Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pathways”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,747 records · Page 97Linked to original sources
PubMed1998

Evidence for the shikimate pathway in apicomplexan parasites.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Nucleolus contains signal molecules that constitute membrane-nucleolus linked pathway.

Since there have been very few studies on nucleolar signaling, an attempt was made to establish nucleolar signal pathways which link the cell membrane to the nucleolus for the transfer of extracellular signals. Two pathways were studied. One was the G alpha s mediated cAMP pathway where two signal molecules were yielded, including RII and protein kinase A. The other was the G alpha q mediated DAG/IP3 pathway which yields two signals including protein kinase C and IP3/Ca2+. By the studying isolated nucleoli from resting liver, regenerating liver or weak carcinogen thioacetamide treated liver, it was possible to detect protein kinase A (PKA), protein kinase C (PKC) and RII subunits. In addition, CK2 was detected. It was found that external signals transmitted through G protein coupled receptors could reach into the nucleolus and that physical translocation of signal molecules was an integral step involved in membrane-nucleolus linked pathways. When an in vitro assay of the above signal molecules was carried out using [gamma-32P]-ATP, most kinase dependent phosphorylation was via the major CK2 (more than 95%). Therefore, it is suggested that the major CK2 dependent pathway is involved in 'house keeping' for nucleolar integrity and the minor pathways, dependent on PKA, PKC and others, are involved in subtle regulatory mechanisms such as 'extra-house-keeping' activities by nucleolar chromosomal remodeling.

Animals↗
PubMed2001

Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways.

Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathway activated by phosphorylating and thereby inhibiting glycogen synthase kinase 3 (GSK3). In contrast, in addition to demonstrating that calcineurin does not mediate IGF-1-induced hypertrophy, we show that IGF-1 unexpectedly acts via Akt to antagonize calcineurin signalling during myotube hypertrophy.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo.

Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude that the activation of the Akt/mTOR pathway and its downstream targets, p70S6K and PHAS-1/4E-BP1, is requisitely involved in regulating skeletal muscle fibre size, and that activation of the Akt/mTOR pathway can oppose muscle atrophy induced by disuse.

Animals↗
PubMed2003

Parallel protein-unfolding pathways revealed and mapped.

Theoretical studies of protein folding suggest that multiple folding pathways should exist, but there is little experimental evidence to support this. Here we demonstrate changes in the flux between different transition states on parallel folding pathways, resulting in unprecedented upward curvature in the denaturant-dependent unfolding kinetics of a beta-sandwich protein. As denaturant concentration increases, the highly compact transition state of one pathway becomes destabilized and the dominant flux of protein molecules shifts toward another pathway with a less structured transition state. Furthermore, point mutations alter the relative accessibility of the pathways, allowing the structure of two transition states on separate, direct folding pathways to be mapped by systematic Phi-value analysis. It has been suggested that pathways with diffuse rather than localized transition states are evolutionarily selected to prevent misfolding, and indeed we find that the transition state favored at high concentrations of denaturant is more polarized than the physiologically relevant one.

Connectin↗
PubMed2003

Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells.

Activated Ras utilises several downstream pathways, including the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway and the phosphoinositide 3-kinase (PI-3k)/Akt pathway, to promote cell proliferation and to inhibit apoptosis. To investigate which pathway plays a major role in Ras-induced drug resistance to chemotherapeutic agents in breast cancer cells, we transfected MCF7 breast cancer cells with a constitutively active H-RasG12V and examined the toxicities of three commonly used breast cancer chemotherapeutic agents, paclitaxel, doxorubicin, and 5-fluorouracil in these cells under the conditions that PI-3K or MEK were selectively inhibited by their respective specific inhibitors or dominant negative expression vectors. We found that Ras-mediated drug resistance is well correlated with resistance to apoptosis induced by anticancer agents in MCF7 breast cancer cells. Although inhibition of MEK/MAPK or PI-3K/Akt can each enhance the cytotoxicity of paclitaxel, doxorubicin, or 5-fluorouracil, inhibition of the PI-3K/Akt pathway seems to have a greater effect than inhibition of the MEK/MAPK pathway in reversing Ras-mediated drug resistance. Our results indicate that the PI-3K pathway may play a more important role in receptor tyrosine kinase-mediated resistance to chemotherapy and suggest that PI-3K/Akt might be a critical target molecule for anticancer intervention in breast cancer.

Antineoplastic Agents↗
PubMed1997

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide.

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNgamma) 100 u ml(-1), interleukin-1beta (IL-1beta) 1 u ml(-1) and lipopolysaccharide (LPS) 10 microg ml(-1) induced nitrite formation which could be inhibited by the competitive NOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME). IL-1beta alone (1-50 u ml(-1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNgamma 100 u ml(-1), IL-1beta 1 u ml(-1) and LPS 10 microg ml(-1) to induce both the iNOS and COX-2 pathways, and IL-1beta 3 u ml(-1) to induce COX-2 without iNOS activity. 3. Cells treated with IFNgamma 100 u ml(-1), IL-1beta I u ml(-1) and LPS 10 microg ml(-1) for 48 h either alone, or with the addition of L-NAME (0 to 10(-2) M), demonstrated inhibition by L-NAME of PGE2 (3.61 +/- 0.55 to 0.51 +/- 0.04 pg/l0(4) cells; P<0.001) and nitrite (34.33 +/- 8.07 to 0 pmol/10(4) cells; P<0.001) production. Restoration of the PGE2 response (0.187 +/- 0.053 to 15.46 +/- 2.59 pg/10(4) cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10(-6) M, but with the addition of the NOS substrate L-arginine (0 to 10(-5) M). 4. Cells incubated with IL-1beta 3 u ml(-1) for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10(-4) M), demonstrated increased PGE2 formation (1.23 +/- 0.03 to 2.92 +/- 0.19 pg/10(4) cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 microM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1beta 3 u ml(-1) for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10(-3) M) also demonstrated an increased PGE2 response (2.56 +/- 0.21 to 4.53 +/- 0.64 pg/10(4) cells; P<0.05). 5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

Cytokines↗
PubMed2006

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

Animals↗
PubMed1999

Ultraviolet radiation mutagenesis of hedgehog pathway genes in basal cell carcinomas.

The identification of mutations in Hedgehog (HH) pathway genes in some basal cell carcinomas (BCC) and the detection of HH pathway dysregulation in almost all BCC confirms the importance of this developmental regulatory pathway in human BCC tumorigenesis. Moreover, the occurrence of UVB signature mutations in key HH pathway genes in BCC provides the first genetic evidence that UV radiation (UVR) may be the principal mutagen involved in BCC tumorigenesis. We review herein current advances in the understanding of the role of the HH pathway in BCC tumorigenesis including transgenic and knock-out animal models of HH pathway dysregulation. Furthermore, we summarize abnormalities in other tumor suppressors and oncogenes including ras and p53 and evidence for interactions between these regulatory genes and the HH pathway.

Animals↗
PubMed1998

The roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway; a potential route to radio- and chemo-sensitization of tumor cells resulting in the induction of apoptosis and loss of clonogenicity.

During the last 10 years, multiple signal transduction pathways within cells have been discovered. These pathways have been linked to the regulation of many diverse cellular events such as proliferation, senescence, differentiation and apoptosis. This review will focus upon the many roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway. Recent evidence suggests that signaling by the MAP kinase pathway can both enhance proliferation by increased expression of molecules such as cyclin D1, but also cause growth arrest by increased expression of molecules such as the cyclin kinase inhibitor protein p21(Cip-1/MDA6/WAF1). These differential effects on growth have been correlated to the amplitude and duration of the MAP kinase activity signal. Furthermore several laboratories are reporting data suggesting that inhibition of the MAP kinase pathway, as well as a family of upstream MAP kinase activators, the protein kinase C family, represent an important route to both radio- and chemo-sensitization of tumor cells. Herein, we describe the historical discovery and characterization of the MAP kinase pathway. In addition we describe potential mechanisms by which inhibition of protein kinase C, the MAP kinase pathway, and potentially of p21(Cip-1/MDA6/WAF1) expression, may alter the sensitivities of leukemic and carcinoma cells to cytotoxic insults, leading to increased apoptosis and loss of clonogenicity.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma.

The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases, whereas p19ARF targets MDM2, a p53 inhibitory protein, for degradation. A deletion of CDKN2A would therefore disturb both pathways. To explore the p53 pathway genes as a functional unit in diffuse large B cell non-Hodgkin's lymphomas (DLCL), we wanted to see whether there exists mutually exclusiveness of aberrations of CDKN2A, MDM2 and p53, since this has not been analysed previously. We investigated 37 DLCL for aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigenetic, genetic and/or protein levels. Homozygous deletion of CDKN2A was detected in seven (19%) of 37 tumours, and another three cases were hypermethylated at the 5' CpG island of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue for p16 confirmed these results, as all tumours with alterations of CDKN2A were p16 immunonegative. We found p53 mutations in eight (22%) cases and MDM2 overexpression in 16 (43%) tumours. Twenty-three (62%) tumours had alterations of one or more p53 pathway components (p53, p19ARF and MDM2). Furthermore, 7/9 (78%) p16-immunonegative tumours showed co-aberration of p53 and/or MDM2. The lack of correlation between these aberrations suggests that DLCL acquire additional growth advantage by inactivating both of these critical regulatory pathways.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2000

Concurrent disruption of p16INK4a and the ARF-p53 pathway predicts poor prognosis in aggressive non-Hodgkin's lymphoma.

The INK4a/ARF locus at chromosome 9p21 encodes two structurally and functionally distinct molecules with tumor-suppressive properties. p16INK4a controls cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein (Rb), while ARF prevents MDM2-mediated degradation of p53. By using a panel of PCR-based methods, we have examined the status of the p16INK4a, ARF and p53 genes in 123 cases of non-Hodgkin's lymphoma (NHL) at diagnosis. Alterations of one or more of these genes were detected in seven of 36 (19%) cases with low- to intermediate-grade histology, and in 35 of 87 (40%) cases with aggressive histology. For the aggressive lymphomas, the Kaplan-Meier estimate of overall survival for cases with disruption of either p16INK4a or the ARF-p53 pathway was not different from cases with retention of both pathways (5 year survival 45% vs 35%; P= 0.85), suggesting that selective inactivation of one of the pathways does not significantly influence overall survival. By contrast, the 5-year survival was only 7% for cases with concurrent disruption of p16INK4a and the ARF-p53 pathway vs 38% for cases with retention of one or both pathways (P = 0.005). Similar results were obtained when the analysis was confined to diffuse large B cell lymphomas (P= 0.019). On stepwise multivariate regression analysis including factors from the international prognostic index, concurrent disruption of p16INK4a and the ARF-p53 pathway was an independent negative prognostic factor in NHL with aggressive histology (P = 0.006). Our results suggest that the compound status of the p16INK4a and ARF-p53 pathways is a major determinant of outcome in NHL.

ADP-Ribosylation Factors↗
PubMed2003

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia.

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

Animals↗
PubMed1998

p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.

p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1971

Importance of the pentose phosphate pathway for D-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis.

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically (14)C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase-transaldolase pathway. 2. From the correlation of the (14)CO(2) liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-(14)C]- and d-[6-(14)C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C(2) fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO(2): about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.

Carbon Dioxide↗
PubMed1981

Control of the flux in the arginine pathway of Neurospora crassa. Modulations of enzyme activity and concentration.

The influence of particular enzyme activities on the flux of metabolites in a pathway can be estimated by 'modulating' enzymes (i.e. changing turnover or concentration) and measuring the response in various parts of the system. By controlling the nuclear ration of two genetically different nuclear types in heterokaryons, the enzyme concentrations at four different steps in the arginine pathway were decreased over a range. This range was extended by the use of bradytrophs, mutant strains specifying enzymes with greatly diminished enzyme activities. Strains altered simultaneously at more than one step were also constructed by genetic recombination. By measuring the outputs of the pathway and the steady-state concentrations of intermediate pools, the fluxes in different parts of the pathway were calculated. This allowed the construction of flux/enzyme relationships, the slope of which is a measure of the sensitivity of a flux to the change in enzyme activity at that step. All fluxes were found to be considerably buffered for quite substantial decreases in the activities of all enzymes. Mass action plays an important part in this phenomenon, as do inhibition and repression. Because of the existence of expansion fluxes in growing systems, we find quantitatively different fluxes in different parts of the single pathway. For the same reason some enzyme modulations given decreased fluxes in one part and increased fluxes in another. The understanding of control in the pathway thus involves consideration of many mechanisms operating simultaneously and the estimation of changes in the whole system. The concept of a 'rate-limiting step' is found to be inadequate and is replaced by a quantitative measure, the Sensitivity Coefficient, which takes account of all the interactions. It is shown that control of the flux is shared among all the enzymes of the pathway. The results are discussed in terms of the theory of flux control.

Arginine↗
PubMed2004

Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells.

Agonists that deplete intracellular Ca2+ stores also activate Ca2+ entry, although the mechanism by which store release and Ca2+ influx are linked is unclear. A potential mechanism involves 'store-operated channels' that respond to depletion of the intracellular Ca2+ pool. Although SOCE (store-operated Ca2+ entry) has been considered to be the principal route for Ca2+ entry during hormonal stimulation of non-electrically excitable cells, recent evidence has suggested that alternative pathways activated by metabolites such as arachidonic acid are responsible for physiological Ca2+ influx. It is not clear whether such messenger-activated pathways exist in all cells, whether they are truly distinct from SOCE and which metabolites are involved. In the present study, we demonstrate that HeLa cells express two pharmacologically and mechanistically distinct Ca2+ entry pathways. One is the ubiquitous SOCE route and the other is an arachidonate-sensitive non-SOCE. We show that both these Ca2+ entry pathways can provide long-lasting Ca2+ elevations, but that the channels are not the same, based on their differential sensitivity to 2-aminoethoxydiphenyl borate, LOE-908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate] and gadolinium. In addition, non-SOCE and not SOCE was permeable to strontium. Furthermore, unlike SOCE, the non-SOCE pathway did not require store depletion and was not sensitive to displacement of the endoplasmic reticulum from the plasma membrane using jasplakinolide or ionomycin pretreatment. These pathways did not conduct Ca2+ simultaneously due to the dominant effect of arachidonate, which rapidly curtails SOCE and promotes Ca2+ influx via non-SOCE. Although non-SOCE could be activated by exogenous application of arachidonate, the most robust method for stimulation of this pathway was application of the widely used calmodulin antagonist calmidazolium, due to its ability to activate phospholipase A2.

Arachidonic Acid↗
PubMed1986

Vitamin K antagonism of coumarin anticoagulation. A dehydrogenase pathway in rat liver is responsible for the antagonistic effect.

In the liver, it appears that there are two different pathways for vitamin K reduction. One pathway is irreversibly inhibited by coumarin anticoagulant drugs. The other pathway has been shown in the present study to be composed of enzymes that are not effected by physiological 'in vivo' concentrations of these drugs. This pathway appears to be responsible for the antidotal effect of vitamin K in overcoming coumarin poisoning. In rat liver the pathway has been shown to be composed of DT-diaphorase (EC.1.6.99.2) and a microsomal dehydrogenase(s). The activity of the microsomal dehydrogenase(s) was 3.6-fold higher with NADH than with NADPH present in the test system. It appears that this enzyme is the physiologically important enzyme in the pathway. In contrast with DT-diaphorase, this enzyme(s) is shown to be tightly associated with the mirosomal membrane. The enzyme(s) is not identical with either of the quinone-reducing enzymes cytochrome P-450 reductase or cytochrome-b5 reductase. Our data thus postulate the existence of an as-yet-unidentified microsomal dehydrogenase that appears to have an important function in the pathway.

4-Hydroxycoumarins↗