Since CHX treatment did not result in the induction of Fas or FasL, and neutralizing anti-Fas and anti-tumor necrosis factor receptor-1 antibodies did not block CHX-mediated apoptosis, these results may also indicate that FADD functions in a receptor-independent manner. Surprisingly, death effector filaments containing FADD and caspase-8 were observed during CHX treatment of Jurkat, Jurkat-FADD-DN, and CEM C7 cells, suggesting that their formation may be necessary, but not sufficient, for cell death.
- Published
- 1999
6. Identification and structure characterization of a Cdk inhibitory peptide derived from neuronal-specific Cdk5 activator.
- Author
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Chin, K T, Ohki, S Y, Tang, D, Cheng, H C, Wang, J H, and Zhang, M
- Abstract
The activation of cyclin-dependent kinase 5 (Cdk5) depends on the binding of its neuronal specific activator Nck5a. The minimal activation domain of Nck5a is located in the region of amino acid residues 150 to 291 (Tang, D., Chun, A. C. S., Zhang, M., and Wang, J. H. (1997) J. Biol. Chem. 272, 12318-12327). In this work we show that a 29-residue peptide, denoted as the alphaN peptide, encompassing amino acid residues Gln145 to Asp173 of Nck5a is capable of binding Cdk5 to result in kinase inhibition. This peptide also inhibits an active phospho-Cdk2-cyclin A complex, with a similar potency. Direct competition experiments have shown that this inhibitory peptide does not compete with Nck5a or cyclin A for Cdk5 or Cdk2, respectively. Steady state kinetic analysis has indicated that the alphaN peptide acts as a non-competitive inhibitor of Cdk5. Nck5a complex with respect to the peptide substrate. To understand the molecular basis of kinase inhibition by the peptide, we determined the structure of the peptide in solution by circular dichroism and two-dimensional 1H NMR spectroscopy. The peptide adopts an amphipathic alpha-helical structure from residues Ser149 to Arg162 which can be further stabilized by the helix-stabilizing solvent trifluoroethanol. The hydrophobic face of the helix is likely to be the kinase binding surface.
- Published
- 1999
7. Cleavage of DFF-45/ICAD by multiple caspases is essential for its function during apoptosis.
- Author
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Tang, D and Kidd, V J
- Abstract
Apoptosis involves the proteolysis of specific cellular proteins by a group of cysteine proteases known as caspases. Many of these cellular targets are either functionally inactivated (e.g. poly(ADP-ribose) polymerase) or activated (e.g. other caspases, gelsolin) by such processing, thereby facilitating the cell death process. Caspase 3 is involved in the processing of many of these proteins. Recently, however, it was reported that caspase 3 is dispensable for the cleavage of a large number of cellular caspase substrates during apoptosis. Among these substrates is DFF-45/ICAD, a subunit of the heterodimeric DNA fragmentation factor (DFF), otherwise known as caspase-activated DNase (CAD), that mediates genomic DNA degradation during apoptosis. Conversely, others have reported that caspase 3 is essential for the cleavage and activation of DFF-45/ICAD. To resolve this controversy we examined DFF-45/ICAD processing during apoptosis in MCF-7 breast carcinoma cells that lack functional caspase 3 and in MCF-7 cells expressing caspase 3. We found that DFF-45/ICAD is cleaved by two distinct caspases, one of which is caspase 3. Furthermore, cleavage of the carboxyl-terminal region of DFF-45/ICAD, which is necessary for activation of the enzyme, requires functional caspase 3. In the absence of caspase 3 cleavage of the amino-terminal region of DFF-45/ICAD by another caspase occurs, but the DFF-45 enzyme remains inactive.
- Published
- 1998
8. Phosphorylation of PITSLRE p110 isoforms accompanies their processing by caspases during Fas-mediated cell death.
- Author
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Tang, D, Gururajan, R, and Kidd, V J
- Abstract
A number of cellular proteins have been identified as caspase targets during cell death, including the PITSLRE protein kinases. These targets generally fall into one of three possible categories: 1) other caspases, 2) proteins that are inactivated during apoptosis, and 3) proteins that are required for execution of the cell death program. However, not all proteins are cleaved by caspases during apoptosis. Why only specific proteins are destined to be processed by caspases during cell death is currently not clear. Here we show that multiple caspase-like activities are involved in the processing of the PITSLRE p110 isoforms during Fas-induced apoptosis in Jurkat T-cells. Three p110 caspase cleavage sites have been mapped to the amino-terminal domain of p110 and verified by site-directed mutagenesis. Curiously, the mutagenesis studies revealed that cleavage of two juxtaposed caspase sites is necessary for the complete processing of this protein during cell death in vivo. Finally, we demonstrate that the PITSLRE p110 protein is rapidly phosphorylated during Fas-induced apoptosis in Jurkat cells and that phosphorylation of an amino-terminal portion of the protein may enhance caspase cleavage in this region.
- Published
- 1998
9. Structure-function relations of smooth muscle calponin. The critical role of serine 175.
- Author
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Tang, D C, Kang, H M, Jin, J P, Fraser, E D, and Walsh, M P
- Abstract
Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin-dependent protein kinase II. To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II. The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively). Tissue-purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective. Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases. These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate.
- Published
- 1996
10. Evidence for the existence of a sodium-dependent glutathione (GSH) transporter. Expression of bovine brain capillary mRNA and size fractions in Xenopus laevis oocytes and dissociation from gamma-glutamyltranspeptidase and facilitative GSH transporters.
- Author
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Kannan, R, Yi, J R, Tang, D, Li, Y, Zlokovic, B V, and Kaplowitz, N
- Abstract
Our laboratory previously has shown apparent carrier-mediated glutathione (GSH) uptake across the blood-brain barrier (BBB) in two animal models. In the present study, when Xenopus oocytes were injected with bovine brain capillary mRNA expression of intact GSH, uptake was observed after 3 days. When total mRNA was converted to cDNA and subfractionated with subsequent cRNA injection into oocytes, three distinct fractions (5, 7-8, and 11-12) expressed carrier-mediated intact GSH transport. Northern blot analysis established the presence of RcGshT, the previously cloned sodium-independent hepatic canalicular transporter, only in fraction 5. GSH transport activity in fraction 7 was significantly inhibited by replacement of NaCl with choline chloride and by sulfobromophthalein-GSH, neither of which affects RcGshT. The Na(+)-dependent GSH uptake kinetics exhibited high affinity (approximately 400 micron) and low affinity (approximately 10 mM) components. Fraction 11 expressed Na(+)-independent transport of intact GSH and also contained the GGT transcript. In conclusion, we have identified three distinct sized transcripts from bovine brain capillary mRNA which express GSH transport: one fraction expresses a novel Na(+)-dependent GSH uptake which can be dissociated unequivocally from both GGT and RcGshT for the first time and which may account for uptake of GSH against its electrochemical gradient at the BBB.
- Published
- 1996
11. The granulin/epithelin precursor abrogates the requirement for the insulin-like growth factor 1 receptor for growth in vitro.
- Author
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Xu, S Q, Tang, D, Chamberlain, S, Pronk, G, Masiarz, F R, Kaur, S, Prisco, M, Zanocco-Marani, T, and Baserga, R
- Abstract
3T3 cells null for the type 1 insulin-like growth factor receptor are refractory to stimulation by a variety of purified growth factors that are known to be required for the stimulation of other 3T3 cells. However, these cells, known as R- cells, grow in serum-supplemented medium and also in media conditioned by certain cell lines. We report here the purification of a growth factor that stimulates DNA synthesis (and growth) of R- cells. The growth factor, purified to homogeneity by SDS-polyacrylamide gel electrophoresis, was identified as the granulin/epithelin precursor by an accurate determination of the masses of endoproteinase Lys-C peptides using matrix-assisted laser desorption ionization mass spectrometry, followed by a data base search. The granulin/epithelin precursor is a little known growth factor, secreted by a variety of epithelial and hemopoietic cells. It is at present the only purified growth factor that can stimulate the growth of mouse embryo fibroblasts null for the type 1 insulin-like growth factor receptor.
- Published
- 1998
12. Cyclin-dependent kinase 5 (Cdk5) activation domain of neuronal Cdk5 activator. Evidence of the existence of cyclin fold in neuronal Cdk5a activator.
- Author
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Tang, D, Chun, A C, Zhang, M, and Wang, J H
- Abstract
Neuronal Cdk5 activator (Nck5a) differs from other cyclin-dependent kinase (Cdk) activators in that its amino acid sequence is only marginally similar to the cyclin consensus sequence. Nevertheless, computer modeling has suggested that Nck5a contains the cyclin-fold motif recently identified in the crystal structure of cyclin A. In the present study, a number of truncation mutants and substitution mutants of the Nck5a were produced and tested for the Cdk5 activation and Cdk5 binding activity. The active domain of Nck5a determined by using the truncation mutants consists of the region spanning residues 150 to 291. The size of Nck5a active domain is essentially the same as that of cyclin A required for Cdk2 activation (Lees, E. M., and Harlow, E. (1993) Mol. Cell. Biol. 13, 1194-1201). The change, or the lack of change, in Cdk5 activation activity observed with a number of substitution mutants may be understood on the basis of structure and function relationship of cyclin A. These results provide support to the previous suggestion (Brown, N. R., Noble, M. E. M., Endicott, J. A., Garman, E. F., Wakatsuki, S., Mitchell, E., Rasmussen, B., Hunt, T., and Johnson, L. N. (1995) Structure 3, 1235-1247) that the activation domain of Nck5a adopts a conformation similar to that of cyclin A. They also provide a partial answer to the question of how Nck5a, a non-cyclin, activates a cyclin-dependent kinase.
- Published
- 1997
13. Association of neurofilament proteins with neuronal Cdk5 activator.
- Author
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Qi, Z, Tang, D, Zhu, X, Fujita, D J, and Wang, J H
- Abstract
Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J.H. (1996) J. Biol. Chem. 271, 1538-1543). The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a.
- Published
- 1998
14. Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain.
- Author
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Lee, K Y, Rosales, J L, Tang, D, and Wang, J H
- Abstract
Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J., and Wang, J. H. (1995) Trends Biochem. Sci. 20, 33-37). The regulatory subunit is an N-terminal truncated derivative of a 35-kDa protein expressed specifically in brain, hence the name neuronal Cdk5 activator, p25/p35nck5a. In this study, we probe the relationship between the two different forms of Nck5a and their interaction with and activation of Cdk5 in bovine brain extract. Using protein fractionation procedures in combination with Western blot analysis and protein kinase assay, three forms of Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be activated by bacterially expressed GST-p21nck5a, a heterodimer of Cdk5 and p25nck5a that displays high kinase activity, and a Cdk5.p35nck5a complex that is inactive and refractory to GST-p21nck5a activation. Analysis of the Cdk5.p35nck5a complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of approximately 670 kDa. When the macromolecular complex was subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35nck5a and Cdk5, although eluting at the same position as control, displayed high kinase activity. The result is compatible with the suggestion that the macromolecular complex contained a kinase inhibitory factor that dissociated from the complex in 10% ethylene glycol.
- Published
- 1996
15. An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator.
- Author
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Tang, D, Yeung, J, Lee, K Y, Matsushita, M, Matsui, H, Tomizawa, K, Hatase, O, and Wang, J H
- Abstract
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit that is derived from a 35-kDa brain- and neuron-specific protein called the neuronal Cdk5 activator (p35/p25nck5a) (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426; Tsai, L. H., Delalle, I., Caviness, V. S., Jr., Chae, T., and Harlow, E. (1994) Nature 371, 419-423). Upon screening of a human hippocampus library with a bovien Nck5a cDNA, we uncovered a distinct clone encoding a 39-kDa isoform of Nck5a. The isoform, designated the neuronal Cdk5 activator isoform (p39nck5ai), showed a high degree of sequence similarity to p35nck5a with 57% amino acid identity. Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined. In situ hybridization showed that Nck5ai was enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation. Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa truncated form of Nck5ai expressed as a glutathione S-transferase fusion protein in Escherichia coli] was found to associate with Cdk5 to form an active Cdk5 kinase. Thus, the isoform shares many common characteristics with p35nck5a, including Ckd5 activating activity and brain- and neuron-specific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins.
- Published
- 1995
16. Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels.
- Author
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Tang D, Xu J, Li Y, Zhao P, Kong X, Hu H, Liang S, Tang C, and Liu Z
- Subjects
- Animals, Chilopoda enzymology, Chilopoda metabolism, HEK293 Cells, Humans, Mutagenesis, Site-Directed, Potassium Channel Blockers pharmacology, Potassium Channels, Inwardly Rectifying antagonists & inhibitors, Toxins, Biological toxicity, Potassium Channels, Inwardly Rectifying metabolism, Toxins, Biological metabolism
- Abstract
Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin-channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K
+ "knock-off" effect, suggesting the toxin functions as an out pore blocker physically occluding the K+ -conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel-specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
17. Engineering of highly potent and selective HNTX-III mutant against hNa v 1.7 sodium channel for treatment of pain.
- Author
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Zhang Y, Wang L, Peng D, Zhang Q, Yang Q, Li J, Li D, Tang D, Chen M, Liang S, Liu Y, Wang S, and Liu Z
- Subjects
- Animals, Humans, Molecular Docking Simulation, NAV1.4 Voltage-Gated Sodium Channel drug effects, NAV1.4 Voltage-Gated Sodium Channel genetics, NAV1.5 Voltage-Gated Sodium Channel drug effects, NAV1.5 Voltage-Gated Sodium Channel genetics, NAV1.7 Voltage-Gated Sodium Channel chemistry, NAV1.7 Voltage-Gated Sodium Channel drug effects, Nociceptive Pain genetics, Nociceptive Pain pathology, Peptides chemistry, Peptides pharmacology, Spider Venoms genetics, NAV1.7 Voltage-Gated Sodium Channel genetics, Nociceptive Pain drug therapy, Peptides genetics, Spider Venoms chemistry
- Abstract
Human voltage-gated sodium channel Na
v 1.7 (hNav 1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav 1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav 1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav 1.7. Site-directed mutagenesis analysis indicated key residues on hNav 1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav 1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 μM) and >1000-fold selectivity against Nav 1.4 and Nav 1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)- Published
- 2021
- Full Text
- View/download PDF
18. Naja atra venom peptide reduces pain by selectively blocking the voltage-gated sodium channel Nav1.8.
- Author
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Zhang F, Zhang C, Xu X, Zhang Y, Gong X, Yang Z, Zhang H, Tang D, Liang S, and Liu Z
- Subjects
- Analgesics adverse effects, Analgesics pharmacology, Analgesics therapeutic use, Animals, Cell Line, Dose-Response Relationship, Drug, Humans, Mice, Naja naja, Neuralgia drug therapy, Patch-Clamp Techniques, Rats, Rats, Sprague-Dawley, Elapid Venoms chemistry, NAV1.8 Voltage-Gated Sodium Channel drug effects, Sodium Channel Blockers pharmacology
- Abstract
The voltage-gated sodium channel Nav1.8 is preferentially expressed in peripheral nociceptive neurons and contributes to inflammatory and neuropathic pain. Therefore, Nav1.8 has emerged as one of the most promising analgesic targets for pain relief. Using large-scale screening of various animal-derived toxins and venoms for Nav1.8 inhibitors, here we identified μ-EPTX-Na1a, a 62-residue three-finger peptide from the venom of the Chinese cobra ( Naja atra ), as a potent inhibitor of Nav1.8, exhibiting high selectivity over other voltage-gated sodium channel subtypes. Using whole-cell voltage-clamp recordings, we observed that purified μ-EPTX-Na1a blocked the Nav1.8 current. This blockade was associated with a depolarizing shift of activation and repolarizing shift of inactivation, a mechanism distinct from that of any other gating modifier toxin identified to date. In rodent models of inflammatory and neuropathic pain, μ-EPTX-Na1a alleviated nociceptive behaviors more potently than did morphine, indicating that μ-EPTX-Na1a has a potent analgesic effect. μ-EPTX-Na1a displayed no evident cytotoxicity and cardiotoxicity and produced no obvious adverse responses in mice even at a dose 30-fold higher than that producing a significant analgesic effect. Our study establishes μ-EPTX-Na1a as a promising lead for the development of Nav1.8-targeting analgesics to manage pain., (© 2019 Zhang et al.)
- Published
- 2019
- Full Text
- View/download PDF
19. Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping.
- Author
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Vielemeyer O, Yuan H, Moutel S, Saint-Fort R, Tang D, Nizak C, Goud B, Wang Y, and Perez F
- Subjects
- Animals, Antibody Affinity immunology, Antibody Specificity immunology, Antigens, Bacterial immunology, Epitope Mapping, Golgi Matrix Proteins, Immunoglobulin Variable Region immunology, Membrane Proteins immunology, Models, Immunological, Phosphorylation, Rats, Antibodies, Monoclonal analysis, Antibodies, Monoclonal immunology, Antibodies, Phospho-Specific analysis, Antibodies, Phospho-Specific immunology, Phosphoamino Acids analysis, Protein Engineering methods
- Abstract
In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.
- Published
- 2009
- Full Text
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20. Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue.
- Author
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Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, Kurz M, Jarrott R, Shouldice SR, Hiniker A, Martin JL, Heras B, and Bardwell JC
- Subjects
- Amino Acid Sequence, Escherichia coli Proteins metabolism, Hydrogen-Ion Concentration, Isoleucine chemistry, Kinetics, Molecular Conformation, Molecular Sequence Data, Oxidation-Reduction, Oxidoreductases chemistry, Proline chemistry, Protein Conformation, Protein Disulfide-Isomerases metabolism, Protein Folding, Protein Structure, Secondary, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Protein Disulfide-Isomerases chemistry, Thioredoxins chemistry
- Abstract
The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.
- Published
- 2009
- Full Text
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21. Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay.
- Author
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Tang D, Mar K, Warren G, and Wang Y
- Subjects
- Adenosine Triphosphatases metabolism, Animals, CDC2 Protein Kinase metabolism, Cell Cycle Proteins metabolism, Cell-Free System metabolism, Cell-Free System ultrastructure, Coat Protein Complex I metabolism, Golgi Apparatus ultrastructure, Golgi Matrix Proteins, HeLa Cells, Humans, Membrane Proteins metabolism, N-Ethylmaleimide-Sensitive Proteins metabolism, Nuclear Proteins metabolism, Phosphorylation, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Rats, Polo-Like Kinase 1, Golgi Apparatus metabolism, Membrane Fusion physiology, Mitosis physiology
- Abstract
In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.
- Published
- 2008
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22. An insight into the mechanism of human cysteine dioxygenase. Key roles of the thioether-bonded tyrosine-cysteine cofactor.
- Author
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Ye S, Wu X, Wei L, Tang D, Sun P, Bartlam M, and Rao Z
- Subjects
- Amino Acid Sequence, Animals, Cloning, Molecular, Coenzymes chemistry, Consensus Sequence, Conserved Sequence, Crystallography, X-Ray, Cysteine Dioxygenase genetics, Cysteine Dioxygenase isolation & purification, Humans, Kinetics, Mass Spectrometry, Molecular Sequence Data, Mutagenesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Coenzymes metabolism, Cysteine, Cysteine Dioxygenase chemistry, Cysteine Dioxygenase metabolism, Tyrosine
- Abstract
Cysteine dioxygenase is a non-heme mononuclear iron metalloenzyme that catalyzes the oxidation of cysteine to cysteine sulfinic acid with addition of molecular dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolic pathways related to diverse sulfurate compounds. Cysteine dioxygenase is therefore very important for maintaining the proper hepatic concentration of intracellular free cysteine. Mechanisms for mouse and rat cysteine dioxygenases have recently been reported based on their crystal structures in the absence of substrates, although there is still a lack of direct evidence. Here we report the first crystal structure of human cysteine dioxygenase in complex with its substrate L-cysteine to 2.7A, together with enzymatic activity and metal content assays of several single point mutants. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly associated with a thioether-bonded tyrosine-cysteine cofactor involving Tyr-157 and Cys-93. This cross-linked protein-derived cofactor plays several key roles different from those in galactose oxidase. This report provides a new potential target for therapy of diseases related to human cysteine dioxygenase, including neurodegenerative and autoimmune diseases.
- Published
- 2007
- Full Text
- View/download PDF
23. Simvastatin attenuates expression of cytokine-inducible nitric-oxide synthase in embryonic cardiac myoblasts.
- Author
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Madonna R, Di Napoli P, Massaro M, Grilli A, Felaco M, De Caterina A, Tang D, De Caterina R, and Geng YJ
- Subjects
- Animals, Cell Line, Embryo, Mammalian anatomy & histology, Humans, I-kappa B Kinase, Interleukin-1 pharmacology, Mevalonic Acid metabolism, NF-kappa B metabolism, Nitric Oxide Synthase genetics, Nitric Oxide Synthase Type II, Nitrites metabolism, Polyisoprenyl Phosphates metabolism, Protein Serine-Threonine Kinases metabolism, RNA, Messenger metabolism, Rats, Tumor Necrosis Factor-alpha pharmacology, rho GTP-Binding Proteins antagonists & inhibitors, rho GTP-Binding Proteins metabolism, Embryo, Mammalian physiology, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Myoblasts, Cardiac drug effects, Myoblasts, Cardiac enzymology, Nitric Oxide Synthase metabolism, Simvastatin pharmacology
- Abstract
Cardiac stem cells or myoblasts are vulnerable to inflammatory stimulation in hearts with infarction or ischemic injury. Widely used for the prevention and treatment of atherosclerotic heart disease, the cholesterol-lowering drugs statins may exert anti-inflammatory effects. In this study, we examined the impact of inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase with simvastatin on the expression of inducible nitric-oxide synthase (iNOS) in embryonic cardiac myoblasts stimulated with the proinflammatory cytokines, interleukin-1 or tumor necrosis factor. Treatment with simvastatin significantly reduced the levels of iNOS mRNA and protein in cytokine-treated rat H9c2 cardiac embryonic myoblasts. Addition of the HMG-CoA reductase product, L-mevalonate, and the by-product of cholesterol synthesis, geranylgeranyl pyrophosphate, could reverse the statin inhibitory effect on iNOS expression. Simvastatin treatment lowered the Rho GTPase activities, whereas the Rho-associated kinase inhibitor Y27632 partially blocked the statin inhibitory effect on nitrite production in the cytokine-treated H9c2 cells. Treatment with simvastatin led to inactivation of NF-kappaB by elevation of the NF-kappaB inhibitor IkappaB and reduction of the NF-kappaB nuclear contents in the cytokine-stimulated H9c2 cells. Hence, treatment with simvastatin can attenuate iNOS expression and NO synthesis in cytokine-stimulated embryonic cardiac myoblasts. The statin inhibitory effect may occur through isoprenoid-mediated intracellular signal transduction, which involves several key signal proteins, such as Rho kinase and IkappaB/NF-kappaB. These data suggest that statin therapy may protect the cardiac myocyte progenitors against the cytotoxicity of cytokine-induced high output of NO production in infarcted or ischemic hearts with inflammation.
- Published
- 2005
- Full Text
- View/download PDF
24. TDAG51 is induced by homocysteine, promotes detachment-mediated programmed cell death, and contributes to the cevelopment of atherosclerosis in hyperhomocysteinemia.
- Author
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Hossain GS, van Thienen JV, Werstuck GH, Zhou J, Sood SK, Dickhout JG, de Koning AB, Tang D, Wu D, Falk E, Poddar R, Jacobsen DW, Zhang K, Kaufman RJ, and Austin RC
- Subjects
- Animals, Blotting, Northern, Cell Adhesion, Cell Nucleus metabolism, Cells, Cultured, Cloning, Molecular, Crosses, Genetic, DNA, Complementary metabolism, Endoplasmic Reticulum metabolism, Endothelium, Vascular cytology, Fluorescent Antibody Technique, Indirect, Gene Expression Profiling, Gene Library, Homocysteine metabolism, Humans, Hyperhomocysteinemia complications, In Situ Nick-End Labeling, Mice, Mice, Inbred C57BL, Mice, Transgenic, Phosphorylation, Protein Binding, Protein Structure, Tertiary, RNA, Messenger metabolism, Subcellular Fractions, Time Factors, Tissue Distribution, Transcription Factors metabolism, Transcriptional Activation, Transfection, Tunicamycin pharmacology, Umbilical Veins, Apoptosis, Arteriosclerosis pathology, Homocysteine chemistry, Hyperhomocysteinemia pathology, Transcription Factors chemistry
- Abstract
Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and accelerates atherosclerosis in apoE-/- mice. Despite the observations that homocysteine causes endoplasmic reticulum (ER) stress and programmed cell death (PCD) in cultured human vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherogenesis have not been completely elucidated. We report here that homocysteine induces the expression of T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. This effect was observed for other ER stress-inducing agents, including dithiothreitol and tunicamycin. TDAG51 expression was attenuated in homozygous A/A mutant eukaryotic translation initiation factor 2 alpha mouse embryonic fibroblasts treated with homocysteine or tunicamycin, suggesting that ER stress-induced phosphorylation of eukaryotic translation initiation factor 2 alpha is required for TDAG51 transcriptional activation. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion, and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoE-/- mice fed hyperhomocysteinemic diets, compared with mice fed a control diet. Collectively, these findings provide evidence that TDAG51 is induced by homocysteine, promotes detachment-mediated PCD, and contributes to the development of atherosclerosis observed in hyperhomocysteinemia.
- Published
- 2003
- Full Text
- View/download PDF
25. Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress.
- Author
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Zhao M, Tang D, Lechpammer S, Hoffman A, Asea A, Stevenson MA, and Calderwood SK
- Subjects
- 3' Untranslated Regions, Animals, Blotting, Northern, Blotting, Western, Cell Nucleus metabolism, Cells, Cultured, DNA-Binding Proteins metabolism, Heat Shock Transcription Factors, Hot Temperature, Mice, Phosphorylation, Plasmids metabolism, Promoter Regions, Genetic, Protein Biosynthesis, RNA metabolism, RNA, Messenger metabolism, Temperature, Time Factors, Transcription Factors, Transcription, Genetic, Transfection, HSP70 Heat-Shock Proteins metabolism, RNA, Double-Stranded metabolism, eIF-2 Kinase physiology
- Abstract
We have investigated the role of the double-stranded RNA-dependent protein kinase gene (pkr) in the regulation of the heat shock response. We show that the pkr gene is essential for efficient activation of the heat shock response and that pkr disruption profoundly inhibits heat shock protein 70 (HSP70) synthesis and blocks the development of thermotolerance. Despite these profound effects, pkr disruption did not markedly affect the activation of heat shock factor 1 by heat and did not reduce the rate of transcription of the HSP70 gene after heat shock. However, despite the lack of effect of pkr disruption on HSP70 gene transcription, we found a significant decrease in the expression of HSP70 mRNA in pkr-/- cells after heat shock. Kinetic studies of mRNA turnover suggested a block in the thermal stabilization of HSP70 mRNA in pkr-/- cells. As the thermal stabilization of HSP70 mRNA is thought to involve cis-acting A+U rich (ARE) elements in the 3'-untranslated region (UTR), we examined a potential role for pkr in this process. We found that a reporter beta-galactosidase mRNA destabilized by introduction of a functional ARE into the 3'-UTR became stabilized by heat but only in cells containing an intact pkr gene. Our studies suggest therefore that pkr plays a significant role in the stabilization of mRNA species containing ARE destruction sequences in the 3'-UTR and through this mechanism, contributes to the regulation of the heat shock response and other processes.
- Published
- 2002
- Full Text
- View/download PDF
26. ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53.
- Author
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Tang D, Wu D, Hirao A, Lahti JM, Liu L, Mazza B, Kidd VJ, Mak TW, and Ingram AJ
- Subjects
- 3T3 Cells, Animals, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Line, Transformed, Cyclin-Dependent Kinase Inhibitor p21, Cyclins biosynthesis, DNA-Binding Proteins, Enzyme Activation, Etoposide pharmacology, Humans, Mice, Mitogen-Activated Protein Kinases physiology, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases physiology, Tumor Suppressor Protein p53 physiology, Tumor Suppressor Proteins, Apoptosis physiology, Cell Cycle physiology, DNA Damage, Mitogen-Activated Protein Kinases metabolism
- Abstract
In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.
- Published
- 2002
- Full Text
- View/download PDF
27. Apoptotic release of histones from nucleosomes.
- Author
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Wu D, Ingram A, Lahti JH, Mazza B, Grenet J, Kapoor A, Liu L, Kidd VJ, and Tang D
- Subjects
- 3T3 Cells, Animals, Blotting, Western, Caspase 3, Caspases metabolism, Cell Line, Cell Nucleus metabolism, Chromatin metabolism, DNA Fragmentation, Enzyme Inhibitors pharmacology, Etoposide pharmacology, HeLa Cells, Humans, In Situ Nick-End Labeling, Jurkat Cells, Mice, Protein Binding, Retroviridae genetics, Staurosporine pharmacology, Tetrazolium Salts metabolism, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha metabolism, fas Receptor metabolism, Apoptosis, Histones metabolism, Nucleosomes metabolism
- Abstract
Chromatin structure is influenced by histone modification, and this may help direct chromatin behavior to facilitate transcription, DNA replication, and DNA repair. Chromatin condensation and DNA fragmentation are the classic nuclear features but remain poorly characterized. It is highly probable that nucleosomal structure must be altered to allow these features to become apparent, but data to support this construct are lacking. We report here that in response to apoptotic signals from a death receptor (CD95 and tumor necrosis factor-alpha) or mitochondrial (staurosporine) apoptotic stimulus, the core nucleosomal histones H2A, H2B, H3, and H4 become separated from DNA during apoptosis in Jurkat and HeLa cells and are consequently detectable in the cell lysate prepared using a non-ionic detergent. The timing of this histone release from DNA correlates well with the progression of apoptosis. We also show expression of a caspase cleavage-resistant form of ICAD (ICAD-DM) in Jurkat and HeLa cells abolished DNA fragmentation and also dramatically reduced histone release in apoptotic cells. However, we demonstrate that apoptotic histone release is not an inevitable consequence of CAD/DFF-40-mediated DNA destruction as DNA fragmentation but not histone release occurs efficiently in tumor necrosis factor-alpha- and etoposide-treated NIH3T3 cells. Furthermore, in an in vitro apoptotic assay, incubation of apoptotic Jurkat cellular extract with non-apoptotic Jurkat nuclei led to nuclear DNA fragmentation without obvious histone release. Taken together, these data demonstrate that CAD/DFF-40 functions indirectly in mediating nucleosomal destruction during apoptosis.
- Published
- 2002
- Full Text
- View/download PDF
28. Akt is activated in response to an apoptotic signal.
- Author
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Tang D, Okada H, Ruland J, Liu L, Stambolic V, Mak TW, and Ingram AJ
- Subjects
- 3T3 Cells, Androstadienes pharmacology, Animals, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors pharmacology, Etoposide pharmacology, Immunoblotting, Mice, Nucleic Acid Synthesis Inhibitors pharmacology, Phosphatidylinositol 3-Kinases metabolism, Poly(ADP-ribose) Polymerases metabolism, Protein Binding, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins c-bcl-2 metabolism, Staurosporine pharmacology, Time Factors, Transfection, Wortmannin, bcl-X Protein, Apoptosis, Protein Serine-Threonine Kinases, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins physiology
- Abstract
Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.
- Published
- 2001
- Full Text
- View/download PDF
29. Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles.
- Author
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Word RA, Tang DC, and Kamm KE
- Subjects
- Animals, Calcium metabolism, Cattle, Electric Stimulation, Enzyme Activation, Female, Humans, In Vitro Techniques, Isometric Contraction physiology, Muscle Relaxation physiology, Muscle, Smooth enzymology, Myometrium enzymology, Phosphorylation, Trachea enzymology, Muscle Contraction physiology, Muscle, Smooth physiology, Myometrium physiology, Myosin-Light-Chain Kinase metabolism, Trachea physiology
- Abstract
In intact smooth muscle, myosin light chain kinase (MLCK) is phosphorylated at its regulatory site by Ca2+/calmodulin-dependent protein kinase II resulting in an increase in the concentration of Ca2+/calmodulin required for half-maximal activation of the enzyme (KCaM). We investigated the physiological significance of MLCK phosphorylation during cycles of contraction and relaxation in tonic (tracheal) and phasic (uterine) smooth muscles. MLCK phosphorylation and dephosphorylation occurred at rates sufficient to modulate the Ca2+ sensitivity of light chain phosphorylation. In contractions of both smooth muscles (though using different sources of activating Ca2+), increases in [Ca2+]i preceded light chain phosphorylation; but, the rate of increase in light chain phosphorylation was significantly greater than the rate of increase in [Ca2+]i. The onset of MLCK phosphorylation with the resultant increase in KCaM coincided with the diminished rate of light chain phosphorylation. During spontaneous contractions of uterine smooth muscle, the Ca2+ transient was characterized by an initial rapid increase, a sustained plateau, and rapid decline. During the sustained phase of the Ca2+ transient, MLCK phosphorylation increased and coincided with dephosphorylation of light chain and relaxation. These results indicate that MLCK is sensitive to small increases in intracellular Ca2+ during the initiation of contraction and that the enzyme subsequently becomes desensitized to Ca2+/calmodulin, thereby limiting the extent of light chain phosphorylation.
- Published
- 1994
30. Identification of PECAM-1 in solid tumor cells and its potential involvement in tumor cell adhesion to endothelium.
- Author
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Tang DG, Chen YQ, Newman PJ, Shi L, Gao X, Diglio CA, and Honn KV
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Antigens, Differentiation, Myelomonocytic biosynthesis, Antigens, Differentiation, Myelomonocytic metabolism, Base Sequence, Cell Adhesion Molecules metabolism, Cell Line, Cells, Cultured, DNA Primers, DNA, Neoplasm isolation & purification, DNA, Neoplasm metabolism, Fluorescent Antibody Technique, Humans, Immunohistochemistry, Mice, Molecular Sequence Data, Oligonucleotides, Antisense, Platelet Endothelial Cell Adhesion Molecule-1, Polymerase Chain Reaction, Rats, Tumor Cells, Cultured cytology, Tumor Cells, Cultured metabolism, Antigens, Differentiation, Myelomonocytic analysis, Cell Adhesion, Cell Adhesion Molecules analysis, Endothelium, Vascular physiology, Tumor Cells, Cultured physiology
- Abstract
PECAM-1 (CD31/EndoCAM) is an adhesion molecule in the immunoglobulin supergene family that is expressed on endothelial cells, platelets, and some hematopoietic lineage cells. In this paper, using several polyclonal and monoclonal antibodies against PECAM-1, we identified PECAM-1 molecules on human, rat, and murine solid tumor cell lines. Immunocytochemical labeling and flow cytometric analysis using either polyclonal, monoclonal, or Fab portion of the antibodies against PECAM-1 detected a distinct distribution on tumor cell surface. Immunoblotting revealed proteins ranging from 120 to 130 kDa in tumor cells derived from different species. Immunoprecipitation and subcellular fractionation studies indicated that PECAM-1 is constitutively expressed on the surface of human tumor cells (i.e. colon adenocarcinoma). The specificity of a major polyclonal anti-PECAM-1 used in the current study (i.e. SEW-3) was confirmed by the preabsorption studies. PECAM-1 molecules on tumor cells appear to bear terminal carbohydrate moieties (i.e. sialic acid residues) different from those on platelets, since neuraminidase treatment of tumor cells, unlike platelets, did not result in a mobility shift. Polymerase chain reaction (PCR) analysis of genomic DNA derived from tumor cell lines of different species revealed the presence of PECAM-1 gene in the genome. The mRNAs of PECAM-1 in tumor cells were detected by reverse transcription-PCR followed by Southern hybridization. Screening of more than 20 human, rat, and murine solid tumor cell lines indicated that PECAM-1 is widely expressed, although the level of expression varies considerably among different cell lines. The expression of PECAM-1 message in tumor cells was confirmed by Northern blotting. DNA sequencing of the PCR fragment revealed that human tumor cell PECAM-1 matches 100% to the human endothelial cell counterpart. Finally, it was demonstrated that tumor cell PECAM-1 is involved in mediating tumor cell adhesion to endothelium, as evidenced by the ability of anti-PECAM-1 antibodies to decrease the adhesion of unstimulated tumor cells to microvascular endothelial cells.
- Published
- 1993
31. Regulation of the Ca2+ dependence of smooth muscle contraction.
- Author
-
Tang DC, Stull JT, Kubota Y, and Kamm KE
- Subjects
- Animals, Carbachol pharmacology, Cations, Divalent, Cattle, In Vitro Techniques, Isoproterenol pharmacology, Membrane Potentials, Muscle, Smooth drug effects, Myosin-Light-Chain Kinase metabolism, Myosins metabolism, Phosphorylation, Potassium Chloride pharmacology, Calcium metabolism, Muscle Contraction drug effects, Muscle, Smooth metabolism
- Abstract
Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.
- Published
- 1992
32. Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis.
- Author
-
Gibson BW, Tang DZ, Mandrell R, Kelly M, and Spindel ER
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Base Sequence, Hemolysis, Humans, Mass Spectrometry, Microbial Sensitivity Tests, Molecular Sequence Data, Peptides chemistry, Peptides pharmacology, Sequence Alignment, Skin metabolism, Amphibian Proteins, Anti-Bacterial Agents isolation & purification, Antimicrobial Cationic Peptides, Anura, Peptides isolation & purification, Skin chemistry
- Abstract
The structures and hemolytic and bactericidal activities of three bombinin-like peptides, or BLP-1-3, from the skin of Bombina orientalis are described. The peptides were isolated from the skin of B. orientalis and sequenced by tandem mass spectrometry and are amphipathic, cationic peptides of 25-27 amino acids in length. The sequence of the most abundant member (BLP-1) is: Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu- Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NH2. All three peptides were found to share considerable, but not complete, homology with bombinin, an antimicrobial, hemolytic peptide first isolated by Michl and Csordas (Csordas, A., and Michl, A. (1970) Monatsh. Chem. 101, 182-189) from the skin of Bombina variegata. The BLPs have been assayed for antibiotic and hemolytic activity and found to be more potent than magainin 2 (a related antimicrobial peptide from Xenopus laevis) in their ability to kill bacteria. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic over eukaryotic membranes. The molecular basis for antibacterial activity is presumed to be due to their predicted amphipathic alpha-helical structures which is supported by circular dichroism measurements that found significant helical content (63-69% alpha-helix) in 40% trifluoroethanol. Last, a cDNA library was constructed from the skin of B. orientalis and screened with an oligonucleotide probe complementary to the COOH terminus of BLP-1. Several clones were isolated and sequenced that encode BLP-1 and BLP-3, as well as an additional peptide (BLP-4) that differs by two amino acid substitutions from BLP-3.
- Published
- 1991
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