Role of opioid delta1 receptors, mitochondrial K(ATP) channels, and protein kinase C during cardiocyte apoptosis.
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Retraction Notice↗
Biomedical subjects
Z Yao
Publications and source records attributed to Z Yao.
Metabolism and disposition of gemfibrozil in Wistar and multidrug resistance-associated protein 2-deficient TR- rats.
1. The roles of multidrug resistance-associated protein (Mrp) 2 deficiency and Mrp3 up-regulation were evaluated on the metabolism and disposition of gemfibrozil. 2. Results from in vitro studies in microsomes showed that the hepatic intrinsic clearance (CLint) for the oxidative metabolism of gemfibrozil was slightly higher (1.5-fold) in male TR- rats, which are deficient in Mrp2, than in wild-type Wistar rats, whereas CLint for glucuronidation was similar in both strains. 3. The biliary excretion of intravenously administered [14C]gemfibrozil was significantly impaired in TR-) rats compared with Wistar rats (22 versus 93% of the dose excreted as the acyl glucuronides over 72 h). Additionally, the extent of urinary excretion of radioactivity was much higher in TR- than in Wistar rats (78 versus 2.6% of the dose). 4. There were complex time-dependent changes in the total radioactivity levels and metabolite profiles in plasma, liver and kidney, some of which appeared to be related to the up-regulation of Mrp3. 5. Overall, it was demonstrated that alterations in the expression of the transporters Mrp2 and Mrp3 significantly affected the excretion as well as the secondary metabolism and distribution of [14C]gemfibrozil.
ATP-Binding Cassette Transporters↗
Genomic organization of human TCF12 gene and spliced mRNA variants producing isoforms of transcription factor HTF4.
The human TCF12 gene, mapping to 15q21, encodes the helix-loop-helix transcription factor 4 (HTF4). A detailed analysis of this genomic region established the organization of the TCF12 gene. The gene includes 21 exons and is significantly larger than an average human gene. Preceding the second exon, two alternative acceptor sites for mRNA splicing yield two distinguishable transcripts (HTF4a and HTF4b) which differ in their 5' untranslated region but share identical coding sequences. Differential utilization of exon 15 in the TCF12 gene may reflect a mechanism producing a cell-type-specific protein (HTF4c). In addition, intron 5 in the TCF12 gene corresponds to the region involved in a translocation, t(9;15)(q22;q21), that results in a form of extraskeletal myxoid chondrosarcoma.
5' Flanking Region↗
Non-regulated and stimulated mechanisms cooperate in the nuclear accumulation of MEK1.
MEKs, which operate within the ERK cascade, shuttle into the nucleus, but are rapidly exported from this location, forming an apparent cytosolic distribution both before and after stimulation. Two different mechanisms have been proposed for the nuclear translocation of MEKs. One of them involves a constant and non-regulated shuttling of MEKs into the nucleus operating both before and after mitogenic stimulation. The other mechanism seems to require the activity of MEKs and is facilitated in response to mitogenic stimulation. Here we show that these two mechanisms may coexist in the same cells. We found that leptomycin B (LMB), a potent inhibitor of nuclear export, induces a nuclear accumulation of MEKs, and this was significantly facilitated by stimulation of LMB-treated cells with EGF, TPA and peroxovanadate. The EGF-stimulated, but not the LMB-induced translocation was attenuated by MEK inhibitors and by using inactive forms of MEK1. We also show that LMB slightly activates the ERK cascade, but this activity only partially induces the nuclear accumulation of MEKs in cells treated by LMB alone. Thus, MEKs translocate into the nucleus by a combination of non-regulated and stimulated processes that contribute to the nuclear translocation of MEKs either in resting cells or upon mitogenic stimulation.
Active Transport, Cell Nucleus↗
The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.
The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.
Animals↗
Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a).
We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.
Alanine↗
Altered regulation of ERK1b by MEK1 and PTP-SL and modified Elk1 phosphorylation by ERK1b are caused by abrogation of the regulatory C-terminal sequence of ERKs.
ERK1b is an alternatively spliced form of ERK1, containing a 26-amino acid insertion between residues 340 and 341 of ERK1. Although under most circumstances the kinetics of ERK1b activation are similar to that of ERK1 and ERK2, we have previously found several conditions under which the activation of ERK1b by extracellular stimuli differs from that of other ERKs. We studied the molecular mechanisms that cause this differential regulation of ERK1b and found that ERK1b is altered in its ability to interact with MEK1 and this influenced its subcellular localization but not its kinetics of activation. ERK1b had a decreased ability to phosphorylate Elk1, but this did not change much the transcriptional activity of the latter. Importantly, the interaction of ERK1b with PTP-SL, which can act as a MAPK phosphatase, shortly after mitogenic stimulation, was significantly affected as well. Using mutants of ERK1b we found that the differential interaction of ERK1b with the three effectors is caused by the site of insertion that abrogates the cytosolic retention sequence/common docking motif of ERKs, and is not dependent on the actual sequence of the insert. Prolonged epidermal growth factor stimulation of Rat1 cells resulted in a differential inactivation and not activation of ERK1b as compared with ERK1 and ERK2. The reduced sensitivity to phosphatases without major differences in the kinetics of activation or activation of substrates, suggests that ERK1b plays a role in the transmission of extracellular signals under conditions of persistent stimulation, where ERK1b and MAPK phosphatases are induced, and the activity of ERK1 and ERK2 is suppressed.
Alternative Splicing↗
Carbon nanotube single-electron transistors at room temperature.
Room-temperature single-electron transistors are realized within individual metallic single-wall carbon nanotube molecules. The devices feature a short (down to approximately 20 nanometers) nanotube section that is created by inducing local barriers into the tube with an atomic force microscope. Coulomb charging is observed at room temperature, with an addition energy of 120 millielectron volts, which substantially exceeds the thermal energy. At low temperatures, we resolve the quantum energy levels corresponding to the small island. We observe unconventional power-law dependencies in the measured transport properties for which we suggest a resonant tunneling Luttinger-liquid mechanism.
Journal Article↗
Identification of putative downstream genes of Oct-4 by suppression-subtractive hybridization.
As a step toward understanding how toti/pluripotence is maintained by Oct-4, we have first constructed a cell model with differentially expressed Oct-4 in embryonic stem cells, and then used suppression-subtractive hybridization (SSH) method to identify the downstream genes of Oct-4. Among the 384 clones we screened, 40 clones were detected as differentially expressed genes with colony hybridization, and 13 clones were confirmed as the putative downstream genes of Oct-4 by Northern blot analysis. Sequencing showed 12 different genes, 8 known genes (Oct-4, Rex-1, Sox-2, Creatine kinase B, Makorin 1, Importin beta, Histone H2A.Z, Ribosomal protein S7) and 4 new genes. Except Oct-4 and Rex-1, the other genes have not been reported to be regulated by Oct-4. These results showed that SSH provides a very efficient means to identify the downstream genes of transcription factor. Some known genes identified may provide new insight of the function of Oct-4 in stem cells.
Animals↗
Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2.
Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.
Animals↗
The insulin-stimulated cell surface presentation of low density lipoprotein receptor-related protein in 3T3-L1 adipocytes is sensitive to phosphatidylinositide 3-kinase inhibition.
The regulation of low density lipoprotein receptor-related protein (LRP) activity by insulin was studied using 3T3-L1 adipocytes. The LRP mRNA and protein expression were independent of differentiation state of the cells and of insulin treatment. In differentiated cells, insulin treatment acutely stimulated the cell surface presentation of LRP (approximately 2-fold) as evidenced by methylamine-activated alpha(2)-macroglobulin binding and by biotinylation of cell surface LRP. The increased cell surface presentation was accompanied by a 39% decrease in LRP level in the low density microsomes. The magnitude of insulin-stimulated cell surface presentation of LRP was similar to that of transferrin receptor but was much less than that of GLUT4. Both the increases in LRP and GLUT4 cell surface presentation upon insulin treatment were abolished by inhibition of phosphatidylinositide 3-kinase. The increased cell surface presentation of LRP was associated with proportionally increased endocytic activity, and the internalization rate constant (K(e)) was not decreased by insulin treatment. Thus, insulin treatment most likely stimulates recycling of LRP from an endosomal pool to the plasma membrane, which is regulated in a phosphatidylinositide 3-kinase-dependent manner in 3T3-L1 adipocytes.
3T3 Cells↗
Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide.
We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR-cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 microM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.
Amino Acid Sequence↗
The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from beta-amyloid-induced cell death by inhibiting the formation of beta-amyloid-derived diffusible neurotoxic ligands.
beta Amyloid (Abeta) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Abeta protein prevented, in a dose-dependent manner, the Abeta-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Abeta-induced toxicity although it prevented the Abeta-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Abeta-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Abeta. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Abeta-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Abeta-induced neurotoxicity, we examined whether EGb 761 interacts directly with Abeta. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of beta-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer's disease.
Amyloid beta-Peptides↗
Morphine mimics preconditioning via free radical signals and mitochondrial K(ATP) channels in myocytes.
BACKGROUND: We tried to determine whether morphine mimics preconditioning (PC) to reduce cell death in cultured cardiomyocytes and whether opioid delta(1) receptors, free radicals, and K(ATP) channels mediate this effect. METHODS AND RESULTS: Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, and O(2) and CO(2) tension were controlled. Cardiomyocyte viability was quantified with propidium iodide (5 micromol/L), and production of free radicals was measured with 2',7'-dichlorofluorescin diacetate. PC with 10 minutes of simulated ischemia before 10 minutes of reoxygenation or morphine (1 micromol/L) or BW373U86 (10 pmol/L) infusion for 10 minutes followed by a 10-minute drug-free period before 1 hour of ischemia and 3 hours of reoxygenation reduced cell death to the same extent (*P:<0.05) (PC, 20+/-1%, n=7*; morphine, 32+/-4%, n=8*; BW373U86, 21+/-6%; controls, 52+/-5%, n=8). Like PC, morphine and BW373U86 increased free radical production 2-fold before ischemia (0.35+/-0.10, n=6*; 0.41+/-0.08, n=4* versus controls, 0.15+/-0.05, n=8, arbitrary units). Protection and increased free radical signals during morphine infusion were abolished with either the thiol reductant 2-mercaptopropionyl glycine (400 micromol/L), an antioxidant; naloxone (10 micromol/L), a nonselective morphine receptor antagonist; BNTX (0.1 micromol/L), a selective opioid delta(1) receptor antagonist; or 5-hydroxydecanoate (100 micromol/L), a selective mitochondrial K(ATP) channel antagonist. CONCLUSIONS: These results suggest that direct stimulation of cardiocyte opioid delta(1) receptors leads to activation of mitochondrial K(ATP) channels. The resultant increase of intracellular free radical signals may be an important component of the signaling pathways by which morphine mimics preconditioning in cardiomyocytes.
Adenosine Triphosphate↗
Role of opioid delta1 receptors, mitochondrial K(ATP) channels, and protein kinase C during cardiocyte apoptosis.
Opioids attenuate cardiac injury after ischemia and reperfusion. We wanted to determine whether the protection of opioids is mediated by blocking cardiocyte apoptosis, and if so, to describe the role of opioid delta1 receptors and protein kinase C (PKC) in this effect. Chick embryonic cardiomyocytes were subjected to 12 h of simulated ischemia and then 12 h of re-oxygenation, which resulted in 54+/-3% (n=6) of cell apoptosis (n=6) as measured by flow cytometry. This result was consistent with DNA laddering and TUNEL assay. Preconditioning, elicited with three cycles of 1 min of simulated ischemia separated by 5 min of reoxygenation before prolonged simulated ischemia, reduced apoptosis (36+/-4%, n=6*). Pretreatment with BNTX (0.1 micromol/l), a selective opioid delta1 receptor blocker, abolished the effects of preconditioning (57+/-5%, n=6). The selective opioid delta receptor agonist BW373U86 (20 pmol/l) also attenuated apoptosis (39+/-3%, n=6* v control). These effects were abolished by 5-hydroxydecanoate (100 microm), a selective mitochondrial K(ATP) channel blocker (50+/-5%, n=6) and by Go-6976 (0.1 micromol/l), a specific PKC inhibitor. Both preconditioning and BW373U86 activated the PKC delta isoform of particulate fraction before simulated ischemia without effect on total and cytosolic fractions. Stimulation of opioid delta1 receptors activates mitochondrial K(ATP) channels and the PKC delta isoform in cultured ventricular myocytes. This is one important signal transduction pathway through which ischemic preconditioning blocks apoptosis and preserves cardiac function.
Adenosine Triphosphate↗
Enhanced sensitivity in RIACT/MQ-MAS NMR experiments using rotor assisted population transfer.
The rotor assisted population transfer (RAPT) sequence is used to enhance the sensitivity of the RIACT(II) experiment for spin-3/2 quadrupolar nuclei. A detailed theoretical analysis of the polarizations that contribute to different types of MQ-MAS experiments is provided. In particular, two polarization pathways are distinguished for the creation of triple-quantum coherence. The existence of these pathways is experimentally demonstrated by comparing the sensitivities of different sequences with and without RAPT preparation.
Journal Article↗
The impact of chronic estrogen deprivation on immunologic parameters in the ovariectomized rhesus monkey (Macaca mulatta) model of menopause.
A large clinical literature suggests that estradiol (E(2)) plays a critical role in immune function. To further explore the relationship between E(2) and immune function, we examined a variety of immunological parameters in a rhesus monkey model of menopause and hormone replacement therapy. Rhesus monkeys (Age, 13.7+/-2.6 years) were ovariectomized and received either sham (n=10) or estradiol (n=10) replacement implants. Nine months post-ovariectomy, a variety of immunologic parameters were measured. E(2)-deprivation reduced natural killer cell activity and increased serum soluble gp130 levels. There was a trend for an increased proportion of CD8(+) (P=0.12) and HLA-DR(+)CD3(+) cells (P=0.15) and decreased proportion of eosinophils (P=0.11) in the E(2)-deprived monkeys. There was no difference in leukocyte distribution, CD28, CD56, CD4, CD8/CD45, colony forming units-granulocyte/monocytes formation, peripheral blood mononuclear cell apoptotic rate, or serum TNF, TNF-R1, TNF-R2, IL-6, soluble IL-6R, and IL-1 between the groups. These data demonstrate that E(2)-deprivation affects several aspects of immune function. These findings may have implications for menopause-associated changes of immune function that occur in women.
Animals↗