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At least 1,603 records · Page 89Linked to original sources
PubMed2003

Adenosine A2A receptor agonists as anti-inflammatory agents.

Stressed or injured tissues release endogenous adenosine, which blocks potentially destructive inflammatory cascades by binding to A2A adenosine receptors (ARs) and decreasing activation of platelets, leukocytes and endothelial cells. In these tissues, adenosine acts by reducing expression of adhesion molecules and release of pro-inflammatory mediators (e.g., reactive oxygen species, elastase and tumor necrosis factor-alpha). Synthetic A2A selective AR agonists are currently undergoing preclinical testing for the treatment of allergen-induced inflammation, ischemia-reperfusion injury, sepsis and autoimmune diseases. This review discusses potential disease targets for A2A AR agonist treatment, mechanisms of A2A AR agonist action and considerations for synthetic A2A AR agonist design.

Adenosine↗
PubMed1987

[New therapeutic aspects in the treatment of diffuse peritonitis].

The diffuse peritonitis is a syndrome during the course of which biochemical reactive chains are activated according to a cascade principle. Most of these biochemical reactive chains develop within an acid environment. The solutions applied for abdominal lavage so far had neutral pH value. The present acute peritonitis experiment has shown that a peritoneal pH increase during lavage therapy stopped the septic reactive chain and thus improved significantly the rate of survival in the test animals. These experimental findings ar now subject for clinical examination.

Animals↗
PubMed1998

Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis.

In plants, a number of MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) homologues have been reported. However, there have been no reports of protein-protein interactions between these kinases or molecular analysis of MAPK cascades in higher plants. To analyze a possible MAPK cascade in Arabidopsis thaliana, we took two molecular approaches. One is the two-hybrid screening of ATMEKK1 (a MAPKKK)-interacting proteins; the other is an analysis of physical and functional interactions among isolated MAPK, MAPKK, and MAPKKK homologues from Arabidopsis. In two-hybrid screening using ATMEKK1 as bait, we isolated a novel MAPKK homologue, ATMKK2, a MAPK homologue, ATMPK4, and an unknown protein. ATMKK2 has high sequence similarity with MEK1 (a MAPKK) in Arabidopsis. Based on yeast two-hybrid analysis, we detected protein-protein interactions between ATMEKK1 and ATMKK2/MEK1 (MAPKKs), between ATMKK2/MEK1 and ATMPK4 (a MAPK), and between ATMPK4 and ATMEKK1. ATMPK4 and ATMKK2/MEK1 interacted with two distinct regions of ATMEKK1, the N-terminal regulatory domain and the C-terminal kinase domain, respectively. Coexpression of ATMEKK1 increased the ability of two closely related MAPKKs, ATMKK2 and MEK1, to complement a growth defect of the yeast pbs2 mutant. Coexpression of ATMPK4 and MEK1 complemented a growth defect of the yeast mpk1 and bck1 mutants. By contrast, other combinations of MAPKs and MAPKKs did not suppress these yeast mutations. These results suggest that ATMEKK1, ATMKK2/MEK1, and ATMPK4 may constitute a MAP kinase cascade.

Alanine↗
PubMed2004

RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences.

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.

Amino Acid Sequence↗
PubMed2002

Metalloproteinases stimulate ErbB-dependent ERK signaling in human skin organ culture.

To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5-24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97-98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41-65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade.

Blotting, Western↗
PubMed1999

A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor.

Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of beta-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1Delta sho1Delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade.

Amino Acid Sequence↗
PubMed2001

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator.

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.

Amino Acid Sequence↗
PubMed2001

Insulin induces specific interaction between insulin receptor and protein kinase C delta in primary cultured skeletal muscle.

Certain protein kinase C (PKC) isoforms, in particular PKCs beta II, delta, and zeta, are activated by insulin stimulation. In primary cultures of skeletal muscle, PKCs beta II and zeta, but not PKC delta, are activated via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. The purpose of this study was to investigate the possibility that PKC delta may be activated upstream of PI3K by direct interaction with insulin receptor (IR). Experiments were done on primary cultures of newborn rat skeletal muscle, age 5--6 days in vitro. The time course of insulin-induced activation of PKC delta closely paralleled that of IR. Insulin stimulation caused a selective coprecipitation of PKC delta with IR, and these IR immunoprecipitates from insulin-stimulated cells displayed a striking induction of PKC activity due specifically to PKC delta. To examine the involvement of PKC delta in the IR signaling cascade, we used recombinant adenovirus constructs of wild-type (W.T.) or dominant negative (D.N.) PKC delta. Overexpression of W.T.PKC delta induced PKC delta activity and coassociation of PKC delta and IR without addition of insulin. Overexpression of D.N.PKC delta abrogated insulin- induced coassociation of PKC delta and IR. Insulin-induced tyrosine phosphorylation of IR was greatly attenuated in cells overexpressing W.T.PKC delta, whereas in myotubes overexpressing D.N.PKC delta, tyrosine phosphorylation occurred without addition of insulin and was sustained longer than that in control myotubes. In control myotubes IR displayed a low level of serine phosphorylation, which was increased by insulin stimulation. In cells overexpressing W.T.PKC delta, serine phosphorylation was strikingly high under basal conditions and did not increase after insulin stimulation. In contrast, in cells overexpressing D.N.PKC delta, the level of serine phosphorylation was lower than that in nonoverexpressing cells and did not change notably after addition of insulin. Overexpression of W.T.PKC delta caused IR to localize mainly in the internal membrane fractions, and blockade of PKC delta abrogated insulin-induced IR internalization. We conclude that PKC delta is involved in regulation of IR activity and routing, and this regulation may be important in subsequent steps in the IR signaling cascade.

Acetophenones↗
PubMed1995

Modulation of cardiac sodium channel isoform by cyclic AMP dependent protein kinase does not depend on phosphorylation of serine 1504 in the cytosolic loop interconnecting transmembrane domains III and IV.

Both the neuronal IIA as well as the cardiac SkM2 isoform of the pore forming alpha-subunit of voltage dependent sodium channels are modulated by Protein Kinase A. While alphaIIA becomes attenuated upon PKA stimulation, alphaSkM2 becomes upregulated. PKC dependent phosphorylation of a serine, located in the highly conserved cytoplasmatic region between the third and the fourth transmembraneous domain has been found to be a prerequisite for PKA modulation of the alphaIIA isoform. We used site-directed mutagenesis, expression in Xenopus laevis oocytes and the two-electrode voltage clamp technique to test, whether phosphorylation of the corresponding serine in alphaSkM2 is required for the PKA modulation of also the cardiac isoform. The results clearly indicate that serine 1504 does not play a significant role in the PKA modulation of the cardiac sodium channel isoform, further underlining the differential modulation of the two isoforms by identical signal transduction cascades.

Animals↗
PubMed1992

Inhibition of alpha 2/alpha 3 sodium pump isoforms potentiates glutamate neurotoxicity.

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na+,K(+)-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (alpha 1) and high (alpha 2/alpha 3) affinity for ouabain, we were able to inhibit selectively the alpha 2 (principally glial) and alpha 3 (neuronal) catalytic subunits without affecting the alpha 1 isoform. Brief exposures (5-60 min) to high ouabain concentrations (1-10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the alpha 2/alpha 3 isoforms (by 1 microM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [3H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reductions in alpha 2/alpha 3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude that inhibition of neuronal NaP with high affinity for ouabain is not lethal to unstimulated cells, but markedly increases susceptibility to glutamate excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Influence of burn-induced lipid-protein complex on IL1 secretion by PBMC in vitro.

The lipid-protein complex (LPC) formed by thermal injury to skin, which has been shown to have a toxic effect on mice, and which suppresses the immune response, was tested for its specific influence on monocytes. Growth of bacterial endotoxin-stimulated peripheral blood mononuclear cells (PBMC) was inhibited in the presence of LPC, however, the inhibition was less at the time of the optimal rate of cell proliferation. Inhibition was proportional to LPC concentration. ConA-stimulated PBMC were also inhibited by LPC in a dose-related manner. PBMC, in the presence of LPC, secreted interleukin 1 (IL1) at an increasing rate as LPC concentration rose from 5 to 40 micrograms/ml, and the levels of IL1 which could be induced by endotoxin were increasingly amplified in the presence of LPC. In comparison to LPC, the native tissue proteins which were isolated from unburned skin by the same techniques which produced LPC from burned skin, were tested for their effect on PBMC. Native proteins had no effect on IL1 secretion, whether on background or endotoxin-stimulated levels. Thus, the thermally induced change in skin proteins has a specific effect on monocyte IL1 secretion which is not matched by the native proteins, indicating that burn injury to skin specifically affects the lymphokine cascade and consequent immune function.

Animals↗
PubMed2006

Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats.

Human truncated tau protein is an active constituent of the neurofibrillary degeneration in sporadic Alzheimer's disease. We have shown that modified tau protein, when expressed as a transgene in rats, induced AD characteristic tau cascade consisting of tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau complexes. These pathological changes led to the functional impairment characterized by a variety of neurobehavioural symptoms. In the present study we have focused on the behavioural alterations induced by transgenic expression of human truncated tau. Transgenic rats underwent a battery of behavioural tests involving cognitive- and sensorimotor-dependent tasks accompanied with neurological assessment at the age of 4.5, 6 and 9 months. Behavioural examination of these rats showed altered spatial navigation in Morris water maze resulting in less time spent in target quadrant (p<0.05) and fewer crossings over previous platform position (p<0.05) during probe trial. Spontaneous locomotor activity and anxiety in open field was not influenced by transgene expression. However beam walking test revealed that transgenic rats developed progressive sensorimotor disturbances related to the age of tested animals. The disturbances were most pronounced at the age of 9 months (p<0.01). Neurological alterations indicating impaired reflex responses were other added features of behavioural phenotype of this novel transgenic rat. These results allow us to suggest that neurodegeneration, caused by the non-mutated human truncated tau derived from sporadic human AD, result in the neuronal dysfunction consequently leading to the progressive neurobehavioural impairment.

Alzheimer Disease↗
PubMed1997

SH3 domain-dependent association of huntingtin with epidermal growth factor receptor signaling complexes.

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Bistability analyses of a caspase activation model for receptor-induced apoptosis.

Apoptosis is an important physiological process crucially involved in development and homeostasis of multicellular organisms. Although the major signaling pathways have been unraveled, a detailed mechanistic understanding of the complex underlying network remains elusive. We have translated here the current knowledge of the molecular mechanisms of the death-receptor-activated caspase cascade into a mathematical model. A reduction down to the apoptotic core machinery enables the application of analytical mathematical methods to evaluate the system behavior within a wide range of parameters. Using parameter values from the literature, the model reveals an unstable status of survival indicating the need for further control. Based on recent publications we tested one additional regulatory mechanism at the level of initiator caspase activation and demonstrated that the resulting system displays desired characteristics such as bistability. In addition, the results from our model studies allowed us to reconcile the fast kinetics of caspase 3 activation observed at the single cell level with the much slower kinetics found at the level of a cell population.

Apoptosis↗
PubMed1999

Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma.

Whether p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascades are required for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in RAW 264.7 murine macrophages exposed to lipopolysaccharide (LPS) plus recombinant interferon-gamma (rIFN-gamma) was investigated. By use of Western blotting for iNOS detection and ELISA for quantitation of TNF secretion, three selective inhibitors of these pathways were tested (the p38 inhibitors SB202190 and SB203580 and the MEK 1,2/ERK inhibitor PD98059). Dose-related inhibition of iNOS production was demonstrated when inhibitors were added 1 h before, simultaneously with, or 1 h after LPS plus rIFN-gamma stimulation. In contrast, inhibition of TNF secretion was observed only when cells were preincubated with these agents. Thus, both the p38 and ERK pathways are involved in the up-regulation of iNOS and TNF production by murine macrophages, and specific inhibitors of these pathways block macrophage iNOS production even when added 1 h after activation of these cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1979

Ineffectiveness of aprotinin on psoralen-UVA-(PUVA)-induced erythema.

Because bradykinin constitutes a possible candidate for mediation of topical 8-methoxypsoralen-UVA-(PUVA)-induced erythema, aprotinin (Trasylol), inhibitor of kallikrein and interrupter of the cascade leading to kinin production was assessed in guinea pigs. Response was assessed at 24, 48, and 72 hr after topical PUVA and there was no significant difference between normal saline and aprotinin by intradermal or intraperitoneal routes of administration. The results of this study indicate that intradermal and intraperiotoneal aprotinin, in the dose and method tested, is not capable of significantly decreasing erythema induced by topical PUVA in guinea pigs.

Animals↗
PubMed1996

Integrin-dependent induction of early growth response genes in capillary endothelial cells.

Studies were carried out to explore how extracellular matrix molecules, such as fibronectin (FN), promote capillary endothelial (CE) cell growth. When G0-synchronized cells were plated on FN-coated dishes, expression of the immediate-early mRNAs, c-fos, c-myc and c-jun, was rapidly induced, even in the absence of serum or soluble growth factors. Moreover, plating cells on different FN densities (5-200 micrograms/150 mm dish), resulted in a dose-dependent increase in the steady state levels of these mRNAs. Addition of FGF potentiated gene activation and was required for maximal DNA synthesis, however, the overall steady-state level of gene induction was dictated primarily by the density of immobilized FN. Expression of junB also was induced when suspended cells bound RGD-peptide coated microbeads that promote integrin clustering, but not when the suspended cells bound beads coated with other receptor ligands (e.g. acetylated low density protein) or when they were stimulated by soluble FN or FGF in the absence of substrate adhesion. c-Jun exhibited a similar requirement for gene induction except that it also was partially induced by binding to soluble FN alone. In contrast, c-fos expression was induced by all stimuli tested. Interestingly, inhibition of Na+/H+ exchange using hexamethylene-amiloride prevented most of the FN-induced increase in c-jun expression whereas it was relatively ineffective when cells were simultaneously stimulated by both FN and FGF. These data demonstrate that cell adhesion to extracellular matrix and associated integrin binding can directly activate signaling cascades in quiescent CE cells that lead to induction of immediate-early genes associated with the G0/G1 transition and thereby, stimulate these cells to reenter the growth cycle.

Animals↗