Back to the Dark Ages.
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Facility Regulation and Control↗
Biomedical subjects
M Carlson
Publications and source records attributed to M Carlson.
STD1 (MSN3) interacts directly with the TATA-binding protein and modulates transcription of the SUC2 gene of Saccharomyces cerevisiae.
STD1 (MSN3) was isolated independently as a multicopy suppressor of mutations in the TATA-binding protein and in SNF4, suggesting that STD1 might couple the SNF1 kinase signaling pathway to the transcriptional machinery. We report here a direct physical interaction between STD1 and the TATA-binding protein (TBP), observed in vivo by the two-hybrid system and in vitro by binding studies. STD1 bound both native TBP in yeast cell-free extracts and purified recombinant TBP. This interaction was altered when TBP delta 57 was used, suggesting a role for the non-conserved N-terminal domain of TBP in mediating protein-protein interactions. We also show that perturbation of STD1-TBP stoichiometry alters SUC2 expression in vivo and that this effect is dependent on the N-terminal domain of TBP. The activation of SUC2 expression by increased copy number of STD1 occurs at the level of mRNA accumulation and it requires the same TATA element and uses the same transcription start site as does activation of SUC2 by glucose limitation. Taken together, these results suggest that STD1 modulates SUC2 transcription through direct interactions with TBP.
Base Sequence↗
Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.
The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.
Amino Acid Sequence↗
Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein.
The SSN6-TUP1 protein complex represses transcription of diversely regulated genes in the yeast Saccharomyces cerevisiae. Here we present evidence that MIG1, a zinc-finger protein in the EGR1/Zif268 family, recruits SSN6-TUP1 to glucose-repressed promoters. DNA-bound LexA-MIG1 represses transcription of a target gene in glucose-grown cells, and repression requires SSN6 and TUP1. We also show that MIG1 and SSN6 fusion proteins interact in the two-hybrid system. Unexpectedly, we found that LexA-MIG1 activates transcription strongly in an ssn6 mutant and weakly in a tup1 mutant. Finally, LexA-MIG1 does not repress transcription in glucose-deprived cells, and MIG1 is differentially phosphorylated in response to glucose availability. We suggest a role for phosphorylation in regulating repression.
Bacterial Proteins↗
Definition of the best prediction criteria of the time domain signal-averaged electrocardiogram for serious arrhythmic events in the postinfarction period. The Cardiac Arrhythmia Suppression Trial/Signal-Averaged Electrocardiogram (CAST/SAECG) Substudy Investigators.
OBJECTIVES: The goal of this study was to establish guidelines for the prognostic use of the time domain signal-averaged electrocardiogram (ECG) after myocardial infarction. BACKGROUND: Previous studies of the prognostic use of the signal-averaged ECG in postinfarction patients had one or more of the following limitations: a small study group, empiric definition of an abnormal recording and possible bias in the selection of high risk groups or classification of arrhythmic events, or both. To correct for these limitations, a substudy was conducted in conjunction with the Cardiac Arrhythmia Suppression Trial (CAST). METHODS: Ten centers recruited 1,211 patients with acute myocardial infarction without application of the ejection fraction or Holter criteria restrictions of the main CAST protocol. Several clinical variables, ventricular arrhythmias on the Holter recording, ejection fraction and six signal-averaged ECG variables were analyzed. Patients with bundle branch block were excluded from the analysis, and the remaining 1,158 were followed for up to 1 year after infarction. The classification of arrhythmic events was reviewed independently by the CAST Events Committee. RESULTS: During an average (+/- SD) follow-up of 10.3 +/- 3.2 months, 45 patients had a serious arrhythmic event (nonfatal ventricular tachycardia or sudden cardiac arrhythmic death). A Cox regression analysis with only the six signal-averaged ECG variables indicated that the filtered QRS duration at 40 Hz > or = 120 ms (QRSD-40 Hz) at a cutpoint > or = 120 ms was the most predictive criterion of arrhythmic events. In a regression analysis that included all clinical, Holter and ejection fraction variables, a QRSD-40 Hz > or = 120 ms was the most significant predictor (p < 0.0001). The positive, negative and total predictive accuracy and odds ratio for QRSD-40 Hz > or = 120 ms were 17%, 98%, 88% and 8.4, respectively, and improved to 32%, 97%, 94% and 16.7, respectively, after combination with ejection fraction < or = 40% and complex ventricular arrhythmias on the Holter recording. CONCLUSIONS: The signal-averaged ECG predicts serious arrhythmic events in the first year after infarction better than do clinical, ejection fraction and ventricular arrhythmia variables, and QRSD-40 Hz > or = 120 ms provides the best predictive criterion in this clinical setting.
Arrhythmias, Cardiac↗
Structural and functional effects of apolar mutations of the distal valine in myoglobin.
High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
High-resolution, low-energy dissociative recombination spectrum of H3eH+
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Journal Article↗
Molecular analysis of the SNF8 gene of Saccharomyces cerevisiae.
Mutations in the SNF8 gene impair derepression of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26,936-dalton product. Disruption of the chromosomal locus caused a five-fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related.
Amino Acid Sequence↗
Time to arrhythmic, ischemic, and heart failure events: exploratory analyses to elucidate mechanisms of adverse drug effects in the Cardiac Arrhythmia Suppression Trial.
In this study we investigated the time to the first arrhythmic, ischemic, or failure event for encainide-flecainide and moricizine versus their respective placebo comparison groups in the Cardiac Arrhythmia Suppression Trial. The purpose was to explore possible mechanisms for the excessive deaths associated with active therapy that have been previously reported. Differences were noted between the active drugs. In particular, encainide-flecainide appeared to convert an ischemic event into death in more cases and more promptly than moricizine. However, the excessive deaths noted on encainide-flecainide were as likely to occur subsequent to a failure event as an ischemic event; for both encainide-flecainide and moricizine, the vast majority of excess deaths appeared to be the result of an increase in arrhythmia events without any protective effect of the drug. We were unable to identify any specific mechanism to explain the adverse effect of encainide and flecainide.
Anti-Arrhythmia Agents↗
Continuous subcutaneous insulin infusion (CSII) in children and adolescents with chronic poorly controlled type 1 diabetes mellitus.
This study was undertaken to determine if continuous subcutaneous insulin infusion (CSII) could improve control, diminish episodes of diabetic ketoacidosis (DKA), decrease number of hospitalizations and save health care expenditure in children and adolescents with long-standing poorly controlled diabetes mellitus. A retrospective analysis was done of six patients with type 1 diabetes for 1-8 years, of whom 4 were non-adherent to the diabetic regimen (ages 12-16.5 years) and 2 of whom had brittle diabetes (ages 8.5 and 10 years). These patients were non-randomly placed on the MiniMed (Sylmar, CA) CSII system. The year prior to CSII was compared with the year during pump use. Glycoslyated hemoglobin (HbA1c), spot urinary microalbumin, total cholesterol, insulin dose, growth velocity, number of convulsions and hypoglycemic events, number of episodes of DKA, number of hospitalizations and total inpatient costs were compared for the 2 years. The year prior to CSII, mean HbA1c was 9.02% (S.D. = 0.86%), mean number of hospitalizations was 5.2/patient (S.D. = 4.6), mean number of hospital days was 20.8/patient (S.D. = 14.7) and mean cost was $29330/patient (S.D. = $22804). During 1 year of CSII, mean number of hospital days decreased to 5 days/patient (S.D. = 0.8, P = 0.016), mean number of hospitalizations (including DKA and pump initiation) decreased to 1.7/patient (S.D. = 0.7, P = 0.31), mean inpatient costs decreased to $12762/patient (S.D. = $5.950, P = 0.047). HbA1c, urinary microalbumin, cholesterol, insulin dose and growth velocity did not change in a statistically significant manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.
Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.
Animals↗
SNF11, a new component of the yeast SNF-SWI complex that interacts with a conserved region of SNF2.
The yeast SNF-SWI complex is required for transcriptional activation of diverse genes and has been shown to alter chromatin structure. The complex has at least 10 components, including SNF2/SWI2, SNF5, SNF6, SWI1/ADR6, and SWI3, and has been widely conserved in eukaryotes. Here we report the characterization of a new component. We identified proteins that interact in the two-hybrid system with the N-terminal region of SNF2, preceding the ATPase domain. In addition to SWI3, we recovered a new 19-kDa protein, designated SNF11. Like other SNF/SWI proteins, SNF11 functions as a transcriptional activator in genetic assays. SNF11 interacts with SNF2 in vitro and copurifies with the SNF-SWI complex from yeast cells. Using a specific antibody, we showed that SNF11 coimmunoprecipitates with members of the SNF-SWI complex and that SNF11 is tightly and stoichiometrically associated with the complex. Furthermore, SNF11 was detected in purified SNF-SWI complex by staining with Coomassie blue dye; its presence previously went unrecognized because it does not stain with silver. SNF11 interacts with a 40-residue sequence of SNF2 that is highly conserved, suggesting that SNF11 homologs exist in other organisms.
Adenosine Triphosphatases↗
Further thoughts on the pitfalls of partition: a response to Mosey.
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Humans↗
A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.
The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.
AMP-Activated Protein Kinases↗
The yeast BDF1 gene encodes a transcription factor involved in the expression of a broad class of genes including snRNAs.
While screening for genes that affect the synthesis of yeast snRNPs, we identified a thermosensitive mutant that abolishes the production of a reporter snRNA at the non-permissive temperature. This mutant defines a new gene, named BDF1. In a bdf1-1 strain, the reporter snRNA synthesized before the temperature shift remains stable at the non-permissive temperature. This demonstrates that the BDF1 gene affects the synthesis rather than the stability of the reporter snRNA and suggests that the BDF1 gene encodes a transcription factor. BDF1 is present in single copy on yeast chromosome XII, and is important for normal vegetative growth but not essential for cell viability. bdf1 null mutants share common phenotypes with several mutants affecting general transcription and are defective in snRNA production. BDF1 encodes a protein of 687 amino-acids containing two copies of the bromodomain, a motif also present in other transcription factors as well as a new conserved domain, the ET domain, also present in Drosophila and human proteins.
Amino Acid Sequence↗
Prognostic significance of signal-averaged electrocardiogram after thrombolytic therapy and/or angioplasty during acute myocardial infarction (CAST substudy). Cardiac Arrhythmia Suppression Trial (CAST) SAECG Substudy Investigators.
Thrombolytic therapy and angioplasty during the early phase of an acute myocardial infarction (AMI) have been shown to improve prognosis. Time-domain analysis of the signal-averaged electrocardiogram (SAECG) provides strong, independent prediction of arrhythmic events (arrhythmic death/resuscitated cardiac arrest) after AMI. To determine whether the prognostic significance of an abnormal SAECG (QRS duration > or = 120 ms) measured after AMI is influenced by thrombolytic therapy/angioplasty given in the AMI period, the predictive value of SAECG was compared in patients with and without prior thrombolysis/angioplasty in a substudy of the Cardiac Arrhythmia Suppression Trial. Information was available in 787 patients. The average follow-up was 10 +/- 3 months and arrhythmic events occurred in 33 patients (4.2%). The prevalence of abnormal SAECG in patients with and without thrombolytic therapy/angioplasty was 9.4% (34 of 363 patients) and 14.9% (63 of 424 patients), respectively (p < 0.02). The arrhythmic event rate for patients with abnormal SAECG with and without thrombolytic therapy/angioplasty was 20.6% (7 of 34 patients) and 20.6% (13 of 63 patients), respectively. The arrhythmic event rate for patients with normal SAECG with and without thrombolytic therapy/angioplasty was 0.9% (3 of 329 patients) and 2.8% (10 of 361 patients), respectively. It is concluded that in patients with an AMI (1) the use of thrombolytic therapy/angioplasty is associated with a significantly decreased prevalence of abnormal SAECG, (2) thrombolytic therapy/angioplasty associated with a normal SAECG portends an excellent prognosis, and (3) an abnormal SAECG is predictive of an increased incidence of arrhythmic events in all patients regardless of prior thrombolytic therapy/angioplasty.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo.
The product of the SNF1 gene is a protein kinase whose activity is essential for transcriptional activation of glucose repressed genes in Saccharomyces cerevisiae. We have cloned a mammalian AMP-activated protein kinase (AMPK) that is 46% identical to the deduced amino acid sequence of SNF1 (Carling, D., Aguan, K., Woods, A., Verhoeven, A.J.M., Beri, R., Brennan, C.H., Sidebottom, C., Davison, M.D., and Scott, J. (1994) J. Biol. Chem. 269, 11442-11448). Mammalian AMPK plays a major role in the control of lipid metabolism and phosphorylating, thereby inactivating both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, key regulatory enzymes in the synthesis of fatty acids and cholesterol, respectively. We present evidence indicating that, in common with its mammalian homologue, SNF1 forms part of a protein kinase cascade. SNF1 is inactivated in vitro by treatment with protein phosphatase 2A and can be reactivated using a partially purified preparation of mammalian AMPK kinase. SNF1 undergoes a time-dependent increase in activity during growth in glucose-derepressing conditions, providing the first evidence that SNF1 activity is regulated by the level of available glucose. In wild-type yeast, but not in a snf1 deletion mutant, acetyl-CoA carboxylase shows a reciprocal change in activity compared with SNF1 under glucose derepressing conditions, indicating that SNF1 regulates acetyl-CoA carboxylase in vivo. These results suggest that, in addition to their structural similarity, the role of SNF1 and AMPK in the regulation of fatty acid synthesis has been highly conserved throughout evolution.
AMP-Activated Protein Kinases↗
The development of an assay for human neutrophil lipocalin (HNL)--to be used as a specific marker of neutrophil activity in vivo and vitro.
Human neutrophil lipocalin (HNL) is a newly discovered protein from human neutrophil secretory granules. A double-antibody radioimmunoassay (RIA) was developed for the measurement of HNL in various body fluids and its high specificity was confirmed by the absence of cross-reaction with other granulocyte granule proteins. The RIA measures HNL within the range of 4-256 micrograms/l. The intra- and interassay coefficients of variation were less than 6% and 10%, respectively. When HNL was added to serum samples full recovery was obtained. Sera and plasma from 100 apparently healthy individuals revealed a mean level of 78.40 micrograms/l (range 37.95-190.87 micrograms/l) in serum and a mean level of 50.65 micrograms/l (range 30.51-105.8 micrograms/l) in EDTA-plasma. The distribution of HNL after gel filtration indicated that HNL exists mainly in two major forms, dimer and monomer. This, in addition to the excellent recovery, suggests that these major forms of HNL do not bind to compounds in serum or plasma that would interfere with the assay. The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the measurement of HNL in blood and other body fluids.
Acute-Phase Proteins↗