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At least 631 records · Page 35Linked to original sources
PubMed2006

Antimutagenic effect of essential oil of sage (Salvia officinalis L.) and its monoterpenes against UV-induced mutations in Escherichia coli and Saccharomyces cerevisiae.

Mutagenic and antimutagenic potential of essential oil (EO) of cultivated sage (S. officinalis L.) and its monoterpenes: thujone, 1,8-cineole, camphor and limonene against UVC-induced mutations was studied with Salmonella/microsome, E. coli WP2, E. coli K12 [Simić, D., Vuković-Gacić, B., Knezević-Vukcević, J., 1998. Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system. Mutat. Res. 402, 51-57] and S. cerevisiae D7 reversion assays. The toxicity of EO differed, depending on the strain used. The most sensitive were permeable strains TA100, TA102, E. coli K12 IB112 and non-permeable WP2. Mutagenic potential of EO and monoterpenes was not detected, with or without S9. EO reduced the number of UV-induced revertants in a concentration-dependent manner, reaching 50-70% of inhibition at the maximum non-toxic concentrations: 3 microl/plate (TA102), 5 microl/plate (WP2), 7.5 microl/plate (IB112), 30 microl/plate (E. coli K12 SY252) and 60 microl/plate (D7). The metabolic activation had no effect on antimutagenic potential of EO. Similar toxicity of monoterpenes was observed in TA100, E. coli SY252 and D7, with the exception of limonene (less toxic to D7). Reduction of UV-induced revertants by non-toxic concentrations of monoterpenes, tested with SY252 and D7, reached 40-50% at 15-20 microl/plate of thujone, 10 microl/plate of cineole and 1-10 microg/plate of camphor. Limonene showed antimutagenic effect only in D7. Our data recommend sage monoterpenes for further chemoprevention studies.

Antimutagenic Agents↗
PubMed2004

Glycosylation of asparagines 136 and 184 is necessary for the alpha2delta subunit-mediated regulation of voltage-gated Ca2+ channels.

The CaValpha2delta auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage-gated Ca2+ channels. One of the most prominent roles of CaValpha2delta is to increase whole-cell Ca2+ current amplitude. Using N-glycosidase F and truncated forms of CaValpha2delta, earlier studies suggested an important role for N-linked glycosylation in current stimulation. Here, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to examine the impact of individual glycosylation sites within the CaValpha2delta subunit on the regulation of Ba2+ currents through recombinant Ca2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular alpha2 domain of the protein that are functional. Substitution of asparagines for glutamines at amino acid positions 136 and 184 rendered these sites non-functional as shown by patch-clamp experiments. These results corroborate that N-glycosylation is required for the CaValpha2delta subunit-induced current stimulation and suggest that sites N136 and N184 are directly involved in this action. Likewise, N136Q and N184Q mutations prevented whole-cell current stimulation without altering its kinetic properties, suggesting a regulation on the number of functional channels at the plasma membrane.

Amino Acid Motifs↗
PubMed2006

The insulin-like growth factor-I receptor gene: a downstream target for oncogene and tumor suppressor action.

The insulin-like growth factor-I receptor (IGF-IR) mediates the biological actions of both IGF-I and IGF-II. The IGF-IR is expressed in most transformed cells, where it displays potent antiapoptotic and cell-survival activities. IGF-IR levels are tightly regulated by the concerted action of secreted (e.g. peptide and steroid hormones, growth factors and cytokines) and cellular (e.g. transcription factors, oncogenes and tumor suppressors) factors. The mode of action of many tumor suppressors involves transcriptional suppression of the IGF-IR gene, with a subsequent reduction in cell-surface IGF-IR concentrations and IGF-I action. Loss-of-function mutation of tumor suppressors, a common theme in human cancer, can lead to aberrant regulation of IGF-IR gene expression.

Biomarkers, Tumor↗
PubMed1986

Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp.

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.

Aspartic Acid↗
PubMed1998

DNA binding of the glucocorticoid receptor is not essential for survival.

Transcriptional regulation by the glucocorticoid receptor (GR) is essential for survival. Since the GR can influence transcription both through DNA-binding-dependent and -independent mechanisms, we attempted to assess their relative importance in vivo. In order to separate these modes of action, we introduced the point mutation A458T into the GR by gene targeting using the Cre/loxP system. This mutation impairs dimerization and therefore GRE-dependent transactivation while functions that require cross-talk with other transcription factors, such as transrepression of AP-1-driven genes, remain intact. In contrast to GR-/- mice, these mutants termed GRdim are viable, revealing the in vivo relevance of DNA-binding-independent activities of the GR.

Amino Acid Sequence↗
PubMed2001

MiRP2 forms potassium channels in skeletal muscle with Kv3.4 and is associated with periodic paralysis.

The subthreshold, voltage-gated potassium channel of skeletal muscle is shown to contain MinK-related peptide 2 (MiRP2) and the pore-forming subunit Kv3.4. MiRP2-Kv3.4 channels differ from Kv3.4 channels in unitary conductance, voltage-dependent activation, recovery from inactivation, steady-state open probability, and block by a peptide toxin. Thus, MiRP2-Kv3.4 channels set resting membrane potential (RMP) and do not produce afterhyperpolarization or cumulative inactivation to limit action potential frequency. A missense mutation is identified in the gene for MiRP2 (KCNE3) in two families with periodic paralysis and found to segregate with the disease. Mutant MiRP2-Kv3.4 complexes exhibit reduced current density and diminished capacity to set RMP. Thus, MiRP2 operates with a classical potassium channel subunit to govern skeletal muscle function and pathophysiology.

Animals↗
PubMed2001

Genetic evidence for noradrenergic control of long-term memory consolidation.

Memory formation involves dynamic interactions among many brain structures and their linking pathways. The noradrenaline (NA) system in the CNS plays an important role in a wide variety of neurological and psychological functions. Alteration in the NA system is implicated in the pathological states of some neuropsychiatric disorders. Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme for the biosynthesis of catecholamines. The regulatory mechanism of the TH reaction is generally considered to play a key role in controlling the catecholaminergic actions. Mice heterozygous for the mutation of the gene encoding TH exhibit the reduced TH activity in tissues. These mice have a moderate reduction in NA accumulation and release in brain regions. The mutant mice exhibit deficits in the water-finding task associated with latent learning performance, suggesting the impairment in memory formation. Spatial learning performance measured by the water maze task is normal in the mutants. However, they display deficits in long-term memory formation of conditioned learning evaluated with three distinct behavioral paradigms, including active avoidance, cued fear conditioning, and conditioned taste aversion, without affecting short-term memory. These memory deficits are restored by the drug-induced stimulation of NA activity at the postconditioning phase. Analysis of the mutant mice indicates that the central NA system is essential for the consolidation process in long-term memory of conditioned learning. The process appears to be implicated in the NA activity in the cerebral cortex and/or amygdaloid complex.

Amygdala↗
PubMed2002

Activation of bradykinin B1 receptor by ACE inhibitors.

ACE or kininase II inhibitors are very important, widely used therapeutic agents for the treatment of a variety of diseases. Although they inhibit ACE, thus, angiotensin II release and bradykinin (BK) inactivation, this inhibition alone does not suffice to explain their successful application in medical practice. Enalaprilat and other ACE inhibitors at nanomolar concentrations activate the BK B1 receptor directly in the absence of ACE and the peptide ligands, des-Arg-kinins. The inhibitors activate at the Zn-binding pentameric consensus sequence HEXXH (195 -199) of B1, a motif also present in the active centers of ACE but absent from the BK B2 receptor. ACE inhibitors, when activating the B1 receptor, elevate intracellular calcium [Ca2+]i and release NO from cultured cells. Activation by ACE inhibitor was abolished by Ca-EDTA, a B1 receptor antagonist, by a synthetic undecapeptide representing the 192-202 sequence in the B1 receptor, and by site-directed mutagenesis of H195 to A. With the exception of the B1 receptor blocker, these agents and the mutation did not affect the actions of the peptide ligand des-Arg10-Lys1-BK. Ischemia and inflammatory cytokines induce B1 receptors and elevate its expression. Direct activation of the B1 receptor by ACE inhibitors can contribute to their therapeutic efficacy, for example, by releasing NO in vascular beds, or to some of their side effects.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

p27 deregulation in breast cancer: prognostic significance and implications for therapy.

p27 is a key regulator of G1-to-S phase progression. It prevents premature activation of cyclin E-cdk2 in G1 and promotes the assembly and activation of D-type cyclin-cdks. While the p27 gene is rarely mutated in human cancers, the action of p27 is impaired in breast and other human cancers through accelerated p27 proteolysis, sequestration by cyclin D-cdks, and by p27 mislocalization in tumor cell cytoplasm. Reduced p27 protein is strongly associated with high histopathologic tumor grade, reflecting a lack of tumor differentiation. Loss of p27 is also an indicator of poor patient outcome in a majority of breast cancer studies, including node negative disease. The broad application of p27 in the clinical evaluation of breast cancer prognosis will require a consensus on methods of tumor fixation, staining, and scoring. This review will focus on mechanisms of p27 regulation in normal cells and how deregulation of p27 may arise in breast and other human cancers. The prognostic significance of p27 in human breast cancer and the possible therapeutic implications of these findings will also be reviewed.

Breast Neoplasms↗
PubMed2005

Blockade of HERG cardiac K+ current by antifungal drug miconazole.

1. Miconazole, an imidazole antifungal agent, is associated with acquired long QT syndrome and ventricular arrhythmias. Miconazole increases the plasma concentration of QT-prolonging drugs by inhibiting the hepatic cytochrome P450 metabolic pathway, but whether it has direct effects on cardiac ion channels has not been elucidated. 2. To determine the mechanism underlying these clinical findings, we investigated the effect of miconazole on human ether-a-go-go-related gene (HERG) K+ channels. 3. HERG channels were heterologously expressed in human embryonic kidney 293 (HEK293) cells and whole-cell currents were recorded using a patch-clamp technique (23 degrees C). 4. Miconazole inhibited HERG peak tail current in a concentration-dependent manner (0.4-40 microM) with an IC50 of 2.1 microM (n=3-5 cells at each concentration, Hill coefficient 1.2). HERG block was not frequency-dependent. It required channel activation, occurred rapidly, and had very slow dissociation properties. 5. The activation curve was shifted in a negative direction (V(1/2): -9.5+/-2.3 mV in controls and -15.3+/-2.4 mV after 4 microM miconazole, P<0.05, n=6). Miconazole did not change other channel kinetics (activation, deactivation, onset of inactivation, recovery from inactivation, steady-state inactivation). 6. The S6 domain mutation, F656C, abolished the inhibitory action of miconazole on HERG current indicating that miconazole preferentially binds to an aromatic amino-acid residue within the pore-S6 region. 7. Our findings indicate that miconazole causes HERG channel block by binding to a common drug receptor, and this involves preferential binding to activated channels. Thus, miconazole prolongs the QT interval by direct inhibition of HERG channels.

Action Potentials↗
PubMed2006

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis.

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.

DNA Polymerase III↗
PubMed1999

Ras stimulates DNA topoisomerase II alpha through MEK: a link between oncogenic signaling and a therapeutic target.

Topoisomerase II alpha (topo II alpha) is a major target of antitumor treatments. In an effort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might effect the levels of expression of the topo II alpha gene. In support of this idea, it was found that topo II alpha was elevated following microinjection of oncogenic Ras protein. Oncogenic ras was further shown to stimulate the topo II alpha promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo II alpha by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct confirmation that both ERK and SAPK were involved in topo II alpha regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo II alpha promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo II alpha promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Site-directed mutagenesis further demonstrated that an Ets-like binding site near the 5' end (-480 to -475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo II alpha expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.

3T3 Cells↗
PubMed2006

Modelling insecticide-binding sites in the voltage-gated sodium channel.

A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides, their mechanism of action and the role of mutations in the channel that are known to confer insecticide resistance. The sodium channel was modelled in an open conformation with the insecticide-binding site located in a hydrophobic cavity delimited by the domain II S4-S5 linker and the IIS5 and IIIS6 helices. The binding cavity is predicted to be accessible to the lipid bilayer and therefore to lipid-soluble insecticides. The binding of insecticides and the consequent formation of binding contacts across different channel elements could stabilize the channel when in an open state, which is consistent with the prolonged sodium tail currents induced by pyrethroids and DDT. In the closed state, the predicted alternative positioning of the domain II S4-S5 linker would result in disruption of pyrethroid-binding contacts, consistent with the observation that pyrethroids have their highest affinity for the open channel. The model also predicts a key role for the IIS5 and IIIS6 helices in insecticide binding. Some of the residues on the helices that form the putative binding contacts are not conserved between arthropod and non-arthropod species, which is consistent with their contribution to insecticide species selectivity. Additional binding contacts on the II S4-S5 linker can explain the higher potency of pyrethroid insecticides compared with DDT.

Amino Acid Sequence↗
PubMed1999

Pseudomonas syringae pv tomato induces the expression of tomato EREBP-like genes pti4 and pti5 independent of ethylene, salicylate and jasmonate

The tomato genes Pti4 and Pti5 encode ethylene-responsive element binding protein-like transcription factors that bind to the GCC box, a conserved cis-element in many defense-related genes. The Pti proteins have previously been shown to interact with the tomato disease resistance protein Pto. Here we report that the expression of both Pti4 and Pti5 are induced by a virulent strain of Pseudomonas syringae pv tomato. The expression of Pti5 is further enhanced by the interaction of the Pto gene in tomato and the corresponding avrPto gene in the bacterium. The enhancement of Pti5 expression by Pto-avrPto interaction requires a functional Prf gene in the plant. Pti5 appears to be expressed specifically during biotic stresses, suggesting a specific role in plant defense. Pti4 and several EREBP-like genes are induced by ethylene, salicylate and wounding. However, the Pseudomonas bacterium induced a wild-type level of Pti4 and Pti5 transcripts in tomato plants carrying the nahG transgene, the Nr mutation, or the def1 mutation. In addition, the ethylene action inhibitor norbornadiene did not inhibit the induction of Pti4 and Pti5 either in the compatible or incompatible interactions. The results suggest that the Pseudomonas bacterium induces Pti4 and Pti5 expression through a pathway independent of salicylic acid, ethylene and jasmonic acid.

Journal Article↗
PubMed1995

Inhibition of insulin receptor phosphorylation by PC-1 is not mediated by the hydrolysis of adenosine triphosphate or the generation of adenosine.

Individuals with insulin resistance show increased levels of PC-1 expression in skeletal muscle and fibroblasts, and in transfected cell lines that overexpress PC-1 there is a reduction in the insulin-stimulated insulin receptor tyrosine phosphorylation. As PC-1 is a type II transmembrane protein with extracellular phosphodiesterase and pyrophosphatase activity, increased expression of PC-1 at the cell surface will decrease extracellular adenosine triphosphate levels and increase extracellular adenosine levels. Consequently it is possible that PC-1-mediated insulin resistance could be caused either by a decrease in adenosine triphosphate or an indirect increase in adenosine levels. We have tested this hypothesis and find that the PC-1-mediated inhibition of insulin-stimulated insulin receptor autophosphorylation is not altered by agents that alter the level or action of adenosine. Further, a mutated PC-1 with a single amino acid change that abolishes the phosphodiesterase and pyrophosphatase activities is still able to inhibit insulin-stimulated insulin receptor phosphorylation. The results of these experiments indicate that the phosphodiesterase activity of PC-1 is not involved in the inhibition of insulin receptor autophosphorylation.

Adenosine↗
PubMed1998

Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit.

Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.

Adenosine Triphosphatases↗
PubMed2004

Protein kinase C activation promotes the internalization of the human cationic amino acid transporter hCAT-1. A new regulatory mechanism for hCAT-1 activity.

The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gräf, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.

Animals↗
PubMed2006

Mechanisms of two modulatory actions of the channel-binding protein Slob on the Drosophila Slowpoke calcium-dependent potassium channel.

Slob57 is an ion channel auxiliary protein that binds to and modulates the Drosophila Slowpoke calcium-dependent potassium channel (dSlo). We reported recently that residues 1-39 of Slob57 comprise the key domain that both causes dSlo inactivation and shifts its voltage dependence of activation to more depolarized voltages. In the present study we show that removal of residues 2-6 from Slob57 abolishes the inactivation, but the ability of Slob57 to rightward shift the voltage dependence of activation of dSlo remains. A synthetic peptide corresponding in sequence to residues 1-6 of Slob57 blocks dSlo in a voltage- and dose-dependent manner. Two Phe residues and at least one Lys residue in this peptide are required for the blocking action. These data indicate that the amino terminus of Slob57 directly blocks dSlo, thereby leading to channel inactivation. Further truncation to residue Arg(16) eliminates the modulation of voltage dependence of activation. Thus these two modulatory actions of Slob57 are independent. Mutation within the calcium bowl of dSlo greatly reduces its calcium sensitivity (Bian, S., I. Favre, and E. Moczydlowski. 2001. Proc. Natl. Acad. Sci. USA. 98:4776-4781). We found that Slob57 still causes inactivation of this mutant channel, but does not shift its voltage dependence of activation. This result confirms further the independence of the inactivation and the voltage shift produced by Slob57. It also suggests that the voltage shift requires high affinity Ca(2+) binding to an intact calcium bowl. Furthermore, Slob57 inhibits the shift in the voltage dependence of activation of dSlo evoked by Ca(2+), and this inhibition by Slob57 is greater at higher free Ca(2+) concentrations. These results implicate distinct calcium-dependent and -independent mechanisms in the modulation of dSlo by Slob.

Amino Acids↗