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PubMed1994

The synergy of pathways and algorithms: two tools work better than one.

BACKGROUND: Clinical quality improvement efforts at the Medical Center Hospital of Vermont (MCHV) led to the development of critical pathways, which show the ideal plan of care, and algorithms, which help clinicians make one of many complicated decisions within a plan of care. A synergy appears to develop when pathways and algorithms are used together. DEVELOPMENT OF PATHWAYS AND ALGORITHMS: A steering committee supports and oversees pathway and algorithm efforts. A quarterly tracking report updating progress for all pathways and algorithms is circulated to all nurse managers, medical staff, and administrators. When combining pathways and algorithms, the pathway is created first. Algorithms are developed for trouble spots within a pathway. CORONARY ARTERY BYPASS GRAFT (CABG) PATIENTS: Having developed the pathway for CABG patients, the CABG case management team meets monthly to review outcomes and variances. For example, an algorithm for managing atrial arrhythmias--the chief cause of variance for one month's results--was developed. The combination of pathways and algorithms for CABG patients has resulted in a reduction of 2.5 days for total length of stay (including 1 day on the surgical intensive care unit [SICU]), for a mean cost savings of $3,500. Re-admission to the SICU, reintubation, and mortality rates have all decreased. CONCLUSION: The idea of reaping the benefits of both pathways and algorithms is becoming more popular at MCHV, where teams use algorithms to improve on complicated processes underlying the pathways.

Algorithms↗
PubMed2001

Beta-carotene redox reactions in photosystem II: electron transfer pathway.

A carotenoid (Car), a chlorophyll (Chl(Z)), and cytochrome b(559) (Cyt b(559)) are able to donate electrons with a low quantum yield to the photooxidized chlorophyll, P680(+), when photosystem II (PSII) is illuminated at low temperatures. Three pathways for electron transfer from Cyt b(559) to P680(+) are considered: (a) the "linear pathway" in which Cyt b(559) donates via Chl(Z) to Car, (b) the "branched pathway" in which Cyt b(559) donates via Car and where Chl(Z) is also able to donate to Car, and (c) the "parallel pathway" where Cyt b(559) donates to P680 without intermediate electron carriers and electron donation from Chl(Z) and Car occurs by a competing pathway. Experiments were performed using EPR and spectrophotometry in an attempt to distinguish among these pathways, and the following observations were made. (1) Using PSII with an intact Mn cluster in which Cyt b(559) was preoxidized, Car oxidation was dominant upon illumination at < or =20 K, while electron donation from Chl dominated at >120 K. (2) When Cyt b(559) was prereduced, its light-induced oxidation occurred at < or =20 K in what appeared to be all of the centers and without the formation of a detectable Car(+) intermediate. The small and variable quantity of Car(+) photoinduced in these experiments can be attributed to the residual centers in which Cyt b(559) remained oxidized prior to illumination. (3) The relative rates for irreversible electron donation from Cyt b(559) and Car were determined indirectly at 20 K by monitoring the flash-induced loss of charge separation (i.e., the accumulation of Cyt b(559)(+)Q(A)(-) or Car(+)Q(A)(-)). Similar yields per flash were observed (13% for Cyt b(559) and 8% for Car), indicating similar donation rates. The slightly lower yield with Car as a donor is attributed at least in part to slow charge recombination occurring from the Car(+)Q(A)(-) radical pair in a fraction of centers. (4) Light-induced oxidation of Cyt b(559) and Car at 20 K was monitored directly by EPR, and the rates were found to be indistinguishable. The parallel pathway predicts that when both Cyt b(559) and Car are prereduced, the relative amounts of Cyt b(559)(+) and Car(+) produced upon illumination at 20 K should depend directly on their relative electron donation rates. The measured similarity in the donation rates thus predicts comparable yields of oxidation for both donors. However, what is observed experimentally is that Cyt b(559) oxidation occurs almost exclusively, and this argues strongly against the parallel pathway. The lack of Car(+) as a detectable intermediate is attributed to rapid electron transfer from Cyt b(559) to Car(+). The trapping of Car(+) at low temperature when Cyt b(559) is preoxidized but its absence when Cyt b(559) is prereduced is taken as an argument against the simple linear pathway. Overall, the data reported here and previously favor the branched pathway over the linear pathway, while the parallel pathway is thought to be unlikely. Structural considerations provide further arguments in favor of the branched model.

Carotenoids↗
PubMed2005

Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.

In the absence of subunit III the aa(3)-type cytochrome c oxidase exhibits a shortened catalytic life span (total number of turnovers) due to an increased probability of undergoing irreversible inactivation during steady-state turnover. Inactivation results from structural alteration of the heme a(3)-Cu(B) active site in subunit I [Hosler (2004) Biochim. Biophys. Acta 1655, 332-339]. The absence of subunit III also dramatically slows proton uptake to the active site via the D proton pathway, as well as inhibiting the proton backflow/exit pathway that connects the active site/proton pump with the outer surface of the oxidase complex. Here we demonstrate that these phenomena are linked: slow proton delivery to the active site through these pathways induces suicide inactivation, thus shortening the catalytic life span of the enzyme. Mutations that inhibit the D pathway, but not the K pathway, increase the probability of suicide inactivation. Strong inhibition of the D pathway allows suicide inactivation to occur even in the presence of subunit III. Arachidonic acid, which stimulates proton uptake by the D pathway, retards suicide inactivation. Steady-state turnover in the presence of DeltaPsi and DeltapH, which inhibits proton uptake from the inner surface of the protein, enhances suicide inactivation. Simultaneous inhibition of proton uptake from both sides of the protein by a double mutation affecting the D pathway and the proton backflow/exit pathway greatly shortens the catalytic life span of the oxidase even in the presence of subunit III. Thus, maintenance of rapid proton transfer through the D pathway and the backflow/exit pathway is one mechanism by which subunit III normally functions to prevent suicide inactivation of cytochrome c oxidase. The experiments suggest that increased lifetimes of the heme a(3) oxoferryl intermediates as well as the anionic form of Glu286 of the D pathway cause suicide inactivation in the active site.

Animals↗
PubMed2003

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention.

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

Drug Design↗
PubMed1987

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.

Animals↗
PubMed1996

Galpha12 and Galpha13 regulate extracellular signal-regulated kinase and c-Jun kinase pathways by different mechanisms in COS-7 cells.

Many growth factors and agonists for G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways, including the extracellular signal-regulated kinase (ERK) pathway and the c-Jun kinase (JNK) pathway. Transient transfection of dominant negative and constitutively active pathway components in COS-7 cells shows that two G protein subunits, Galpha12 and Galpha13, inhibit the ERK pathway and stimulate the JNK pathway. Constitutively active (GTPase-deficient) Galpha12 and Galpha13 both inhibit ERK pathway activation by epidermal growth factor. A Galpha13/alphaz chimera, which responds to stimulation by Gi-coupled receptors, mediates inhibition of ERK via such a receptor, the dopamine-2 receptor. In addition, expression of a dominant negative mutant of the GTPase, Cdc42, blocks activation of the JNK pathway by Galpha12 and Galpha13 but does not alter inhibition of ERK activation by the same Galpha proteins; conversely, mutationally activated Cdc42 stimulates the JNK pathway but has no effect on the ERK pathway. Our results show that different mechanisms mediate two effects of Galpha12 and Galpha13: the ERK pathway inhibition is mediated at the level of MAP kinase kinase in a Ras- and Raf-independent fashion, whereas the JNK pathway stimulation is mediated by Cdc42.

Animals↗
PubMed2006

Nonparametric pathway-based regression models for analysis of genomic data.

High-throughout genomic data provide an opportunity for identifying pathways and genes that are related to various clinical phenotypes. Besides these genomic data, another valuable source of data is the biological knowledge about genes and pathways that might be related to the phenotypes of many complex diseases. Databases of such knowledge are often called the metadata. In microarray data analysis, such metadata are currently explored in post hoc ways by gene set enrichment analysis but have hardly been utilized in the modeling step. We propose to develop and evaluate a pathway-based gradient descent boosting procedure for nonparametric pathways-based regression (NPR) analysis to efficiently integrate genomic data and metadata. Such NPR models consider multiple pathways simultaneously and allow complex interactions among genes within the pathways and can be applied to identify pathways and genes that are related to variations of the phenotypes. These methods also provide an alternative to mediating the problem of a large number of potential interactions by limiting analysis to biologically plausible interactions between genes in related pathways. Our simulation studies indicate that the proposed boosting procedure can indeed identify relevant pathways. Application to a gene expression data set on breast cancer distant metastasis identified that Wnt, apoptosis, and cell cycle-regulated pathways are more likely related to the risk of distant metastasis among lymph-node-negative breast cancer patients. Results from analysis of other two breast cancer gene expression data sets indicate that the pathways of Metalloendopeptidases (MMPs) and MMP inhibitors, as well as cell proliferation, cell growth, and maintenance are important to breast cancer relapse and survival. We also observed that by incorporating the pathway information, we achieved better prediction for cancer recurrence.

Adult↗
PubMed1996

Pathways of glycosphingolipid biosynthesis in SW13 cells in the presence and absence of vimentin intermediate filaments.

We reported previously that the incorporation of sugars into glycosphingolipids (GSL) is diminished in SW13 cells that lack a vimentin intermediate filament (IF) network (vim-) compared to vim+ cells. To further analyze the nature of this abnormality, we double-labeled cells with 3H-serine and 14C-sugars. There was no difference between vim+ and vim- cells in the incorporation of serine into GSL, although the usual difference in sugar incorporation was observed. This indicated that the defect in vim- cells was not in the incorporation of sugars into ceramide synthesized de novo by acylation of sphinganine (pathway 1). Sugars can also be incorporated into ceramide synthesized from sphingosine that is derived from catabolism of sphingolipids (pathway 2), and into GSL that recycle through the Golgi apparatus from endosomes (pathway 3). The amount of galactose and glucosamine incorporated into GSL in these three pathways was analyzed by the use of two inhibitors of sphingolipid biosynthesis. beta-Chloroalanine inhibits the de novo synthesis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2). We were surprised to observe that in both vim+ and vim- cells only 20-40% of sugar incorporation into GSL took place in pathway 1, and 60-80% of sugar incorporation took place in the recycling pathways. Moreover, in contrast to larger GSL, GlcCer was not synthesized in pathway 3. Our observations indicate that vimentin IF facilitate the recycling of GSL and sphingosine, and that the differences between vim+ and vim- cells are predominantly in pathways 2 and 3. Furthermore, although it is generally believed that virtually all GSL are synthesized in the de novo pathway, these data indicate that the recycling pathways predominate in the incorporation of sugars into GSL in SW13 cells.

Acylation↗
PubMed1994

The electrophysiologic effects of volatile anesthetics and sufentanil on the normal atrioventricular conduction system and accessory pathways in Wolff-Parkinson-White syndrome.

BACKGROUND: The effects of volatile agents and sufentanil anesthesia on the electrophysiologic properties of the accessory pathway and on the incidence of intraoperative tachyarrhythmias in patients with Wolff-Parkinson-White syndrome are unknown. Therefore, we studied these agents for their use in patients undergoing ablative procedures or requiring a general anesthetic for other surgeries. METHODS: Twenty-one patients with Wolff-Parkinson-White syndrome undergoing surgical ablation were anesthetized with sufentanil (20 micrograms/kg), lorazepam (0.06 mg/kg), and vecuronium (20 mg). After sternotomy, the electrophysiologic study during antegrade stimulation consisted of the effective refractory period of the right atrium, atrioventricular node, and accessory pathway; the shortest cycle length of the atrioventricular node and accessory pathway; and the coupling interval. During retrograde stimulation, the effective refractory period of the right ventricle and accessory pathway and the shortest cycle length of the accessory pathway were measured and compared to preoperative electrophysiologic values. Patients then were randomized to receive 1 MAC of halothane, isoflurane, or enflurane, and the electrophysiologic study was repeated. RESULTS: Sufentanil-lorazepam caused mild prolongation (P < 0.05) of the effective refractory period of the accessory pathway and the shortest cycle length of the atrioventricular node. Enflurane and isoflurane significantly prolonged all parameters related to refractoriness during antegrade conduction, with enflurane having the largest effect. During retrograde conduction, isoflurane prolonged the effective refractory period of the right ventricle and accessory pathway and the shortest cycle length of the accessory pathway, whereas enflurane prolonged only the accessory pathway effective refractory period and shortest cycle length. Halothane had the least effect on refractoriness, causing significant prolongation of the atrioventricular node effective refractory period and the shortest cycle length of the accessory pathway only during antegrade conduction. The coupling interval, a measure of the period of vulnerability to supraventricular tachycardia, was prolonged only by halothane and isoflurane. Supraventricular tachycardia was still obtainable in all patients. CONCLUSIONS: Sufentanil-lorazepam has no clinically significant effect on the electrophysiologic expression of the accessory pathway. Of the volatile agents, enflurane most, isoflurane next, and halothane least increased refractoriness within the accessory and atrioventricular pathways. Therefore, administration of these volatile agents during ablative procedures may confound interpretation of postablative studies used to determine the success of ablation treatment. Conversely, in patients with preexcitation syndrome requiring general anesthesia for nonablative procedures, volatile agents may reduce the incidence of perioperative tachyarrhythmias because of their effects on refractoriness. Enflurane would be the agent of choice because it increases refractoriness the most without prolonging the coupling interval.

Adolescent↗
PubMed1998

Differential effect of adenosine on anterograde and retrograde fast pathway conduction in patients with atrioventricular nodal reentrant tachycardia.

INTRODUCTION: Several studies have shown that the fast pathway is more responsive to adenosine than the slow pathway in patients with AV nodal reentrant tachycardia. Little information is available regarding the effect of adenosine on anterograde and retrograde fast pathway conduction. METHODS AND RESULTS: The effects of adenosine on anterograde and retrograde fast pathway conduction were evaluated in 116 patients (mean age 47 +/- 16 years) with typical AV nodal reentrant tachycardia. Each patient received 12 mg of adenosine during ventricular pacing at a cycle length 20 msec longer than the fast pathway VA block cycle length and during sinus rhythm or atrial pacing at 20 msec longer than the fast pathway AV block cycle length. Anterograde block occurred in 98% of patients compared with retrograde fast pathway block in 62% of patients (P < 0.001). Unresponsiveness of the retrograde fast pathway to adenosine was associated with a shorter AV block cycle length (374 +/- 78 vs 333 +/- 74 msec, P < 0.01), a shorter VA block cycle length (383 +/- 121 vs 307 +/- 49 msec, P < 0.001), and a shorter VA interval during tachycardia (53 +/- 23 vs 41 +/- 17 msec, P < 0.01). CONCLUSION: Although anterograde fast pathway conduction is almost always blocked by 12 mg of adenosine, retrograde fast pathway conduction is not blocked by adenosine in 38% of patients with typical AV nodal reentrant tachycardia. This indicates that the anterograde and retrograde fast pathways may be anatomically and/or functionally distinct. Unresponsiveness of VA conduction to adenosine is not a reliable indicator of an accessory pathway.

Adenosine↗
PubMed2006

A modelling approach to quantify dynamic crosstalk between the pheromone and the starvation pathway in baker's yeast.

Cells must be able to process multiple information in parallel and, moreover, they must also be able to combine this information in order to trigger the appropriate response. This is achieved by wiring signalling pathways such that they can interact with each other, a phenomenon often called crosstalk. In this study, we employ mathematical modelling techniques to analyse dynamic mechanisms and measures of crosstalk. We present a dynamic mathematical model that compiles current knowledge about the wiring of the pheromone pathway and the filamentous growth pathway in yeast. We consider the main dynamic features and the interconnections between the two pathways in order to study dynamic crosstalk between these two pathways in haploid cells. We introduce two new measures of dynamic crosstalk, the intrinsic specificity and the extrinsic specificity. These two measures incorporate the combined signal of several stimuli being present simultaneously and seem to be more stable than previous measures. When both pathways are responsive and stimulated, the model predicts that (a) the filamentous growth pathway amplifies the response of the pheromone pathway, and (b) the pheromone pathway inhibits the response of filamentous growth pathway in terms of mitogen activated protein kinase activity and transcriptional activity, respectively. Among several mechanisms we identified leakage of activated Ste11 as the most influential source of crosstalk. Moreover, we propose new experiments and predict their outcomes in order to test hypotheses about the mechanisms of crosstalk between the two pathways. Studying signals that are transmitted in parallel gives us new insights about how pathways and signals interact in a dynamical way, e.g., whether they amplify, inhibit, delay or accelerate each other.

Monte Carlo Method↗
PubMed2006

Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400.

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the DeltabenABCD::kan mutant), the boxC pathway (the DeltaboxABC::kan mutant), and both pathways (the DeltabenABCDDelta boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the DeltabenABCD::kan and DeltabenABCD DeltaboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the DeltabenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

Bacterial Proteins↗
PubMed2004

Regulatory network connecting two glucose signal transduction pathways in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

Base Sequence↗
PubMed1974

Two pathways of glutamate fermentation by anaerobic bacteria.

Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.

Acetates↗
PubMed1999

SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions.

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Base Sequence↗
PubMed2003

Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1.

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C(1) transfer between formaldehyde and formate, the tetrahydrofolate (H(4)F)-linked pathway and the tetrahydromethanopterin (H(4)MPT)-linked pathway. Both pathways are required for growth on C(1) substrates; however, mutants defective for the H(4)MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H(4)MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H(4)F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD(+) can successfully substitute for the H(4)MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H(4)MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H(4)MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.

Carbon↗
PubMed1991

Linking in accessory pathways. Functional loss of antegrade preexcitation.

BACKGROUND: Concealed retrograde activation has been proposed as a mechanism for antegrade conduction block in the bundle branches and atrioventricular accessory pathways. We studied this hypothesis (linking) in 10 patients with the Wolff-Parkinson-White syndrome in whom antegrade preexcitation could be persistently blocked by overdrive atrial pacing. METHODS AND RESULTS: An atrial pacing protocol, with a decremental ramp followed by an incremental ramp, defined a range of atrial paced cycle lengths (linking window) associated with both persistent conduction and block in the accessory pathway. Within the limits of the linking window, the ability of an atrial impulse to conduct over the accessory pathway was dependent on the preceding state (i.e., conduction or block). The observed linking window ranged from 70 to 290 msec (mean, 185 +/- 68 msec) and closely approximated the measured delay in retrograde activation of the accessory pathway during persistent antegrade block. The mean antegrade effective refractory period of the accessory pathways was long (486 +/- 156 msec), and in each case, it exceeded the antegrade refractory period of the normal atrioventricular pathway. Critically timed premature ventricular extrastimuli, delivered while linking was maintained in the accessory pathway, were able to interrupt the linking and restore antegrade accessory pathway conduction. CONCLUSIONS: These observations suggest that accessory pathway linking is associated with bidirectional block in the accessory pathway. The ability to initiate linking (and the stability of the phenomenon) depends on a critical relation between antegrade accessory pathway refractoriness and the magnitude of retrograde accessory pathway activation delay.

Adult↗
PubMed1992

Selective transcatheter ablation of the fast and slow pathways using radiofrequency energy in patients with atrioventricular nodal reentrant tachycardia.

BACKGROUND: The safety and efficacy of selective fast versus slow pathway ablation using radiofrequency energy and a transcatheter technique in patients with atrioventricular nodal reentrant tachycardia (AVNRT) were evaluated. METHODS AND RESULTS: Forty-nine consecutive patients with symptomatic AVNRT were included. There were 37 women and 12 men (mean age, 43 +/- 20 years). The first 16 patients underwent a fast pathway ablation with radiofrequency current applied in the anterior/superior aspect of the tricuspid annulus. The remaining 33 patients initially had their slow pathway targeted at the posterior/inferior aspect of the right interatrial septum. The fast pathway was successfully ablated in the initial 16 patients and in three additional patients after an unsuccessful slow pathway ablation. A mean of 10 +/- 8 radiofrequency pulses were delivered; the last (successful) pulse was at a power of 24 +/- 7 W for a duration of 22 +/- 15 seconds. Four of these 19 patients developed complete atrioventricular (AV) block. In the remaining 15 patients, the post-ablation atrio-His intervals prolonged from 89 +/- 30 to 138 +/- 43 msec (p less than 0.001), whereas the shortest 1:1 AV conduction and effective refractory period of the AV node remained unchanged. Ten patients lost their ventriculoatrial (VA) conduction, and the other five had a significant prolongation of the shortest cycle length of 1:1 VA conduction (280 +/- 35 versus 468 +/- 30 msec, p less than 0.0001). Slow pathway ablation was attempted initially in 33 patients and in another two who developed uncommon AVNRT after successful fast pathway ablation. Of these 35 patients, 32 had no AVNRT inducible after 6 +/- 4 radiofrequency pulses with the last (successful) pulse given at a power of 36 +/- 12 W for a duration of 35 +/- 15 seconds. After successful slow pathway ablation, the shortest cycle length of 1:1 AV conduction prolonged from 295 +/- 44 to 332 +/- 66 msec (p less than 0.0005), the AV nodal effective refractory period increased from 232 +/- 36 to 281 +/- 61 msec (p less than 0.0001), and the atrio-His interval as well as the shortest cycle length of 1:1 VA conduction remained unchanged. No patients developed AV block. Among the last 33 patients who underwent a slow pathway ablation as the initial attempt and a fast pathway ablation only when the former failed, 32 (97%) had successful AVNRT abolition with intact AV conduction. During a mean follow-up of 6.5 +/- 3.0 months, none of the 49 patients had recurrent tachycardia. Forty patients had repeat electrophysiological studies 4-8 weeks after their successful ablation, and AVNRT could not be induced in 39 patients. CONCLUSIONS: These data suggest that both fast and slow pathways can be selectively ablated for control of AVNRT: Slow pathway ablation, however, by obviating the risk of AV block, appears to be safer and should be considered as the first approach.

Adult↗