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PubMed2004

Genetic selection scheme for isolation of signal transduction pathway mutants.

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5'-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5'-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.

Cyanobacteria↗
PubMed2006

Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.

Cells, Cultured↗
PubMed2000

Two independent signaling pathways mediate the antiapoptotic action of macrophage-stimulating protein on epithelial cells.

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3'-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis. In contrast to the results with adherent cells, although MSP also prevented apoptosis of cells in suspension (anoikis), its effect was mediated only by the PI3-K/AKT pathway. Despite activation of MAPK by MSP, anoikis was not prevented in suspended cells with a blocked PI3-K/AKT pathway. Thus, activation of MAPK alone is not sufficient to mediate MSP antiapoptotic effects. Cell adhesion generates an additional signal, which is essential for MSP to use MAPK in an antiapoptotic pathway. This may involve translocation of MSP-activated MAPK from the cytoplasm into the nucleus, which occurs only in adherent cells. Our results suggest that there is cross talk between cell matrix adhesion and growth factors in the regulation of cell survival via the MAPK pathway. Growth factors induce MAPK activation, and adhesion mediates MAPK translocation from the cytoplasm into the nucleus.

Animals↗
PubMed1999

Decreased amplitude of left ventricular posterior wall motion with notch movement to determine the left posterior septal accessory pathway in Wolff-Parkinson-White syndrome.

OBJECTIVE: To determine preoperatively, by analysing asynchronous left ventricular wall motion, whether to approach through the right ventricle or the left ventricle when carrying out catheter ablation of the accessory pathway in Wolff-Parkinson-White syndrome, especially in patients with the pathway located on the septum. METHODS: 73 patients with manifest Wolff-Parkinson-White syndrome who underwent successful catheter ablation were studied. Location of accessory pathway was classified as right ventricular side: right anterior paraseptum, right anterior, right lateral, right posterior, anterior septum, midseptum, right posterior septum; left ventricular side: left posterior septum, left posterior, left lateral, left anterior. Asynchronous systolic wall motion was analysed by cross sectional echocardiography. RESULTS: Echocardiography showed that the amplitude of left ventricular posterior systolic wall motion was reduced when the pathway was located on the left ventricular side as opposed to the right ventricular side (mean (SD), 11.1 (1.7) v 12.9 (1.1) mm, p < 0.001), especially in patients with left posterior septal accessory pathway (9.7 (0.8) mm). There were no overlapping values between the left posterior septal accessory pathway and the right ventricular side accessory pathway. Posterior wall notch motion was observed in all patients with a left posterior septal accessory pathway (9/9), but not at all in patients with pathways located on the right ventricular side of the septum. In patients with a septal accessory pathway, an ECG algorithm provided poor information (relatively low sensitivity, specificity, and predictive value) for determining whether the subsite faced either the left (left posterior septum) or the right ventricle (anterior septum, midseptum, right posterior septum). CONCLUSIONS: Decreased amplitude of left ventricular posterior wall motion with notch movement is an important finding for accessory pathways located on the left posterior septum. These findings provided clinically useful information for determining whether to approach catheter ablation from the right or the left ventricle.

Adolescent↗
PubMed2005

Response of the ubiquitin-proteasome pathway to changes in muscle activity.

The ubiquitin-proteasome pathway plays a critical role in the adaptation of skeletal muscle to persistent decreases or increases in muscle activity. This article outlines the basics of pathway function and reviews what we know about pathway responses to altered muscle use. The ubiquitin-proteasome pathway regulates proteolysis in mammalian cells by attaching ubiquitin polymers to damaged proteins; this targets the protein for degradation via the 26S proteasome. The pathway is constitutively active in muscle and continually regulates protein turnover. Conditions of decreased muscle use, e.g., unloading, denervation, or immobilization, stimulate general pathway activity. This activity increase is caused by upregulation of regulatory components in the pathway and leads to accelerated proteolysis, resulting in net loss of muscle protein. Pathway activity is also increased in response to exercise, a two-phase response. An immediate increase in selective ubiquitin conjugation by constitutive pathway components contributes to exercise-stimulated signal transduction. Over hours-to-days, exercise also stimulates a delayed increase in general ubiquitin conjugating activity by inducing expression of key components in the pathway. This increase mediates a late-phase rise in protein degradation that is required for muscle adaptation to exercise. Thus the ubiquitin-proteasome pathway functions as an essential mediator of muscle remodeling, both in atrophic states and exercise training.

Animals↗
PubMed2006

A conserved RAS/mitogen-activated protein kinase pathway regulates DNA damage-induced cell death postirradiation in Radelegans.

Although the epidermal growth factor receptor (EGFR) signaling pathway is overactive in more than half of human cancers and mediates resistance to cytotoxic therapy, the molecular mechanisms of EGFR pathway-mediated resistance have remained elusive in cancer research. This difficulty partly stems from the lack of tissue models enabling clear separation of the many forms of cell death that the downstream signaling pathways of EGFR affect. We have created a model in Caenorhabditis elegans of radiation-induced reproductive cell death ("Radelegans") in isolation of all other forms of cell death. We have employed Radelegans to genetically define the role of the EGFR signaling pathway in protection from reproductive cell death, the primary form of tumor stem or clonogen cell death postirradiation. We have found that the RAS/mitogen-activated protein kinase (MAPK) downstream signal transduction pathway of EGFR is critical for protection from reproductive cell death in Radelegans. In addition, we have shown that RAS/MAPK pathway signaling is genetically linear with the DNA damage response pathway and acts downstream of the DNA damage checkpoint in the radioresponse, implicating this pathway in DNA repair post-cytotoxic therapy. These findings support the hypothesis that enhanced repair is a mechanism of RAS/MAPK pathway-mediated resistance to cytotoxic therapy through its interaction with the DNA damage response pathway postirradiation. We postulate that these findings also help explain why current treatment strategies, based on the presumption that tumors have ineffective repair compared with normal tissues, are ineffective in EGFR/RAS/MAPK pathway-mediated tumors. Radelegans is a platform to further define the genetic basis of the radiation response in tissues.

Animals↗
PubMed1977

An unusual variety of atrioventricular nodal re-entry due to retrograde dual atrioventricular nodal pathways.

Three patients with paroxysmal supraventricular tachycardia (PSVT) had discontinuous ventriculo-artrial conduction curves (V1-V2, A1-A2), suggesting dual A-V nodal pathways. Ventricular echoes occurred simultaneously with sudden increase of V-A interval. These echoes were characterized by retrograde P waves occurring in front of QRS, suggesting utilization of a slow pathway for retrograde conduction and a fast pathway for antegrade conduction. In case one, atropine improved retrograde slow pathway and antegrade fast pathway conduction and made A-V nodal re-entry sustained, resulting in PSVT (with retrograde P in front of the QRS). In cases 2 and 3, atropine markedly shortened retrograde fast pathway refractory period and slightly improved antegrade slow pathway conduction. The discontinuous V1-V2, A1-A2 curves and echoes were no longer demonstrable. However, with improvement of retrograde fast pathway and antegrade slow pathway conduction, A-V nodal re-entrant echoes and PSVT were observed, utilizing the slow pathway for antegrade conduction and the fast pathway for retrograde conduction (P simultaneous with QRS).

Adult↗
PubMed1982

Changes in ventriculoatrial intervals with bundle branch block aberration during reciprocating tachycardia in patients with accessory atrioventricular pathways.

During reciprocating tachycardia in patients with accessory atrioventricular pathways, the observation of changes in ventriculoatrial (VA) intervals with bundle branch block (BBB) aberration has been used to localize the site of the pathway and prove the participation of the pathway in the tachycardia. In this report we present the changes observed during BBB in 93 patients with single atrioventricular pathways in whom the site of their pathways was subsequently proved at the time of their surgical interruption. In patients with left or right free wall pathways, the minimum VA interval (VA min) increased by 61 +/- 19 msec with ipsilateral BBB, whereas no change occurred with contralateral BBB. The smallest increase in the VA interval was 35 msec. In 14 patients, shortening of the AH intervals resulted in changes in overall cycle length that were less than 35 msec. Patients with septal pathways all had changes in VA min of 25 msec or less with either right or left bundle branch block (RBBB or LBBB), which suggests that a clear differentiation between septal and free wall pathways can be made on the basis of changes in VA min. In patients with anteroseptal pathways, VA min intervals frequently prolonged with RBBB (16 +/- 9 msec) but not with LBBB. In patients with posteroseptal pathways, VA min frequently prolonged with LBBB (13 +/- 8 msec) but not with RBBB. Therefore, the observed changes in VA min with BBB may serve as an important indicator of the site of an accessory pathway and may provide guidance in the choice of surgical therapy.

Adolescent↗
PubMed1988

Atriofascicular connection or a nodoventricular Mahaim fiber? Electrophysiologic elucidation of the pathway and associated reentrant circuit.

Accessory pathways showing decremental properties and inserting into the right ventricle have been frequently described as "nodoventricular" or Mahaim pathways. However, conclusive evidence for a nodal origination of such pathways is lacking. The patient in this study had characteristics typical of such a pathway. Antegradely, the pathway showed decremental, nodelike conduction properties. With the aid of right bundle branch recordings, the pathway was demonstrated to insert directly into the right bundle branch. Atrioventricular reciprocating tachycardia could be readily initiated by atrial or ventricular pacing. The QRS morphology was normal during sinus rhythm and demonstrated a left bundle branch block pattern with normal axis during tachycardia. The reentrant circuit involved antegrade conduction over the accessory pathway and retrograde conduction via the bundle branches, His bundle, and the atrioventricular node. More significantly, late atrial stimuli delivered during tachycardia could preexcite the ventricle via the accessory pathway despite their inability to enter the atrioventricular node. Thus, the upper "turn around" of the reentrant circuit involved atrial tissue and the accessory pathway originated directly from the right atrium independent of the atrioventricular node. In view of these new findings and other recent observations during surgical resection of similar pathways, a reassessment of previous descriptions of "nodoventricular" fibers may be necessary. Many of these pathways may actually represent atriofascicular or atrioventricular connections with decremental properties.

Adolescent↗
PubMed1991

Catheter ablation using radiofrequency current to cure symptomatic patients with tachyarrhythmias related to an accessory atrioventricular pathway.

BACKGROUND: Recent investigations have shown that cure of patients with symptomatic tachyarrhythmias related to an accessory atrioventricular pathway may be achieved by closed-chest electrode catheter ablation of the accessory connection. Direct current shocks have primarily been used for this purpose, but its applicability is limited because of the lack of controlled titration of electrical energy, the infliction of barotrauma, and the need for general anesthesia. Radiofrequency current has been proposed as an alternate energy source. METHODS AND RESULTS: Seventy-three symptomatic patients with Wolff-Parkinson-White syndrome and 19 patients with only retrogradely conducting (concealed) pathways underwent ablative therapy with radiofrequency current. There were 71 accessory pathways located on the left side of the heart (57 free-wall and 14 posteroseptal pathways) and 25 on the right side (11 free-wall, seven posteroseptal, and seven midseptal or anteroseptal pathways). In patients with right-sided pathways, ablation was attempted via a catheter positioned at the atrial aspect of the tricuspid annulus. In patients with a left-sided free-wall accessory pathway, a novel approach was used in which the ablation catheter was positioned in the left ventricle directly below the mitral annulus. Accessory pathway conduction was permanently abolished in 79 patients (86%). Growing experience and improved catheter technology resulted in a 100% success rate after the 52nd consecutive patient. Failures were mainly the result of inadequate catheters used initially or an unfavorable approach to left posteroseptal pathways. CONCLUSIONS: Catheter ablation of accessory atrioventricular pathways by the use of radiofrequency current is an effective and safe therapeutic modality for patients with symptomatic tachyarrhythmias mediated by these pathways.

Adult↗
PubMed1993

Radiofrequency endocardial catheter ablation of accessory atrioventricular pathway atrial insertion sites.

BACKGROUND: High rates of success using radiofrequency ablation energy have rapidly transformed catheter ablation from an investigational procedure to the nonpharmacological therapy of choice for symptomatic Wolff-Parkinson-White syndrome. Prior studies of radiofrequency accessory pathway ablation were based on a ventricular approach. Risks associated with prolonged arterial catheter manipulation, retrograde left ventricular catheterization, and production of ventricular lesions required for successful ventricular insertion ablation can be avoided using atrial insertion ablation procedures. The purpose of the present study was to define the safety and efficacy of accessory pathway ablation using radiofrequency energy delivered solely to accessory atrioventricular pathway atrial insertion sites. METHODS AND RESULTS: One hundred fourteen patients with accessory pathway-mediated tachycardia underwent attempted radiofrequency current ablation at the accessory pathway atrial insertion site. All catheters were introduced transvenously. Left-sided accessory pathways were approached using transseptal left atrial catheterization techniques. Retrograde localization of the atrial insertion site during reentrant tachycardia was characterized by 40 +/- 15-msec local ventriculoatrial and 79 +/- 17-msec surface QRS to local atrial electrogram intervals. Presumed accessory pathway potentials were present in only 30% of ablation site electrograms. Successful ablation required 6.2 +/- 5.3 radiofrequency energy applications. Cumulative energy dose required for success was 2,341 +/- 2,233 J. There were no complications associated with transseptal catheterization. Energy delivery to accessory pathway atrial insertion sites was associated with non-life-threatening complications in two patients. Recurrent conduction requiring repeat ablation occurred in 10 of 115 (9%) successfully ablated accessory pathways, all within 1 month of the ablation procedure. After 21.2 +/- 4.6 months of follow-up, 108 of 114 (95%) patients are asymptomatic and without evidence of accessory pathway conduction. CONCLUSIONS: The atrial insertion approach to accessory pathway ablation is safe and highly effective. This approach compares favorably with the retrograde ventricular insertion ablation technique. Atrial insertion ablation eliminates the need to produce ventricular lesions and avoids the risks of prolonged arterial catheter manipulation and retrograde left ventricular catheterization.

Adult↗
PubMed1995

Characterization of atrioventricular nodal behavior and ventricular response during atrial fibrillation before and after a selective slow-pathway ablation.

BACKGROUND: The presence of atrioventricular nodal dual-pathway physiology in patients with atrioventricular nodal reentrant tachycardia (AVNRT) provides an opportunity to characterize the effect of a selective slow-pathway ablation on the ventricular rate during atrial fibrillation (AF). This may have important clinical implications for the nonpharmacological management of AF with a rapid ventricular rate. METHODS AND RESULTS: Selective radiofrequency catheter ablation of the atrioventricular nodal slow pathway was performed with a stepwise approach in patients with documented sustained AVNRT. The AV nodal conduction properties and refractoriness and the ventricular rate during induced AF were assessed at baseline and under autonomic blockade before and after a selective slow-pathway ablation in 18 patients (mean age, 34 +/- 8 years). Sustained AVNRT was induced with a mean cycle length of 339 +/- 58 ms. A slow-pathway ablation was successfully achieved with 5 +/- 4 applications of radiofrequency energy. The shortest cycle length of 1:1 AV conduction and the AV nodal effective refractory period significantly prolonged after ablation (367 +/- 53 versus 403 +/- 55 ms, P < .0001, and 258 +/- 55 versus 292 +/- 74 ms, P < .05, respectively). Selective slow-pathway ablation significantly prolonged the mean (526 +/- 93 versus 612 +/- 107 ms, P < .0001), the shortest (378 +/- 59 versus 423 +/- 73 ms, P < .0001), and the longest (826 +/- 150 versus 969 +/- 226 ms, P < .01) cycle lengths of the ventricular response to AF. Significant slowing of the ventricular rate during AF occurred in 13 patients (72%), including all eight patients in whom AV nodal dual-pathway physiology was abolished. Five patients did not have a significant change in the ventricular rate during AF; a persistent dual AV nodal pathway physiology was demonstrable in four of these patients. Loss of dual-pathway physiology after ablation had a sensitivity of 77%, specificity of 80%, and positive predictive value of 91% for slowing the ventricular rate during AF. CONCLUSIONS: In patients undergoing a slow-pathway ablation for control of AVNRT, selective slow-pathway ablation may cause a significant decrease in the ventricular rate during AF. These effects are primarily due to the prolongation of AV nodal conduction properties and refractory period of the residual AV nodal transmission system. These findings may have important therapeutic implications for the nonpharmacological treatment of AF, particularly in patients with underlying dual AV nodal physiology.

Adult↗
PubMed2003

Stress and radiation-induced activation of multiple intracellular signaling pathways.

Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFalpha and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFbeta and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Animals↗
PubMedDate not supplied

Combining pathway analysis with flux balance analysis for the comprehensive study of metabolic systems.

The elucidation of organism-scale metabolic networks necessitates the development of integrative methods to analyze and interpret the systemic properties of cellular metabolism. A shift in emphasis from single metabolic reactions to systemically defined pathways is one consequence of such an integrative analysis of metabolic systems. The constraints of systemic stoichiometry, and limited thermodynamics have led to the definition of the flux space within the context of convex analysis. The flux space of the metabolic system, containing all allowable flux distributions, is constrained to a convex polyhedral cone in a high-dimensional space. From metabolic pathway analysis, the edges of the high-dimensional flux cone are vectors that correspond to systemically defined "extreme pathways" spanning the capabilities of the system. The addition of maximum flux capacities of individual metabolic reactions serves to further constrain the flux space and has led to the development of flux balance analysis using linear optimization to calculate optimal flux distributions. Here we provide the precise theoretical connections between pathway analysis and flux balance analysis allowing for their combined application to study integrated metabolic function. Shifts in metabolic behavior are calculated using linear optimization and are then interpreted using the extreme pathways to demonstrate the concept of pathway utilization. Changes to the reaction network, such as the removal of a reaction, can lead to the generation of suboptimal phenotypes that can be directly attributed to the loss of pathway function and capabilities. Optimal growth phenotypes are calculated as a function of environmental variables, such as the availability of substrate and oxygen, leading to the definition of phenotypic phase planes. It is illustrated how optimality properties of the computed flux distributions can be interpreted in terms of the extreme pathways. Together these developments are applied to an example network and to core metabolism of Escherichia coli demonstrating the connections between the extreme pathways, optimal flux distributions, and phenotypic phase planes. The consequences of changing environmental and internal conditions of the network are examined for growth on glucose and succinate in the face of a variety of gene deletions. The convergence of the calculation of optimal phenotypes through linear programming and the definition of extreme pathways establishes a different perspective for the understanding of how a defined metabolic network is best used under different environmental and internal conditions or, in other words, a pathway basis for the interpretation of the metabolic reaction norm.

Glucose↗
PubMed2002

Effect of temperature on rates of alternative and cytochrome pathway respiration and their relationship with the redox poise of the quinone pool.

We investigated the effect of short-term changes in temperature on alternative (Alt) and cytochrome (Cyt) pathway respiration, both in intact tissues and isolated mitochondria of 14-d-old cotyledons of soybean (Glycine max L. cv Stevens). We also established the extent to which temperature alters the interaction between the oxidizing pathways and the level of ubiquinone (UQ) reduction (UQ(r)/UQ(t)). No difference was found between the temperature coefficient of respiration (Q(10); proportional change per 10 degrees C) of Alt and Cyt pathway respiration in cotyledon slices (Q(10) = 1.92 and 1.86, respectively). In isolated mitochondria, the Q(10) of the fully activated Alt pathway (Q(10) = 2.24-2.61) was always equal to, or higher than, that of Cyt c oxidase (COX) alone (Q(10) = 2.08) and the complete Cyt pathway (Q(10) = 2.40-2.55). This was true regardless of substrate or whether ADP was present. There was little difference in the Q(10) of the Cyt pathway with or without ADP; however, the Q(10) of COX was substantially lower in the presence of an uncoupler (Q(10) = 1.61) than its absence (Q(10) = 2.08). The kinetics of Alt and Cyt pathway activity in relation to UQ(r)/UQ(t) were not affected by temperature. For a given UQ(r)/UQ(t) value, the proportion of maximum flux taking place was similar at all temperatures for both pathways (+/-ADP). However, the Q(10) of the Alt and the Cyt pathways (+ADP) increased with increasing UQ(r)/UQ(t). We conclude that the Alt pathway is not less temperature sensitive than the Cyt pathway or COX per se and that changes in the degree of control exerted by individual steps in the respiratory apparatus could result in changes in the Q(10) of mitochondrial O(2) uptake.

Adenosine Diphosphate↗
PubMed1992

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMedDate not supplied

[The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons].

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.

Cytochromes↗
PubMedDate not supplied

[Influenza viruses and intracellular signalling pathways].

It has been described in the last years that after influenza virus-infection a variety of intracellular signalling pathways have been induced. There are examples and suggestions how the viral replication cycle leads to the activation of intracellular signalling pathways. A variety of signalling pathways are activated after virus-infection as an alert-response against the invading pathogen that can be considered as an antiviral response of the host cell. Nevertheless, it was also shown, that viruses are able to suppress these cellular responses to assure their own replication. Moreover, viruses are also able to activate and misuse cellular signalling pathways for their own survival. The NF-kappaB signalling pathway is an excellent example of these sceneries. Activation of the NF-kappaB signalling pathway mediated by the virus can partially be blocked by the NS1 protein to suppress a strong antiviral IFN alpha/beta response. At the same time the virus takes advantage of the remaining NF-kappaB activity for virus related apoptosis and for its own replication. This is a highly effective and economic way for the virus to control its replication without the need for specific viral inducers of cellular responses. This demonstrates, that there are no "all or nothing" reactions in the field of interactions of Influenza viruses with intracellular signalling pathways. In one situation cellular antiviral responses can be misused by the virus of its own replication and at another point the same signalling pathway may even be turned into a pro-viral activity. When the impact of a given signalling pathway on viral growth is evaluated these bivalent functions of these pathways should be taken in consideration. Nevertheless, a signalling pathway that supports viral growth is an excellent target for antiviral therapy (Ludwig et al. 2003).

Animals↗