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PubMed1994

Unipolar electrogram models for prediction of outcome in radiofrequency ablation of accessory pathways.

Meticulous catheter positioning close to the accessory pathway is essential for successful radiofrequency ablation. The aim of this study was to identify local unipolar electrogram characteristics predictive of radiofrequency ablation outcome, enabling more accurate accessory pathway localization and catheter positioning. So far mainly bipolar electrogram parameters have been evaluated, stressing the importance of the presence of an accessory pathway potential. However, especially in the absence of this parameter, the unipolar recording mode can be expected to hold several advantages. Nine local unipolar electrogram characteristics were analyzed in preexcited sinus rhythm directly preceding radiofrequency pulses in 35 consecutive patients with a manifest accessory atrioventricular pathway. A total of 1,230 unipolar electrogram complexes were analyzed and recorded at 138 ablation sites. Ablation was successful in 30/35 patients (86%). Multivariate analysis provided two unipolar models for prediction of ablation outcome: in Model I, sites with a suspected accessory pathway potential, local AV interval < or = 30 msec and catheter stability had 76% probability of success, but no more than 1% in their absence. In contrast, using the bipolar recording mode, presence of a suspected accessory pathway potential was the only one of these parameters shown to differentiate between successful and unsuccessful sites, with a predicted chance of success of 48%. Model II, not requiring assessment of possible accessory pathway potentials, showed a 63% probability of success for the combination of initial positivity of the local ventricular signal < or = 0.1 mV, AV interval < or = 30 msec, and catheter stability, but no more than 7% in their absence. Moreover, gradual decrease of initial ventricular positivity and AV interval while approaching a subsequently successful site allows the use of these parameters as dynamic mapping tools. Local unipolar electrogram parameters may thus facilitate precise accessory pathway localization and catheter positioning while offering important information supplementary to the bipolar mode, and enable accurate prediction of ablation outcome at a given site also in the absence of accessory pathway potential recording.

Adolescent↗
PubMed1995

Impact of the local atrial electrogram in AV nodal reentrant tachycardia: ablation versus modification of the slow pathway.

INTRODUCTION: The purpose of this study was to determine the predictors of successful ablation versus modification sites of the slow pathway in patients with AV nodal reentrant tachycardia. Complete elimination of slow pathway conduction ("ablation") is considered to be an appropriate endpoint during radiofrequency (RF) current delivery, whereas the persistence of residual slow pathway conduction with or without single echo beats ("modification") may be indicative of tachycardia recurrence. METHODS AND RESULTS: Of 131 patients, 71 consecutive patients were followed for 15.1 +/- 7.6 months. After elimination of inducible AV nodal reentrant tachycardia in all patients, residual slow pathway conduction (modification) persisted in 38 patients, whereas complete elimination of slow pathway conduction (ablation) was documented in 33 patients. Including electrophysiologic study after 5 to 7 days and after 3 to 6 months, 6 (8.4%) patients had recurrences: 5 with residual slow pathway conduction after the procedure and 1 with complete elimination of slow pathway conduction (P < 0.05). As compared with modulated sites, ablation sites of the slow pathway were characterized as follows: (1) duration of the local atrial electrogram (AEGM) (66.7 +/- 10.2 vs 54.1 +/- 12.6 msec, P < 0.01); (2) interval from the end of the AEGM to onset of His-bundle deflection (4.4 +/- 8.2 vs 16.1 +/- 9.3 msec, P < 0.01); and (3) number of peaks of the AEGM as an indicator of fractionation (4.1 +/- 0.7 vs 3.0 +/- 0.8, P < 0.01). The rate of junctional tachycardias (103.4 +/- 12.1 vs 102.1 +/- 16.9 per min), the AV ratio (0.4 +/- 0.5 vs 0.5 +/- 0.5), the number of RF current deliveries (4.1 +/- 4.4 vs 4.5 +/- 4.4), the duration of the procedure (124.1 +/- 45.3 vs 125.6 +/- 42.3 min), and the fluoroscopy time (15.5 +/- 10.8 vs 16.6 +/- 9.6 min) as well as power and total energy of RF current deliveries and the anatomically calculated catheter position at the successful site were not statistically different. A subset analysis in patients who received only a single RF application showed the same results for both groups. Patients without recurrence (n = 65) were found to have longer duration of the AEGM (61.9 +/- 14.6 msec) and a shorter interval from the end of AEGM to the onset of His-bundle deflection (10.1 +/- 12.2 msec) than patients with recurrence (n = 6) (47.5 +/- 7.5 msec and 20.8 +/- 12.8 msec, respectively). CONCLUSION: Complete ablation of the slow pathway resulted in a lower recurrence rate. The complete ablation approach is feasible using precisely analyzed local AEGMs to guide RF current in AV nodal reentrant tachycardia in a short procedure time.

Adult↗
PubMed1996

PR/RR interval ratio during rapid atrial pacing: a simple method for confirming the presence of slow AV nodal pathway conduction.

INTRODUCTION: Although the AV conduction curve in patients with AV nodal reentrant tachycardia (AVNRT) is usually discontinuous, many patients with this arrhythmia do not demonstrate criteria for dual AV nodal pathways. During rapid atrial pacing, the PR interval often exceeds the pacing cycle length when there is anterograde conduction over the slow pathway and AVNRT is induced. The purpose of this prospective study was to determine the diagnostic value of the ratio of the PR interval to the RR interval during rapid atrial pacing as an indicator of anterograde slow pathway conduction in patients undergoing electrophysiologic testing. METHODS AND RESULTS: The PR and RR intervals were measured during rapid atrial pacing at the maximum rate with consistent 1:1 AV conduction in four study groups: (1) patients with inducible AV nodal reentry and the classical criterion for dual AV nodal pathways during atrial extrastimulus testing (AVNRT Group 1); (2) patients with inducible AV nodal reentry without dual AV nodal pathways (AVNRT Group 2); (3) control subjects < or = 60 years of age without inducible AV nodal reentry; and (4) control subjects > 60 years of age without inducible AV nodal reentry. For both groups of patients with inducible AV nodal reentry, AV conduction was assessed before and after radiofrequency ablation of the slow AV nodal pathway. Before slow pathway ablation, the PR/RR ratio exceeded 1.0 in 12 of 13 AVNRT Group 1 patients (mean 1.27 +/- 0.21) and 16 of 17 AVNRT Group 2 patients (mean 1.18 +/- 0.15, P = NS Group 1 vs Group 2). After slow pathway ablation, the maximum PR/RR ratio was < 1.0 in all AVNRT patients (Group 1 = 0.59 +/- 0.08, P < 0.00001 vs before ablation; Group 2 = 0.67 +/- 0.11; P < 0.00001 vs before ablation). Among both groups of control subjects, the PR/RR ratio was > 1.0 in only 3 of 27 patients with no relation to patient age. CONCLUSION: The ratio of the PR interval to the RR interval during rapid atrial pacing at the maximum rate with consistent 1:1 AV conduction provides a simple and clinically useful method for determining the presence of slow AV nodal pathway conduction. This finding may be particularly useful in patients with inducible AV nodal reentry without dual AV nodal physiology on atrial extrastimulus testing.

Adult↗
PubMed1997

Prevalence of retrograde accessory pathway conduction during atrial fibrillation.

INTRODUCTION: Although atrial fibrillation occurs frequently in patients with the preexcitation syndrome, its pathogenesis remains controversial. The purpose of this study was to test the hypothesis that retrograde conduction over the accessory pathway occurs during atrial fibrillation and can serve as an important source of new wavefronts in atrial fibrillation. METHODS AND RESULTS: Eight patients undergoing surgical division of their accessory pathway(s) were studied. A plaque electrode array containing 56 (7 x 8) bipolar electrodes (5-mm resolution) was placed epicardially at the AV junction over the accessory pathway and atrial fibrillation was electrically induced. Excluding one patient who had only preexcited QRS complexes during atrial fibrillation and another whose accessory pathway was outside the mapped region, 4 of the 6 patients studied showed retrograde conduction over the accessory pathway during atrial fibrillation (mean atrial cycle length 157 +/- 59 msec). In these patients, 186 atrial wavefronts near the accessory pathway were analyzed. Among 67 wavefronts immediately following nonpreexcited QRS complexes, 17 originated from retrograde conduction. This constituted 9% (17/186) of total atrial wavefronts near the accessory pathway. Estimated atrial refractory period during atrial fibrillation ranged from 81 to 165 msec. CONCLUSIONS: (1) In patients with the preexcitation syndrome and atrial fibrillation, retrograde conduction over the accessory pathway contributed up to 9% of total atrial wavefronts near the accessory pathway. (2) The presence of an excitable gap in human atrial fibrillation was suggested by atrial preexcitation during retrograde conduction.

Atrial Fibrillation↗
PubMed1998

Atrioventricular nodal reentry tachycardia in patients with sinus node dysfunction: electrophysiologic characteristics, clinical presentation, and results of slow pathway ablation.

INTRODUCTION: Sinus node dysfunction (SND) is frequently associated with impaired AV conduction. This study investigated the electrophysiologic properties of dual AV nodal pathways in patients suffering from both SND and AV nodal reentrant tachycardia (AVNRT). METHODS AND RESULTS: Two groups of patients with slow-fast AVNRT underwent invasive electrophysiologic testing and catheter ablation of the slow pathway. Group A comprised 10 patients with SND (age 70 +/- 8 years). Group B included 10 age-matched patients without SND (age 69 +/- 7 years; P = NS) who served as controls. Patients of group A exhibited prolongation of the anterograde Wenckebach cycle lengths (WBCLs) of both the fast pathway (559 +/- 96 vs 361 +/- 38 msec; P < 0.01) and the slow pathway (409 +/- 57 vs 339 +/- 32 ms; P < 0.01). However, the delta between the WBCLs of the fast and the slow pathways was larger in patients of group A (150 +/- 80 vs 22 +/- 20 msec; P < 0.01). Retrograde fast pathway conduction was well preserved with no difference in WBCLs (356 +/- 42 vs 330 +/- 47 msec; P = NS). Cycle lengths of AVNRT were longer in group A (468 +/- 46 vs 363 +/- 37 msec; P < 0.01). Clinically, all patients of group A suffered from multiple episodes of AVNRT per week, which was not the case in any patient of group B (P < 0.01). Catheter ablation of the slow pathway eliminated AVNRT in all patients without complications. CONCLUSIONS: Patients with AVNRT and SND exhibit characteristic electrophysiologic alterations of both AV nodal pathways. Clinically, this results in significantly more frequent episodes of tachycardia. Slow pathway ablation appears to be safe and effective in these patients.

Aged↗
PubMed1968

Factors affecting the conductivity of pathways in the cerebral cortex.

1. We have investigated the conductivity of neural pathways in slabs of unanaesthetized, isolated, cerebral cortex, cut from the isolated forebrains of twenty-five cats.2. Neurones within the isolated area were indirectly excited, either by a small electrode thrust into the subcortical white matter, or by remote stimulation of the pial surface. Sometimes a small electrode was employed for intracortical stimulation.3. The response of single neurones to these stimuli was recorded with extracellular micropipettes. Submaximal stimuli produced a stochastic response which was measured from the post-stimulus histogram (PSH) and provided an estimate of the probability of discharge at various times after the stimulus.4. The PSH often displayed several discrete humps of different latencies, indicating several pathways between stimulated and recording point. Conductivity measurements were usually restricted to the pathway of shortest latency.5. The conductivity of a pathway was defined as C = xy/x, averaged over 1 or 2 min, wherex = frequency of afferent test volleys,xy = frequency of response: i.e. of those action potentials contained within a well-defined hump of the PSH,(y = frequency of all discharges of the recorded neurone).6. The progress of conductivity was tested with some constant form of cortical stimulation, repeated at regular intervals of 1-5 sec. Reliable results were obtained for twenty-six pathways subjected to thirty-eight experiments.7. Temporary alteration of x, y or xy (conditioning with Deltax, Deltay or Deltaxy), for a period of 6-25 min, often caused a subsequent change in the conductivity (DeltaC) of a pathway which sometimes attenuated with a time constant of about 10 min, but which could persist without detectable attenuation for 20-30 min.8. Conditioning periods less than 6 min rarely produced changes in conductivity; alterations of conductivity were more likely to be caused by conditioning periods longer than 17 min, than by periods of 6-16 min.9. Changes in conductivity were usually correlatednegatively with temporary changes in xy,negatively with temporary changes in x,positively with temporary changes in y.10. Nineteen of twenty-six pathways tested showed properties consistent with the formula [Formula: see text], whereG(1) lies between -1.0 and -0.16, with mean value -0.50,G(2) lies between 0 and 0.42, with mean value +0.12,G(3) lies between -0.61 and 0 with mean value -0.38,K is a coefficient which is usually different for each experiment. Four out of twenty-six pathways so tested provided results which did not fit this formula; three out of twenty-six pathways did not give adequate information.

Animals↗
PubMed1978

Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway.

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in Saccharomyces cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by (i) measuring feedback inhibition of anthranilate synthase in vivo, (ii) determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and (iii) varying the gene dose of individual enzymes of the pathway at the tetraploid level. We conclude that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influencing on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the "repressed" pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. Our results, therefore, suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.

Anthranilate Synthase↗
PubMed1992

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.

Anaerobiosis↗
PubMed1997

Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12.

We report the complete nucleotide sequence of the gene cluster encoding the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Escherichia coli K-12. Sequence analysis revealed the existence of eight genes that map at min 8 of the chromosome, between the lac and hemB regions. Six enzyme-encoding genes account for a flavin-type monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the meta-cleavage pathway (mhpCDFE). The order of these catabolic genes, with the sole exception of mhpF, parallels that of the enzymatic steps of the pathway. The mhpF gene may encode the terminal acetaldehyde dehydrogenase (acylating) not reported previously in the proposed pathway. Enzymes that catalyze the early reactions of the pathway, MhpA and MhpB, showed the lowest level of sequence similarity to analogous enzymes of other aromatic catabolic pathways. However, the genes mhpCDFE present the same organization and appear to be homologous to the Pseudomonas xyl, dmp, and nah meta-pathway genes, supporting the hypothesis of the modular evolution of catabolic pathways and becoming the first example of this type of catabolic module outside the genus Pseudomonas. Two bacterial interspersed mosaic elements were found downstream of the mhpABCDFE locus and flank a gene, orfT, which encodes a protein related to the superfamily of transmembrane facilitators that might be associated with transport. All of the genes of the 3-HPP cluster are transcribed in the same direction, with the sole exception of mhpR. Inducible expression of the mhp catabolic genes depends upon the presence, in the cis or trans position, of a functional mhpR gene, which suggests that the mhpR gene product is the activator of the 3-HPP biodegradative pathway. The primary structure of MhpR revealed significant similarities to that of members of the IclR subfamily of transcriptional regulators. A 3-HPP catabolic DNA cassette was engineered and shown to be functional not only in enteric bacteria (E. coli and Salmonella typhimurium) but also in Pseudomonas putida and Rhizobium meliloti, thus facilitating its potential application to improve the catabolic abilities of bacterial strains for degradation of aromatic compounds.

Amino Acid Sequence↗
PubMed1999

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli.

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

DNA Polymerase II↗
PubMed2003

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane.

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

Amino Acid Sequence↗
PubMed2004

The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds.

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.

Bacterial Proteins↗
PubMed2005

Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440.

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.

2-Aminoadipic Acid↗
PubMed2005

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400.

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.

Aerobiosis↗
PubMed2006

Lethal mutations in the isoprenoid pathway of Salmonella enterica.

Essential isoprenoid compounds are synthesized using the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.

Erythritol↗
PubMed2001

Activation of the Ral and phosphatidylinositol 3' kinase signaling pathways by the ras-related protein TC21.

TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. We have previously identified the Raf/mitogen-activated protein kinase pathway as a direct TC21 effector pathway required for TC21-induced transformation (M. Rosário, H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270-1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphatidylinositol 3' kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral A and the PI-3K-dependent activation of Akt/protein kinase B. Strong activation of the PI-3K/Akt pathway is seen even with very low levels of TC21 expression, suggesting that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alterations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the other hand, activation of the Ral pathway by TC21 is required for TC21-stimulated DNA synthesis but not transformed morphology. We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cells. Finally, we provide mechanisms for the activation of these pathways, namely, the direct in vivo interaction of TC21 with guanine nucleotide exchange factors for Ral, resulting in their translocation to the plasma membrane, and the direct interaction of TC21 with PI-3K. In both cases, the effector domain region of TC21 is required since point mutations in this region can interfere with activation of downstream signaling.

3T3 Cells↗
PubMed2002

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis.

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.

3T3 Cells↗
PubMed1980

'Dual atrioventricular nodal pathways" in patients with Wolff-Parkinson-White syndrome.

'Dual atrioventricular nodal pathways" were found in five patients who also had the Wolff-Parkinson-White syndrome. All five patients had a re-entrant tachycardia that used the atrioventricular node for conduction in the anterograde direction and an accessory atrioventricular pathway for conduction in the retrograde direction. One of the patients also had a re-entrant tachycardia that originated within the atrium or the atrioventricular node. Dual atrioventricular nodal pathways were identified in three of the five patients during their first electrophysiological study because the effective refractory period of the accessory atrioventricular pathway in the anterograde direction was longer than the effective refractory period of the fast atrioventricular nodal pathway. In the other two patients the dual atrioventricular nodal pathways were found only after operative division of an accessory atrioventricular pathway. Re-entrant tachycardia that uses an accessory pathway may be cured by operative division of the accessory pathway. Tachycardia resulting from re-entry within the atrioventricular node cannot be cured by an operation unless the normal conduction system is divided and a permanent pacemaker implanted. These five patients indicate the importance of determining the aetiology of tachycardia by studying the tachycardia itself and not only the type of atrioventricular conduction present.

Atrioventricular Node↗