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PubMed2003

The balance between pro-apoptotic and anti-apoptotic pathways in the failing myocardium.

The purpose of this review is to highlight those circulating molecules, membrane receptors, and signaling pathways that initiate, potentiate, or conversely, inhibit apoptosis within cardiomyocytes. This review focuses on pathways directly related to the failing heart and discusses the limitations of current methodologies for assessing cardiomyocellular apoptosis. It is important to note that the adrenergic, reactive oxygen species, and proinflammatory cytokine signaling pathways are not the only pro-apoptotic pathways active in the myocardium, nor are IL-6-related cytokine, calcineurin, and IGF-1/PI3K/Akt signaling pathways the only anti-apoptotic pathways active in the myocardium. However, they are among the best-characterized apoptosis-mediating pathways and therefore they may serve as foundation for future studies aimed at identifying novel apoptotic regulating pathways active in cardiomyocytes. Considering the short history of studying cardiomyocellular apoptosis, a tremendous body of knowledge has been collected. Understandably, much more work remains. Tomorrow's studies must (1) continue to examine the signaling pathways mediating cardiomyocellular apoptosis by focusing on the links to the ubiquitous apoptosis effectors, (2) use the expanding body of knowledge to develop more specific inhibitors of apoptosis, and then (3) confirm the causal relationship of cardiomyocellular apoptosis and cardiac dysfunction in physiologic models of cardiac challenge.

Animals↗
PubMed2004

Opposing extracellular signal-regulated kinase and Akt pathways control Schwann cell myelination.

Schwann cells are the myelinating glia of the peripheral nervous system, and their development is regulated by various growth factors, such as neuregulin, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). However, the mechanism of intracellular signaling pathways following these ligand stimuli in Schwann cell differentiation remains elusive. Here, we demonstrate that in cultured Schwann cells, neuregulin and PDGF suppressed the expression of myelin-associated protein markers, whereas IGF-I promoted it. Although these ligands activated common downstream signaling pathways [i.e., extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways], the profiles of activation varied among ligands. To elucidate the function of these pathways and the mechanisms underlying Schwann cell differentiation, we used adenoviral vectors to selectively activate or inactivate these pathways. We found that the selective activation of Erk pathways suppressed Schwann cell differentiation, whereas that of PI3K pathways promoted it. Furthermore, lithium chloride, a modulator of glycogen synthase kinase-3beta (GSK-3beta) promoted Schwann cell differentiation, suggesting the involvement of GSK-3beta as a downstream molecule of PI3K-Akt pathways. Selective activation of PI3K pathways in Schwann cells by gene transfer also demonstrated increased myelination in in vitro Schwann cell-DRG neuron cocultures and in vivo allogenic nerve graft experiments. We conclude that signals mediated by PI3K-Akt are crucial for initiation of myelination and that the effects of growth factors are primarily dependent on the balance between Erk and PI3K-Akt activation. Our results also propose the possibility of augmenting Schwann cell functions by modulating intracellular signals in light of future cell therapies.

Animals↗
PubMed1991

Conduction properties of the antegrade fast and slow AV nodal pathways associated with AV nodal reentrant tachycardia.

To elucidate differences in conduction properties among the normal atrioventricular (AV) node and the antegrade fast and slow dual AV nodal pathways (DAVNPW), AV nodal conduction curves were analyzed quantitatively in 38 patients. Eighteen patients had antegrade DAVNPW with AV nodal reentrant tachycardia (AVNRT) (dual pathways group) and the remaining 20 had smooth AV nodal conduction curves, without evidence of AV nodal dysfunction (control group). The effective refractory period (ERP) of the antegrade fast pathway was longer than that of the normal AV node (at both basic cycle lengths of 700 and 500 msec, p less than 0.01). Although the atrial premature beats were delayed by a longer ERP in the fast pathway, there was no significant difference in the degree of prolongation of AV nodal conduction time related to shortening of the coupling interval (i.e., ratio of A2H2 increment to A1A2 decrement) between these two pathways. On the other hand, the ERP of the antegrade slow pathway was similar to that of the normal AV node. The degree of prolongation of AV nodal conduction time (relative to the shortening of the coupling interval) was greater in the antegrade slow pathway than in the normal AV node. In conclusion, these findings suggest that in DAVNPW with AVNRT: (1) the antegrade fast pathway is similar to the AV node and its conduction properties are unlikely to be better than those of the normal AV node and (2) the antegrade slow pathway has quantitatively poorer conduction properties than the normal AV node, since it has a greater degree of decremental conduction.

Adolescent↗
PubMed1986

Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat.

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6 O2'-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3.5 mmol/l), significantly (P less than 0.01) increased aldosterone output by at least 1.5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P less than 0.01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 +/- 14 vs 105 +/- 19 pmol/10(6) cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion. Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P less than 0.01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine. These observations suggest that: the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

Adrenal Cortex↗
PubMed2026

Integrative Cross-platform Analysis of Kinase Inhibitor Effects on Statin-relevant Cardioprotective Pathways in Human Cardiomyocytes.

BACKGROUND/AIM: Kinase inhibitors (KIs) can cause cardiotoxicity through mechanisms overlapping with statin cardioprotective pathways, yet their effects on these pathways in cardiomyocytes remain uncertain. We evaluated six literature-defined statin-relevant gene sets using transcriptomic and proteomic data. MATERIALS AND METHODS: Pre-ranked gene set enrichment analysis was performed for 23 KIs in primary cardiac cells (GSE146096; n=319) and iPSC-derived cardiomyocytes (GSE217421; n=541), with cross-platform analysis of 21 KIs by shotgun proteomics (PXD014791; n=300). Pathway-specific concordance was assessed by Spearman correlation with Benjamini-Hochberg correction; protein scores were estimated after adjustment for cell line. RESULTS: KI effects were heterogeneous. The anti-fibrotic pathway showed nominal concordance across the two transcriptomic datasets (ρ=0.495, p=0.016, q=0.098; 91% direction concordance) and significant cell-line-adjusted transcriptomic-proteomic concordance (ρ=0.644, p=0.0016, q=0.0081). Nilotinib reproducibly upregulated NF-κB pathway genes [normalized enrichment score (NES)=+2.29 and +2.18 in discovery and validation], with targeted inter-gene-correlation-adjusted testing supporting higher NF-κB expression than under rosuvastatin (CAMERA p=3.54×10-8). No global cross-omics summary remained significant after harmonizing pathway universes and accounting for repeated pathways. CONCLUSION: KI effects on statin-relevant pathways were pathway-specific. Anti-fibrotic concordance and nilotinib-associated NF-κB upregulation are hypothesis-generating candidates for experimental validation.

Humans↗
PubMedDate not supplied

Targeting apoptosis pathways in cancer therapy.

Apoptosis, or programmed cell death, is a mechanism by which cells undergo death to control cell proliferation or in response to DNA damage. The understanding of apoptosis has provided the basis for novel targeted therapies that can induce death in cancer cells or sensitize them to established cytotoxic agents and radiation therapy. These novel agents include those targeting the extrinsic pathway such as tumor necrosis factor-related apoptosis-inducing ligand receptor 1, and those targeting the intrinsic Bcl-2 family pathway such as antisense bcl-2 oligonucleotides. Many pathways and proteins control the apoptosis machinery. Examples include p53, the nuclear factor kappa B, the phosphatidylinositol 3 kinase pathway, and the ubiquitin/proteosome pathway. These can be targeted by specific modulators such as bortezomib, and mammalian target of rapamycin inhibitors such as CCI-779 and RAD 001. Because these pathways may be preferentially altered in tumor cells, there is potential for a selective effect in tumors sparing normal tissue. This article reviews the current understanding of the apoptotic pathways, including the extrinsic (cytoplasmic) and intrinsic (mitochondrial) pathways, and the agents being developed to target these pathways.

Antibodies, Monoclonal↗
PubMed2006

Essential role for activation of the Polycomb group (PcG) protein chromatin silencing pathway in metastatic prostate cancer.

The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.

Adenocarcinoma↗
PubMed1999

The p16-cyclin D1/CDK4-pRb pathway and clinical outcome in epithelial ovarian cancer.

A significant positive association has been reported between p16 expression and clinical outcome for epithelial ovarian cancer patients. However, there is a reciprocal correlation between genetic alterations of single members of the p16-cyclin D1/CDK4-pRb pathway (G1 pathway). Simultaneous evaluation of these four elements may produce a better prognostic factor than p16 alone. We studied the prognostic significance of the G1 pathway in 59 epithelial ovarian cancer patients undergoing surgery and platinum-based chemotherapy by immunohistochemical technique. Abnormal expression of p16 or pRb was defined by negative nuclei staining, and that of CDK4 and cyclin D1 was defined by 50% nuclear staining. An abnormal G1 pathway was indicated in cases that have at least one abnormality among these four elements. Abnormal expression of p16, pRb, and cyclin D1/CDK4 was observed in 33.9, 3.4, and 15.3% of studied cases, respectively. Abnormal G1 pathway was detected in 49.2% (29 of 59) of all cases. The patients with normal G1 pathway tended to achieve a higher complete response rate (81.0%) to chemotherapy, compared with patients with abnormal G1 pathway (55.0%); however, there was no significant difference (P = 0.1001) between the two groups. Univariate analyses identified advanced stage [hazards ratio (HR), 3.665; P = 0.0218], histological low grade (HR, 3.625; P = 0.0066), and abnormal G1 pathway (HR, 2.935; P = 0.03) as prognostic factors for overall survival. The G1 pathway might help as a prognostic factor to select high-risk patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.

Animals↗
PubMed2000

Repolarization abnormalities after catheter ablation of differently located overt accessory pathways.

PURPOSE: Electrical repolarization abnormalities are usually seen after radiofrequency catheter ablation in overt accessory atrio-ventricular pathways. These abnormalities have been recognised as "cardiac memory". The aim of this study was to assess the relationships between electrical repolarization abnormalities and the location of the accessory atrioventricular pathway in the atrio-ventricular junction. We also assessed the relationship between electrical repolarization abnormalities and the degree of ventricular preexcitation. MATERIAL AND METHODS: Our group consisted of 45 patients with overt and persistent preexcitation who were successfully submitted to radiofrequency catheter ablation. Mean age was 38.9 +/- 14.9 years (14 to 64), 28 of whom (62.2%) were male and 17 (37.8%) female. The location of accessory pathways the was left lateral in 14 patients, right posteroseptal in 14 patients, right mesoseptal in eight patients, right anteroseptal in four patients and other locations in the remaining four. Surface electrocardiograms were performed immediately following ablation, 24 hours later and three or more months after the procedure. RESULTS: T wave abnormalities (flattened, inverted or peaked) were detected on the first day in 51% of patients, being more frequent after right accessory pathway ablation (86.9% versus 13.1% in left accessory pathways). These electrocardiographic changes were particularly frequent in patients with right mesoseptal (5/8, 62.5%) and posteroseptal (13/14, 93.3%) pathways, of which 62.5% and 71.5%, respectively, showed marked preexcitation before ablation. These findings were significantly different from those observed in patients with left lateral pathways--only 35.7% showed marked preexcitation and only two of fourteen patients (14.3%) presented T wave abnormalities after ablation. After the third month, most of the patients that had undergone successful ablation presented completed electrocardiographic normalization. CONCLUSIONS: Patients undergoing radiofrequency catheter ablation for right accessory pathways show more frequently repolarization abnormalities than those submitted to ablation of the left lateral accessory pathways. The occurrence of these changes after preexcitation resolution may depend on the degree of previous preexcitation.

Adolescent↗
PubMed2001

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway.

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.

Animals↗
PubMed2003

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Adult↗
PubMed2004

Characterization of the roles of STAT1 and STAT3 signal transduction pathways in mammalian lens development.

PURPOSE: IGF-1 and PDGF are implicated in regulating lens proliferation and/or providing spatial cues that restrict lens proliferation to germinative and transition zones of the lens. However, very little is known about how IGF-1- or PDGF-induced signals are transduced and coupled to gene transcription in lens cells. Therefore, we examined whether these growth factors mediate their effects in the lens through the evolutionarily conserved JAK/STAT signal transduction pathway and if STAT signaling is essential for mammalian lens development. METHODS: Expression of STAT1 and STAT3 was analyzed in mouse lens and lens epithelial cells by RT-PCR and western blot analysis. Activation of the STAT signaling pathway was examined by a combination of gel-shift, super-shift, and western blotting assays. Regulation of lens proliferation and gene transcription by STAT pathways was assessed by 3H-Thymidine incorporation or RT-PCR assays with lens explants treated or untreated with Genistein or the JAK2 and STAT3 inhibitor, AG-490. Mice with targeted deletion of STAT3 in the lens were generated by Cre/lox recombination and STAT1-/-, STAT3-/- deficient as well as normal lenses were characterized by histology. RESULTS: We show that PDGF and IGF1 induce proliferation in 1AMLE6 lens cells and couple their extracellular signals to gene transcription, in part through activation of STAT3 and to a lesser extent STAT1 signal transduction pathways. We further show that targeted deletion of STAT3 in E10.5 lens does not produce overt developmental lens defects. STAT1 knockout mice also exhibit a normal lens phenotype. CONCLUSIONS: Our results showing that deletion of either STAT1 or STAT3 does not affect the normal development of the lens is surprising in view of the fact that STAT pathways are activated in developing chick or mouse lens and inappropriate activation of STAT1 pathway in the lens by ectopic lens expression of IFN? inhibits lens differentiation and induces cataract in transgenic mice. Our data thus suggest that although STAT-signaling pathways may contribute to activation of gene transcription in the lens, it may not be essential for normal lens development or STAT proteins may be functionally redundant during lens development. However, because several growth factors and cytokines present in the lens activate STATs in mouse lens explants and 1AMLE6 lens epithelial cells, it may well be that this evolutionarily conserved signaling pathway is under stringent control in the mammalian lens. Whereas deficiency in any particular STAT pathway can be compensated for by any of the functionally redundant STAT proteins induced by a wide array of growth factors in the lens, chronic or prolonged activation of a particular STAT protein may perturb homeostatic balance in STAT-dependent growth factor signaling, culminating in pathologic lens changes.

Animals↗
PubMed1988

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways.

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.

Adenosine Diphosphate↗
PubMed1989

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01.

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.

Calcium↗
PubMed1995

Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways.

The Ca(2+)-dependent phosphatase calcineurin, a target of FK506 and CsA, synergizes with PKC-induced activation of nuclear factor (NF)-kappa B in T cell lines. We have investigated whether this synergy is present in other cell types and the mechanism(s) by which these two pathways lead to NF-kappa B activation. While this synergy is present in other cell types, in the monocytic cell line U937 calcineurin is also sufficient to activate NF-kappa B. Having previously shown that Ca(2+)- and PKC-dependent pathways synergize by accelerating the degradation of IkB alpha, we focused on the regulation of IkB alpha phosphorylation. While PKC-dependent pathways sequentially result in the phosphorylation and in an incomplete degradation of IkB alpha in T cell lines, co-activation of Ca(2+)-dependent pathways accelerates the rate of IkB alpha phosphorylation and results in its complete degradation. Activation of Ca(2+)-dependent pathways alone do not result in the phosphorylation and/or degradation of IkB alpha in Jurkat T or in U937 cells. Treatment of T cells with the selective PKC inhibitor GF109203X abrogates the PMA-induced IkB alpha phosphorylation/degradation irrespective of activation of Ca(2+)-dependent pathways, but not the phosphorylation and degradation of IkB alpha induced by TNF-alpha, a PKC-independent stimulus. Contrary to the interaction with PKC, Ca(2+)-dependent pathways synergize with TNF-alpha not at the level of IkB alpha phosphorylation, but at the level of its degradation. These results indicate that Ca(2+)-dependent pathways, including the phosphatase calcineurin, participate in the regulation of NF-kappa B in a cell specific fashion and synergize with PKC-dependent and -independent pathways at the level of IkB alpha phosphorylation and degradation.

Animals↗
PubMed1976

Activation of the alternative complement pathway in systemic lupus erythematosus.

Serum factors activating the alternative pathway of complement in vitro, independent of classical pathway activation was demonstrated in six of eleven patients with systemic lupus erythematosus (SLE). These serum factors were detected by lysis of gluthathione-sensitized human erythrocytes and by C3 and factor B conversion in the presence of EGTA (10 mM) and MgCl2 (0-3 mM), conditions which blocked activation of the classical pathway but permitted activation of the alternative pathway. In order to determine if in vivo activation of the alternative pathway of complement was present in SLE, highly-purified factor B was labelled with radioactive iodine (125I), and its metabolism studied in the eleven patients with SLE and in twelve control subjects. All six patients with serum factors capable of activating the alternative pathway in vitro, had in vivo evidence of alternative pathway activation as measured by increased fractional catabolic rate (FCR) of factor B. Two patients without demonstrable alternative pathway activating factors in their sera had an elevated FCR of factor B. Six of the patients with increased FCR of factor B had disease limited to skin or joint and one had lupus nephritis which was inactive at the time of study. One of the four patients who were in clinical remission had elevated FCR. This study demonstrates that a significant number of patients with SLE of relatively mild disease activity had evidence of alternative complement pathway activation. This activation did not appear to be limited to patients with lupus nephritis and raises the possibility that it could also be related to some of the extra-renal manifestations of SLE.

Adolescent↗
PubMed1996

[Ablation of the slow pathway in reciprocating nodal rhythms by radiofrequency].

Radiofrequency ablation is a therapeutic alternative to drug therapy in recurrent reciprocating nodal rhythms. Selective ablation of the slow pathway guided by endocavitary recordings has the advantage of being effective at the price of a very low incidence of atrioventricular block. The authors report their experience with this technique. Fifty consecutive patients with recurrent attacks which were syncopal or uncontrolled by medical therapy, underwent selective ablation of the slow pathway. Firstly, they all underwent electrophysiological investigation to confirm the nodal origin of the reciprocating rhythm before proceeding to ablation itself, guided by the search for the slow pathway potentials. After ablation, it was impossible to trigger reciprocating tachycardia. Interruption of the anterograde slow pathway was achieved in 24 patients and of the retrograde pathway in 1 patient : the other 25 patients went on having dual conduction but with a prolongation of the effective refractory period of the slow pathway (268 +/- 46 ms vs 251 +/- 41 ms : p < 0.01). There were no cases of permanent complete atrioventricular block. Interruption of the slow pathway was associated with shortening of the effective refractory period of the rapid pathway (323 +/- 71 ms vs 348 +/- 80 ms : p < 0.01), which was not observed in cases of persistent dual conduction. No recurrence of tachycardia was observed during follow-up (mean period = 19.2 months) : however, control endocavitary studies in 25 asymptomatic patients after 1 to 3 months showed recurrence in 4 cases, which led to immediate further radiofrequency ablation which was successful. The authors conclude that guided selective radiofrequency of the slow intranodal pathway is a remarkably effective and reliable method of treating poorly tolerated or resistant reciprocating nodal tachycardias. Widening of the clinical indications to patients not wishing to undergo long-term antiarrhythmic therapy is now possible.

Adolescent↗