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At least 1,063 records · Page 59Linked to original sources
PubMed1997

Identification of fiber orientation in left free-wall accessory pathways: implication for radiofrequency ablation.

Previous reports on the anatomic discordance between atrial and ventricular insertion sites of left free-wall accessory pathways were limited and their findings were controversial. The purpose of this study was to explore the fiber orientation and related electrophysiologic characteristics of left free-wall accessory pathways. The study population comprised 96 consecutive patients with a single left free-wall accessory pathway (33 manifest and 63 concealed pathways), who underwent electrophysiologic study and radiofrequency catheter ablation using the retrograde ventricular approach. The atrial insertion site of the accessory pathway was defined from the cinefilms as the site with the earliest retrograde atrial activation bracketed on the coronary sinus catheter during tachycardia, and the ventricular insertion site was defined as the site where successful ablation of the pathway was achieved. Forty-two patients (44%) had their atrial insertion sites 5-20 mm (10 +/- 3 mm) distal to the ventricular insertion sites (proximal excursion), 30 (31%) patients had their atrial insertion sites 5-20 mm (12 +/- 3 mm) proximal to the ventricular insertion sites (distal excursion), and 24 (25%) patients had directly aligned atrial and ventricular insertion sites. Retrograde conduction properties, including 1:1 VA conduction and effective refractory period, were significantly poorer in the pathways with proximal excursion (302 +/- 67, 285 +/- 61 ms respectively) than in those with distal excursion (264 +/- 56, 250 +/- 48 ms respectively) or direct alignment (272 +/- 61, 258 +/- 73 ms respectively). Accessory pathways at the more posterior location had a significantly higher incidence of proximal excursion (P = 0.006), and those at the more anterior location had a higher incidence of distal excursion (P = 0.012). In conclusion, a wide variation in fiber orientations and related electrophysiologic characteristics was found in left free-wall accessory pathways. This may have important clinical implications for radiofrequency ablation.

Adult↗
PubMed2000

Radiofrequency application to the posteroseptal region alters retrograde accessory pathway activation.

INTRODUCTION: Radiofrequency applications to the posteroseptal region can ablate the atrioventricular accessory pathway residing in this area. In conjunction with the adjacent anatomic structures, however, ablative lesions which do not effectively ablate the accessory pathway could markedly alter retrograde atrial activation sequence and confound interpretation of further mapping of an accessory pathway. METHODS AND RESULTS: Electrophysiologic studies, endocardial activation mapping and radiofrequency catheter ablation were undertaken in three patients with recurrent supraventricular tachycardia. Patients were initially thought to have a single posteroseptal accessory pathway; earliest ventrioatrial activation during tachycardias and during ventricular pacing was at the coronary sinus ostium, but initial radiofrequency applications were unsuccessful to ablate the pathway. After initial radiofrequency applications to the posteroseptal region, the earliest retrograde atrial activation changed to the right atrial free wall in two patients. Additional radiofrequency application to the posteroseptal area was able to ablate the single posteroseptal accessory pathway in one patient. Radiofrequency application to the right atrial free wall was required to stop tachycardia initiation in other patient. The third patient was suspected of having a slow-slow atrioventricular nodal reentry tachycardia. Radiofrequency application to the posteroseptal area changed the earliest retrograde atrial activation to the distal coronary sinus recording site, mimicking an accessory pathway at the left atrial free wall. Radiofrequency application to the anteroseptum was able to ablate the concealed accessory pathway. CONCLUSION: Radiofrequency applications to the posteroseptal region can markedly alter retrograde atrial activation, thereby confounding further mapping of the accessory pathway.

Adolescent↗
PubMed1998

Tyrosine kinase receptor-activated signal transduction pathways which lead to oncogenesis.

Oncogenesis is a complicated process involving signal transduction pathways that mediate many different physiological events. Typically, oncogenes cause unregulated cell growth and this phenotype has been attributed to the growth-stimulating activity of oncogenes such as ras and src. In recent years, much research effort has focused on proteins that function downstream of Ras, leading to the identification of the Ras/Raf/MAPK pathway, because activation of this pathway leads to cellular proliferation. Activated receptor tyrosine kinases (RTKs) also utilize this pathway to mediate their growth-stimulating effects. However, RTKs activate many other signaling proteins that are not involved in the cellular proliferation process, per se and we are learning that these pathways also contribute to the oncogenic process. In fact, RTKs and many of the proteins involved in RTK-dependent signal transduction can also function as oncogenes. For example, the catalytic subunit of phosphoinositide 3-kinase (P13-K) was recently identified as an oncogenic protein. The scope of pathways that are activated by oncogenic RTKs is expanding. Thus, not only do RTKs activate Ras-dependent pathways that drive proliferation, RTKs activate P13-K-dependent pathways which also contribute to the oncogenic mechanism. P13-K can initiate changes in gene transcription, cytoskeletal changes through beta-catenin, changes in cell motility through the tumor suppressor, adenomatous polyposis coli (APC), and phosphorylation of BAD, a protein involved in apoptotic and antiapoptotic signaling. There is also cross-talk between RTKs and the oncostatin cytokine receptor which may positively and negatively influence oncogenesis. For this review, we will focus on oncogenic RTKs and the network of cellular proteins that are activated by RTKs because multiple, divergent pathways are responsible for oncogenesis.

Animals↗
PubMed2003

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

Amino Acid Sequence↗
PubMed2005

Linking pathway gene expressions to the growth inhibition response from the National Cancer Institute's anticancer screen and drug mechanism of action.

Novel strategies are proposed to quantitatively analyze and relate biological pathways to drug responses using gene expression and small-molecule growth inhibition data (GI(50)) derived from the National Cancer Institute's 60 cancer cells (NCI(60)). We have annotated groups of drug GI(50) responses with pathways defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta, and functional categories defined by Gene Ontology (GO), through correlations between pathway gene expression patterns and drug GI(50) profiles. Drug-gene-pathway relationships may then be utilized to find drug targets or target-specific drugs. Significantly correlated pathways and the gene products involved represent interesting targets for further exploration, whereas drugs that are significantly correlated with only certain pathways are more likely to be target specific. Separate pathway clustering finds that pathways engaged in the same biological process tend to have similar drug correlation patterns. The biological and statistical significances of our method are established by comparison to known small-molecule inhibitor-gene target relationships reported in the literature and by standard randomization procedures. The results of our pathway, gene expression and drug-induced growth inhibition associations, can serve as a basis for proposing testable hypotheses about potential anticancer drugs, their targets, and mechanisms of action.

Antineoplastic Agents↗
PubMed1969

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative nd non-oxidative reactions and related enzymes of the cycle in adipose tissue.

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.

Adipose Tissue↗
PubMed1998

A clinical-care pathway for decreasing hospital stay after radical prostatectomy.

OBJECTIVES: To evaluate the use of a clinical-care pathway that decreased the stay in hospital after radical retropubic prostatectomy from 3 to 2 days, assessing the costs and quality of care. PATIENTS AND METHODS: Forty-four consecutive men who underwent radical retropubic prostatectomy were evaluated prospectively. The first 22 men were hospitalized under the standard 3-day clinical-care pathway in use at our institution. This pathway was evaluated, shortened to construct a 2-day pathway, and a second group of 22 consecutive men hospitalized under the new pathway. Both groups were evaluated and compared 6 weeks post-operatively. RESULTS: The mean (SD) hospital stay was 2.1 (0.3) days for men in the 2-day and 2.9 (0.4) days for men in the 3-day pathway (P < 0.001). The mean (SD) hospital cost was $8468 (801) in the 2-day and $8806 (630) in the 3-day pathway (P=0.13). None of the men in the 2-day and one of 22 men in the 3-day pathway experienced a major complication (P=0.31). Two of 22 men in the 2-day and one of 22 in the 3-day pathway exceeded the expected stay by one day (P=0.55). CONCLUSION: The hospital stay after radical retropubic prostatectomy can be safely shortened from 3 to 2 days for most men. However, the shorter hospital stay does not result in significant cost savings. The shorter stay does not appear to compromise quality of care. Proper patient education and careful pre- and post-operative supervision are necessary for a successful outcome.

Aged↗
PubMed2001

Risk of development of delayed atrioventricular block after slow pathway modification in patients with atrioventricular nodal reentrant tachycardia and a pre-existing prolonged PR interval.

AIMS: The objective of this prospective study was to assess risk factors for the development of atrioventricular block following slow pathway modification in patients with atrioventricular nodal reentrant tachycardia and a pre-existing prolonged PR interval. METHODS AND RESULTS: Of 346 consecutive patients with atrioventricular nodal reentrant tachycardia undergoing slow pathway modification, 18 patients (62 +/- 7 years; five females) were found to have a prolonged PR interval prior to ablation. Total elimination of the functional slow pathway was assumed if the antegrade effective refractory period following slow pathway modification was longer than the cycle length of atrioventricular nodal reentrant tachycardia. To detect atrioventricular node conduction disturbances, 24-h Holter recordings were performed 1 day prior to slow pathway modification, and 1 day, 1 week, 1, 3 and 6 months after the procedure. Six patients developed late atrioventricular block. The incidence of delayed atrioventricular block following successful slow pathway modification was higher in patients with, compared to patients without, prolonged PR interval at baseline (6/18 vs 0/328, P < .001). In the former group, the antegrade effective refractory period was longer in patients with, compared to those without, a delayed atrioventricular block (492 +/- 150 ms vs 332 +/- 101 ms, P < 0.05). The incidence of delayed atrioventricular block was higher in patients with total elimination of the slow pathway compared to patients without (5/7 vs 1/11, P < 0.01). CONCLUSIONS: Slow pathway modification in patients with atrioventricular nodal reentrant tachycardia and a prolonged PR interval is highly effective. However, there is a significant risk of development of delayed atrioventricular block, particularly when the procedure results in total elimination of the slow pathway.

Case-Control Studies↗
PubMed2001

Impact of radiofrequency catheter ablation of accessory pathways on the frequency of atrial fibrillation during long-term follow-up; high recurrence rate of atrial fibrillation in patients older than 50 years of age.

AIMS: Atrial fibrillation represents a frequent and potentially life-threatening arrhythmia in patients with accessory atrioventricular pathways. Radiofrequency ablation has become the curative treatment of first choice for these patients. Investigations after successful surgical pathway dissection reported an almost complete elimination of paroxysmal atrial fibrillation. However, there are only a few reports which include a small number of patients undergoing radiofrequency ablation. The purpose of this study was to examine whether successful radiofrequency ablation of accessory pathways prevents the occurrence of paroxysmal atrial fibrillation, and to identify possible predictors of atrial fibrillation recurrence. METHODS AND RESULTS: A total of 116 consecutive patients (mean age 42+/-15 years) with manifest or concealed accessory pathways and documented paroxysmal atrial fibrillation underwent radiofrequency catheter ablation. The patients were reexamined at 6 and 12 months. Long-term follow-up information was obtained by questionnaire and/or by consulting the referring physician. Pathway conduction was abolished in 101 cases (87%). Late follow-up information was obtained from 91 of these 101 patients (90%) with successful ablation with a mean follow-up duration of 23.9+/-12.3 months. During follow-up, 25 of 91 patients (27%) experienced arrhythmias. Recurrent episodes of atrial fibrillation were observed in 18 of these 25 cases (i.e. 20% of the 91 patients). Differences between patients with and without recurrences of atrial fibrillation were examined for age, sex, associated cardiac disease, presence of multiple pathways, pathway location, atrial fibrillation inducibility during the procedure and cycle length of the atrioventricular reentrant tachycardia. Only older age was a significant independent predictor of atrial fibrillation recurrence (P=0.02). Eleven of 31 patients (35%) older than 50 years of age had atrial fibrillation recurrences during follow-up compared to seven of 60 patients (12%) under age 50. The recurrence rate of atrial fibrillation was even higher in patients older than 60 years (6 of 11 patients, i.e. 55%). In comparison, the occurrence rate of atrial fibrillation during follow-up in a control group of 100 consecutive patients with successful accessory pathway ablation, who did not have evidence of paroxysmal atrial fibrillation prior to ablation, was 4% and, thus, significantly lower than in the study group of the 91 patients (P=0.001). CONCLUSIONS: The recurrence rate of paroxysmal atrial fibrillation after successful radiofrequency ablation of accessory pathways shows an age-related increase, being low in patients younger than 50 years of age (12%) and high in the older patients: 35% in patients older than 50 years and 55% in patients older than 60. These results have significant therapeutic implications concerning the decisions on pharmacological therapy after successful ablation in patients older than 50 years. Furthermore, these data will help physicians advise older patients properly about their risk of recurrence of atrial fibrillation after ablation.

Adult↗
PubMed2001

Combined analysis of p53 and RB pathways in epithelial ovarian cancer.

Disruptions of the p16-CDK4/cyclin D1-pRb pathway (RB pathway) and the p14ARF-MDM2-p53 pathway (p53 pathway) are important mechanisms in the development of human malignancies. In this study, we investigated RB and p53 pathways in 46 epithelial ovarian cancers (EOCs). In the RB pathway, 16 (34.8%) of 46 cases had p16 gene alterations or loss of expression. The deletion of the p16 gene was a rare event. In 7 cases, we observed methylation in the 5'CpG island in the promoter region of the p16 gene. Abnormal expressions of pRb and CDK4/cyclin D1 were 10.9% and 30.4%, respectively. In the p53 pathway, 10 (21.7%) of 46 cases had p14ARF gene alterations or abnormal expression. In 4 cases, methylation in the 5'CpG island in the promoter region of the p14ARF gene was present. MDM2 overexpression was a rare event. Thirty-six (78.3%) of 46 patients had p53 gene alterations or expression. In our studied cases, p14ARF abnormalities were independent of p16 abnormalities. Abnormal RB and p53 pathways were present in 60.9% and 80.4% of cases, respectively. In conclusion, disruptions of p53 and RB pathways are frequent events and the inverse correlations were present between the abnormality of p16 and p14ARF in EOCs.

Carcinoma↗
PubMed1994

Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway.

Studies presented here show that overall NF-kappa B signal transduction begins with a parallel series of stimuli-specific pathways through which cytokines (tumor necrosis factor alpha), oxidants (hydrogen peroxide and mitomycin C), and phorbol ester (phorbol 12-myristate 13-acetate) individually initiate signaling. These initial pathways culminate in a common pathway through which all of the stimulating agents ultimately signal NF-kappa B activation. We distinguish the stimuli-specific pathways by showing that the oxidative stimuli trigger NF-kappa B activation in only one of two human T-cell lines (Wurzburg but not Jurkat), whereas tumor necrosis factor alpha and phorbol 12-myristate 13-acetate readily stimulate in both lines. We propose the common pathway as the simplest way of accounting for the common requirements and properties of the signaling pathway. We include a redox-regulatory mechanism(s) in this common pathway to account for the previously demonstrated redox regulation of NF-kappa B activation in Jurkat cells (in which oxidants don't activate NF-kappa B); we put tyrosine phosphorylation in the common pathway by showing that kinase activity (inhibitable by herbimycin A and tyrphostin 47) is required for NF-kappa B activation by all stimuli tested in both cell lines. Since internal sites of oxidant production have been shown to play a key role in the cytokine-stimulated activation of NF-kappa B, and since tyrosine kinase and phosphatase activities are known to be altered by oxidants, these findings suggest that intracellular redox status controls NF-kappa B activation by regulating tyrosine phosphorylation event(s) within the common step of the NF-kappa B signal transduction pathway.

Benzoquinones↗
PubMed1996

Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes.

Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway. Growth factors acting through Ras primarily stimulate the Raf/MEK/MAPK cascade of protein kinases. In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pathways. Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways. These studies suggest a high degree of cross-talk apparently not seen in vivo. We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways. We report preferential association between MAP kinases and their activators in eukaryotic cells. Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins. We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation. Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members. The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.

Animals↗
PubMed1999

Plastidic pathway of serine biosynthesis. Molecular cloning and expression of 3-phosphoserine phosphatase from Arabidopsis thaliana.

In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB- mutant. The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence. Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein. Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1. 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E. coli. Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an approximately 1.5-fold abundance in the root compared with the leaf tissues. This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues. This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants.

Amino Acid Sequence↗
PubMed1999

Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor alpha gene expression in T lymphocytes.

Tumor necrosis factor a (TNF-alpha) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-alpha is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-alpha plays a critical role in autoimmune inflammation and superantigen-induced septic shock. However, little is known about the intracellular signaling pathways that regulate TNF expression in T cells. Here we show that extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control the transcription and synthesis of TNF-alpha in A3.01 T cells that produce the cytokine upon T cell activation by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Selective activation of each of the distinct MAPK pathways by expression of constitutively active kinases is sufficient for TNF-alpha promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-alpha promoter. Selective inhibition of one or more MAPK pathways impairs TNF-alpha induction by TPA/ionomycin, indicating a cooperation between these signal transduction pathways. Our approach revealed that the MAPK kinase 6 (MKK6)/p38 pathway is involved in both transcriptional and posttranscriptional regulation of TNF expression. Moreover, analysis of the progressive 5' deletion mutants of the TNF-alpha promoter indicates that distinct promoter regions are targeted by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has been reported for other TNF-alpha-producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of the TNF-alpha gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells.

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.

Androstadienes↗
PubMed2001

Voltage-independent inhibition of P/Q-type Ca2+ channels in adrenal chromaffin cells via a neuronal Ca2+ sensor-1-dependent pathway involves Src family tyrosine kinase.

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.

Animals↗
PubMed2003

Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus.

Rhodothermus marinus responds to fluctuations in the growth temperature and/or salinity by accumulating mannosylglycerate (MG). Two alternative pathways for the synthesis of MG have been identified in this bacterium: a single-step pathway and a two-step pathway. In this work, the genetic and biochemical characterization of the two-step pathway was carried out with the goal of understanding the function of the two pathways and their regulatory mechanisms. Mannosyl-3-phosphoglycerate synthase (MPGS) of the two-step pathway was purified from R. marinus. Sequence information led to the isolation of two contiguous genes, mpgs (encoding MPGS) and mpgp (encoding mannosyl-3-phosphoglycerate phosphatase). The recombinant MPGS had a low specific activity compared with other homologous MPGSs and contained approximately 30 additional residues at the C terminus. Truncation of this extension produced a protein with a 10-fold higher specific activity. Moreover, the activity of the complete MPGS was enhanced upon incubation with R. marinus cell extracts, and protease inhibitors abolished activation. Therefore, the C-terminal peptide of MPGS was identified as a regulatory site for short term control of MG synthesis in R. marinus. The control of gene expression by heat and osmotic stress was also studied; the level of mannosylglycerate synthase involved in the single-step pathway was selectively enhanced by heat stress, whereas MPGS was overproduced in response to osmotic stress. The concomitant changes in the level of MG were assessed as well. We conclude that the two alternative pathways for the synthesis of MG are differently regulated at the level of expression to play specific roles in the adaptation of R. marinus to two different types of aggression. This is the only example of pathway multiplicity being rationalized in terms of the need to respond efficiently to distinct environmental stresses.

Adaptation, Biological↗
PubMed2005

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells.

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.

Breast↗