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Biomedical subjects

F Marcus

Publications and source records attributed to F Marcus.

At least 19 recordsLinked to original sources

Q-wave versus non-Q wave myocardial infarction: a meaningless distinction.

The whole subject can thus be summed up in two statements. 1. Every appropriately designed study comparing first Q and NQMI's has found no difference in post-MI course of the two categories and no foundation for the common notion that the NQMI is a uniquely "unstable" entity, to be classed with unstable angina in terms of prognosis and management. Nine such studies have been published. On the other hand, all studies alleging the "unstable" character of the NQMI have been invalidated by major flaws, chief among them the comparison of undifferentiated mixtures of first and subsequent infarcts with widely differing mortality and morbidity. This confusion is further compounded by the fact that subsequent infarcts generate Qwaves less than half as often as first infarcts. 2. All current studies indicate that there is no benefit to an invasive as compared with a conservative protocol for management of NQMI. Since the characterization of an infarct as "non-Q' conveys no therapeutic implications, the classification becomes irrelevant and should be discarded. Two quotations sum the whole matter succinctly. Moss (63) commented that "The Q-wave versus non-Q-wave categorization does not provide sufficient sensitivity, specificity, or predictive accuracy about the subsequent clinical course of patients with a first myocardial infarction to use it as reliable data in the clinical decision-making process." Surawicz (64) put the matter even more concisely: ". . . a non-Qwave MI is not a unique entity: rather it is a smaller and less extensive MI." In a word, the magnitude of a myocardial infarction should be judged on anatomical and functional considerations rather than on the designation of Qwave versus non-Qwave infarction.

Aged↗

Disulfide bonds of herpes simplex virus type 2 glycoprotein gB.

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.

Amino Acid Sequence↗

Evidence for a phosphorylation site in cytomegalovirus glycoprotein gB.

As part of our vaccine program, we have purified a recombinant form of human cytomegalovirus glycoprotein B that is able to induce high titers of virus-neutralizing antibodies. The isolated protein was found to be phosphorylated at a serine residue in position -7 from the C terminus of the protein. The corresponding synthetic peptide, HLKDSDEEENV, was an efficient in vitro substrate of casein kinase II.

Amino Acid Sequence↗

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458.

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

Amino Acid Sequence↗

Characteristics of the ventricular insertion sites of accessory pathways with anterograde decremental conduction properties.

BACKGROUND: Accessory pathways (APs) with anterograde decremental conduction properties referred to as Mahaim fibers have recently been recognized as originating from the right lateral atrium. Little information is available about their distal insertion. The purpose of this study was to determine the different kinds of APs involved and the characteristics of their distal insertion site. METHODS AND RESULTS: Twenty-one patients (mean age, 28 +/- 13 years) with reciprocating tachycardia or atrial fibrillation were studied. Right-sided atrial and/or ventricular endocardial mapping during tachycardia identified different types of APs. (1) Seventeen patients had long APs originating from the right lateral atrium and coursing several centimeters to the right ventricle. In 10 patients, the AP terminated in or close to the right bundle-branch system (atriofascicular AP) and in 7, the AP terminated in the anterior right ventricle (atrioventricular AP). Patients with atriofascicular APs had narrower QRS complexes (133 +/- 10 versus 165 +/- 26 milliseconds, P = .02) and narrower initial r wave in leads V2 through V4 during maximal preexcitation than patients with atrioventricular APs. In addition, they had earlier His-bundle and right bundle-branch retrograde activation, ie, shorter V-His (16 +/- 5 versus 37 +/- 9 milliseconds, P < .01) and V-right bundle intervals (3 +/- 5 versus 25 +/- 6 milliseconds, P < .01). In 6 patients, minimal preexcitation not readily apparent was present in sinus rhythm despite the appearance of a narrow QRS complex. A wide distal insertion site of 0.5 to 2 cm in diameter consistent with arborization of the AP was found in 10 patients. The distal application of radiofrequency current produced a change in the preexcitation pattern in 4 patients and ablated the AP in 2 patients. In the other patients, radiofrequency current was applied more proximally and successfully ablated the AP bundle (n = 9) or AP proximal insertion (n = 6). No recurrence was observed during a follow-up period of 12 +/- 10 months. (2) Four patients had short paratricuspid atrioventricular APs; in one, the decremental conduction property was acquired as demonstrated by two electrophysiological studies performed 7 years apart. Radiofrequency ablation was successfully accomplished in all 4 patients at the tricuspid annulus. CONCLUSIONS: Different types of APs account for tachycardias previously called Mahaim fibers. Long and short atrioventricular APs are observed in 81% and 19%, respectively. Long APs often have a distal arborization and may have either a fascicular or ventricular insertion. Radiofrequency current is more efficient when applied to the AP bundle or AP proximal insertion rather than to the distal insertion in patients with long APs.

Adult↗

The feasibility of using ultrasound for cardiac ablation.

The feasibility of using ultrasound to induce cardiac tissue necrosis for the treatment of arrhythmias was investigated. A theoretical model was used to optimize the operating frequency for necrosis of highly perfused muscle tissue. From these simulations it appeared that frequencies from 10-15 MHz produce the deepest lesions at ultrasound intensities between 15 and 30 W/cm2. Test catheters with a planar ultrasound transducer (diameter 2.3 mm = 7 F) were also constructed and in vitro and in vivo tests with canine heart muscle were performed. Both of these tests showed that the ultrasound catheters could deliver adequate energy to necrose cardiac tissue. The in vivo lesion depths of 5-9 mm indicated that ultrasound has significant potential for cardiac ablation for the treatment of arrhythmias.

Animals↗

Electrocardiographic characteristics and catheter ablation of parahissian accessory pathways.

BACKGROUND: Accessory pathways may be located in close proximity to the His bundle, resulting in a high risk of heart block during attempted surgical or electrical interruption of these pathways. This study reports the prevalence, ECG characteristics, and results of catheter ablation of parahissian accessory pathways. They were defined on the basis of both the presence of a high amplitude (> 0.1 mV) of His bundle potential at the ablation site and an exclusion of anteroseptal or midseptal location of the accessory pathway. METHODS AND RESULTS: Eight patients with a parahissian accessory pathway were identified among 582 consecutive patients who underwent radiofrequency ablation of an accessory pathway. They were six males and two females with a mean age of 21 +/- 9 years. During maximal preexcitation, the ECG showed a positive delta wave in leads I, II, and a VF in all patients: six had a negative delta wave in leads V1 and V2 instead of the positivity usually observed in anteroseptal accessory pathways. This pattern had a sensitivity of 75%, a specificity of 96%, a positive predictive value of 86%, and a negative predictive value of 93% for a parahissian location in comparison with a group of 28 patients with anteroseptal accessory pathways. At the successful ablation site, the mean amplitude of the His bundle potential was 0.2 +/- 0.1 (0.12 to 0.4 mV). All accessory pathways were successfully ablated without causing heart block using 5 to 20 W of radiofrequency energy. CONCLUSIONS: Parahissian accessory pathways have a preexcitation pattern that is distinctive from that of anteroseptal accessory pathways. Catheter ablation of these pathways is feasible using low energy with preservation of normal atrioventricular conduction.

Adolescent↗

Fructose-1,6-bisphosphatase of the yeast Kluyveromyces lactis.

The fructose-1,6-bisphosphatase [Fru(1,6)P2ase] gene of the budding yeast, Kluyveromyces lactis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354-amino-acid polypeptide. The polypeptide is different from other Fru(1,6)P2ases in that it contains a short amino-acid-insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. lactis enzyme with its closest homolog, the Saccharomyces cerevisiae Fru(1,6)P2ase (74% amino acid identity), reveals that the K. lactis enzyme is significantly less sensitive to AMP (Ki = 540 microM) than the S. cerevisiae enzyme (Ki = 190 microM). However, studies with a K. lactis Fru(1,6)P2ase mutant, in which the insert region (amino acids 152-160) was deleted by site-directed mutagenesis [(des-152-160)Fru(1,6)P2ase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Ki = 280 microM) than the control K. lactis enzyme. Thus, the nine-amino-acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. lactis wild-type enzyme. Catabolite-repression and catabolite-inactivation studies show that, unlike the complete repression of FBP1 mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiae, mRNA levels and enzyme activity of K. lactis Fru(1,6)P2ase decreased only about 2-4-fold due to the presence of glucose in the cell-culture medium.

Amino Acid Sequence↗

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments.

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.

Amino Acid Sequence↗

Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase.

The amino acid sequence of the spinach chloroplast fructose-1,6-bisphosphatase (FBPase) subunit has been determined. Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage. The sequence of spinach chloroplast FBPase shows clear homology (ca. 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose-1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme. The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity. This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines. The recent X-ray crystallographic resolution of pig kidney FBPase (H. Ke, C. M. Thorpe, B. A. Seaton, F. Marcus, and W. N. Lipscomb, 1989, Proc. Natl. Acad. Sci. USA 86, 1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms. It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are conserved in the chloroplast enzyme. The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxin-linked mechanism can be accommodated in the surface of the FBPase molecule.

Amino Acid Sequence↗

Spinach cytosolic fructose-1,6-bisphosphatase. Purification, enzyme properties and structural comparisons.

Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases.

Amino Acid Sequence↗

Structure refinement of fructose-1,6-bisphosphatase and its fructose 2,6-bisphosphate complex at 2.8 A resolution.

The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose 2,6-bisphosphate (Fru-2,6-P2) complexes have been refined to 2.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.021 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-2,6-P2 binding per tetramer have been identified by difference Fourier techniques. The Fru-2,6-P2 site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 12 A. The two Fru-2,6-P2 binding caves of the dimer in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg243 side-chains, indicating the potential for communication between the two sites. The beta, D-fructose 2,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-2,6-P2 interact with Arg243 from the adjacent monomer and the residues of Lys274, Asn212, Tyr264, Tyr215 and Tyr244 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly246 to Met248, as well as the side-chain atoms, Asp121, Glu280 and Lys274. The 2-phosphate group interacts with the side-chain atoms of Ser124 and Lys274. A negatively charged pocket near the 2-phosphate group includes Asp118, Asp121 and Glu280, as well as Glu97 and Glu98. The 2-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn125 and Lys269 are located within a 5 A radius of Fru-2,6-P2. We argue that Fru-2,6-P2 binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-2,6-P2 and the substrate; (2) sequence conservation of the residues directly interacting with Fru-2,6-P2 or located at the negatively charged pocket; (3) a divalent metal site next to the 2-phosphate group of Fru-2,6-P2; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys274, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

The prognostic significance of first myocardial infarction type (Q wave versus non-Q wave) and Q wave location. The Multicenter Diltiazem Post-Infarction Research Group.

The prognostic significance of the type of first acute myocardial infarction (Q wave versus non-Q wave) and Q wave location (anterior versus inferoposterior) was determined from a multicenter data base involving 777 placebo-treated patients who were participants in the Multicenter Diltiazem Post-Infarction Trial. There were 224 patients (29%) with a non-Q wave infarction, 326 (42%) with an inferoposterior Q wave infarction and 227 (29%) with an anterior Q wave infarction. Mean left ventricular ejection fraction was significantly (p less than 0.001) lower in patients with an anterior Q wave infarction than in the other two groups (anterior Q wave 0.39; inferior Q wave 0.52; non-Q wave 0.53). Nevertheless, the total cardiac mortality rate during the follow-up period (average 25 months per patient) was only marginally higher (p = 0.42) in the anterior Q wave group (8.4%) than in the other two groups (inferoposterior Q wave 7.1%; non-Q wave 6.3%). The total first recurrent cardiac event was somewhat higher (p = 0.08) in the anterior Q wave group (18.1%) than in the other two groups (inferoposterior Q wave 11.7%; non-Q wave 15.6%). Survivorship analyses extending over 3 years revealed that electrocardiographic classification of the type of first infarction and Q wave location did not make significant independent contributions to the risk of postinfarction cardiac death or first recurrent cardiac event, either before or after adjustment for baseline clinical variables.

Adult↗

Molecular structure of fructose-1,6-bisphosphatase at 2.8-A resolution.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the cortex of pig kidney and its complexes with either fructose 2,6-bisphosphate (Fru-2,6-P2) or adenosine monophosphate (AMP) have been crystallized in the space group P3(2)21. The three-dimensional structure of the native enzyme has been solved at 3.0-A resolution by the multiple isomorphous replacement method and refined at 2.8-A resolution to a crystallographic R factor of 0.194. A total of 316 of 335 residues, omitting disordered regions 1-5 and 54-67, have been built into the monomer, which has average dimensions of about 30 A by 50 A by 35 A. Four monomeric units aggregate into a molecular tetramer with D2 symmetry, which approximates a disk about 35 A thick. Each monomer consists of about 33% alpha-helix, 23% beta-strand, and 6% beta-turn. Four sites for Fru-2,6-P2 and two major sites for AMP binding per tetramer have been identified by difference Fourier techniques. The binding site for Fru-2,6-P2 is shared by two neighboring monomers and consists of side-chain atoms of Asn-212, Tyr-244, Tyr-264, and Lys-274; backbone atoms of Gly-246 through Met-248; and only Arg-243 from the adjacent subunit. In addition, Asn-125, Tyr-215, and Lys-269 are located within a distance of about 5 A of Fru-2,6-P2. A negatively charged pocket near this binding site includes Asp-118, Asp-121, Glu-280, Glu-97, and Glu-98. The AMP binding site is located near Val-17, Gln-20, Gly-21, Ala-24 through Met-30, Lys-112, Tyr-113, Arg-140, and Met-177.

Animals↗

Partial amino acid sequence of fructose-1,6-bisphosphatase from the blue-green algae Synechococcus leopoliensis.

Purified fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus leopoliensis was S-carboxymethylated and cleaved with trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography and the amino acid sequence of six of the purified peptides was determined by gas-phase microsequencing. The results revealed sequence homology with other fructose-1,6-bisphosphatases. The obtained sequence data provides information required for the design of oligonucleotide hybridization probes to screen existing libraries of cyanobacterial DNA. The determination of the amino acid sequence of cyanobacterial proteins may yield important information with respect to the endosymbiotic theory of evolution.

Amino Acid Sequence↗

Reactivity of the thiol groups of Escherichia coli phosphofructo-1-kinase.

Modification of Escherichia coli phosphofructo-1-kinase (6-phosphofructokinase; EC 2.7.1.11) with several thiol modifying reagents led to a pseudo-first-order loss of activity that was associated with the modification of a single cysteine residue, identified as the cysteine at position 119 in the protein sequence. This cysteine was protected from reaction with vinyl pyridine, bromopyruvate, and dithionitrobenzoic acid by the substrate, fructose-6-P. In the crystal structure of the highly homologous phosphofructokinase from Bacillus stearothermophilus, cysteine 119 is sufficiently distant from the catalytic site to exclude a direct steric inhibition of the binding of substrate as a mechanism of inactivation for the modification. Thus, the inhibition is unlikely to be a direct one but to be the result of interference with the conformational change that is associated with fructose-6-P binding. A second thiol, position 283, was shown to be protected from reaction when the enzyme was in its native conformation. In contrast to the previously published sequence for the E. coli enzyme six cysteines as opposed to seven have been found both in enzyme from strain LE392 and in enzyme produced by a plasmid that was derived from pLC 16-4. The position in question, 75, was identified as phenylalanine.

Chemical Phenomena↗

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis.

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.

Amino Acid Sequence↗