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Biomedical subjects

M Horst

Publications and source records attributed to M Horst.

At least 37 records · Page 2Linked to original sources
PubMed1997

QT dispersion is determined by the extent of viable myocardium in patients with chronic Q-wave myocardial infarction.

BACKGROUND: QT dispersion is lower in patients with successful thrombolysis after acute myocardial infarction, suggesting that QT dispersion may be determined by the extent of viable and scarred myocardium. METHODS AND RESULTS: To test this hypothesis, QT dispersion was measured in a 12-lead resting ECG in 44 patients with chronic Q-wave myocardial infarction. To assess the extent of viable and scarred myocardium, all patients underwent F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET). In addition, all patients had revascularization of the infarct-related artery and repeated angiography 4 months later. QT dispersion was lower (53+/-20 versus 94+/-24 ms, P<.0001) in patients with evidence of a substantial amount of viable myocardium in the infarct region as demonstrated by PET (average FDG uptake > or = 50% of normalized, maximum FDG uptake) than in patients with only minimal residual viability. Average FDG uptake of the infarct region and FDG defect size were significantly related to QT dispersion (r=.64, P<.0001; r=.67, P<.0001), whereas ejection fraction was not (r<.1, P=NS). QT dispersion of < or = 70 ms had a sensitivity of 85% and a specificity of 82% to predict viable myocardium in the infarct region. QT dispersion was also lower in patients with improvement of left ventricular function 4 months after revascularization (54+/-21 versus 88+/-30 ms, P=.0003). QT dispersion of < or = 70 ms had a sensitivity of 83% and a specificity of 71% to predict improvement of left ventricular function. CONCLUSIONS: QT dispersion is determined by the amount of viable myocardium in the infarct region and may serve as a novel, rapidly available marker of substantial viability in the infarct region of patients with chronic Q-wave myocardial infarction.

Angioplasty, Balloon, Coronary↗
PubMed1997

The mitochondrial hsp70 chaperone system. Effect of adenine nucleotides, peptide substrate, and mGrpE on the oligomeric state of mhsp70.

Mitochondrial hsp70 (mhsp70) is a key component in the import and folding of mitochondrial proteins. In both processes, mhsp70 cooperates with the mitochondrial nucleotide exchange factor mGrpE (also termed Mge1p). In this work we have characterized the self-association of purified mhsp70, the interaction of mhsp70 with isolated mGrpE and protein substrate, and the effect of nucleotides on these interactions. mhsp70 can form oligomers that are dissociated by ATP or by a nonhydrolyzable ATP analog. A substrate peptide binds to mhsp70 in the absence of added nucleotides and is released by ATP but not by ADP. Binding of the peptide causes nucleotide-independent dissociation of the mhsp70 oligomers and enhances the mhsp70 ATPase. Purified mGrpE forms a homodimer. In the absence of added nucleotides, one mGrpE dimer binds to one molecule of mhsp70, forming a stable 122 kDa hetero-oligomer. This complex is weakened by ADP and completely dissociated by ATP.

Adenosine Diphosphate↗
PubMed1997

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Adenosine Diphosphate↗
PubMed1997

What is the driving force for protein import into mitochondria?

Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as precursors and then imported into mitochondria. Protein import into the matrix space requires the function of the mitochondrial hsp70 (mhsp70) chaperone. mhsp70 is an ATPase that acts in conjunction with two partner proteins: the Tim44 subunit of the inner membrane import complex, and the nucleotide exchange factor mGrpE. A central question concerns how mhsp70 uses the energy of ATP hydrolysis to transport precursor proteins into the matrix. Recent evidence suggests that mhsp70 is a mechanochemical enzyme that actively pulls precursors across the inner membrane.

Adenosine Triphosphate↗
PubMed1996

The Mas20p and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: evidence for a single hetero-oligomeric receptor.

Protein import into yeast mitochondria is mediated by four integral outer membrane proteins which function as import receptors. These proteins (termed Mas20p, Mas22p, Mas37p and Mas70p) appear to exist as two subcomplexes: a Mas37p-Mas70p heterodimer and a less well characterized Mas20p-Mas22p complex. The subcomplexes interact functionally during protein import, but it has remained uncertain whether they are in direct contact with each other in vivo. Here we show that Mas20p and Mas70p can be cross-linked in intact mitochondria, or co-immunoprecipitated from digitonin-solubilized mitochondria. Furthermore, the cytosolic domains of these two proteins interact in the 'two-hybrid' system. Association of Mas20p and Mas70p is virtually abolished by a mutation in the single tetratricopeptide motif in Mas20p. This mutation specifically inhibits import of precursors that are first recognized by Mas37p-Mas70p and only then transferred to Mas20p-Mas22p. We conclude that the two receptor subcomplexes of the mitochondrial protein import receptor interact in vivo via their Mas20p and Mas70p subunits and that this interaction is functionally important.

Base Sequence↗
PubMed1996

The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis.

During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.

Adenosine Triphosphate↗
PubMed1996

Predictive value of low dose dobutamine transesophageal echocardiography and fluorine-18 fluorodeoxyglucose positron emission tomography for recovery of regional left ventricular function after successful revascularization.

OBJECTIVES: This study was designed to assess the predictive value of myocardial viability diagnosed by dobutamine transesophageal echocardiography and fluorine (F)-18 fluorodeoxyglucose positron emission tomography for left ventricular functional recovery after revascularization in patients with chronic left ventricular dysfunction. BACKGROUND: The identification of akinetic but viable myocardium is of particular importance for the selection of patients with a compromised left ventricle who will benefit from coronary revascularization. METHODS: Multiplane rest and dobutamine transesophageal echocardiography (dobutamine, 5 and 10 microg/min per kg) studies and F-18 fluorodeoxyglucose positron emission tomographic studies at rest were performed in 2 patients with 1) previous myocardial infarction and regional akinesia, 2) a stenosed infarct-related coronary artery, and 3) a patent infarct-related vessel after revascularization. A basally akinetic segment was considered viable by transesophageal echocardiography if dobutamine-induced contractile reserve could be observed. Viability by positron emission tomography was defined as F-18 fluorodeoxyglucose uptake > or = 50% of the maximal uptake in a region with normal wall motion. Recovery of regional left ventricular function 4 to 6 months after revascularization was diagnosed by transesophageal echocardiography if > or = 50% of segments akinetic at baseline had improved wall thickening. RESULTS: Dobutamine transesophageal echocardiography identified viable infarct regions in 25 (59%) of 42 patients, and F-18 fluorodeoxyglucose positron emission tomography in 30 (71%) of 42 patients, yielding diagnostic agreement in 86% of patients. Sensitivity and specificity for prediction of left ventricular functional recovery in individual patients was 92% and 88%, respectively, for dobutamine transesophageal echocardiography versus 96% and 69% for F-18 fluorodeoxyglucose positron emission tomography. Segments remaining akinetic after revascularization had a significantly lower (p < 0.001) F-18 fluorodeoxyglucose uptake (48 +/- 15%) than that (73 +/- 15%) of segments with recovery of regional left ventricular function. CONCLUSIONS: Both dobutamine transesophageal echocardiography and F-18 fluorodeoxyglucose positron emission tomography were highly sensitive in predicting functional recovery of chronically kinetic or dyskinetic myocardium after successful revascularization. Thus, dobutamine transesophageal echocardiography is a clinically valuable alternative to F-18 fluorodeoxyglucose positron emission tomography for assessing residual viability and predicting functional recovery after revascularization.

Adrenergic beta-Agonists↗
PubMed1995

Reconstitution of the initial steps of mitochondrial protein import.

We have reconstituted the initial steps of mitochondrial protein import with a purified precursor protein, a purified, ATP-dependent, cytosolic chaperone selective for mitochondrial precursors (mitochondrial import stimulating factor; MSF), and either intact mitochondria or intact or solubilized mitochondrial outer membranes. We show that the precursor-MSF complex first binds to the Mas37p/Mas70p subunits of the mitochondrial import receptor. After ATP-dependent release of MSF, the precursor is transferred from Mas37p/Mas70p to the Mas20p/Mas22p subunits of the receptor, and finally delivered to the import channel in the outer membrane. Import in the absence of the MSF bypasses Mas37p/Mas70p. The ATP-mediated transfer of a precursor from MSF to specific subunits of the import receptor is similar to the GTP-mediated transfer of precursors from the signal recognition particle to its receptor on the endoplasmic reticulum.

14-3-3 Proteins↗
PubMed1995

Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria.

Mitochondria contain two distinct protein import systems, one in the outer and the other in the inner membrane. These systems can act independently of one another in submitochondrial fractions of if a protein is transported to the outer membrane or to the intermembrane space. It has been proposed that the two systems associate reversibly when a protein is transported across both membranes, but this hypothesis has remained unproven. In order to address this question, we have checked whether antibodies against a subunit of one system can co-immunoprecipitate subunits of the other system. We find that the two systems associate stably if a matrix-targeted precursor is arrested during import; no association is seen in the absence of a stuck precursor. These experiments provide direct evidence that protein import into the mitochondrial matrix is mediated by the reversible interaction of the two translocation systems.

Animals↗
PubMed1995

Internal sequences from proteins digested in polyacrylamide gels.

A simple method for proteolytic digestion of Coomassie blue-stained proteins in a polyacrylamide matrix is presented. It consists of first reducing and alkylating the stained proteins with dithiothreitol and iodoacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequent digestion with the endoproteinase LysC. The reduction and alkylation step was introduced since experiments with lysozyme and ribonuclease A showed that extremely complex peptide patterns were obtained if no precautions were taken to suppress disulfide bond formation during in-gel digestion of proteins. The advantage of this method is that no blotting step is required for generating internal sequences and that extensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate internal sequence data from low microgram quantities of proteins excised from 2-dimensional Coomassie blue-stained gels.

Acrylic Resins↗
PubMed1995

Increases in intra-abdominal pressure affect pulmonary compliance.

OBJECTIVES: To determine the effect of increased intra-abdominal pressure (IAP) on pulmonary compliance and to determine an effective means to measure IAP. DESIGN: A prospective study. SETTING: An urban tertiary care hospital. PATIENTS: Twenty-six adult patients undergoing laparoscopic cholecystectomy. INTERVENTIONS: Intra-operative management of laparoscopic cholecystectomy requiring endotracheal intubation with general anesthesia, nasogastric and urinary bladder catheters, and position changes. Additional interventions included use of a rectal manometer and a respiratory pressure module inserted within the ventilator circuit. MAIN OUTCOME MEASURES: Correlation of changes in IAP with changes in dynamic pulmonary compliance, measured as tidal volume/(end inspiratory pressure--end expiratory pressure) and comparison of three different measurement techniques (bladder, rectal, and gastric) with a standard technique (insufflation pressure) in three different positions (supine, Trendelenburg's, and reverse Trendelenburg's). RESULTS: Compliance was significantly related to insufflation pressure (P < .001) by analysis of variance. In the gas insufflation model, the mean increment in bladder pressure reflected most closely the IAP increment in the supine position (5.7 vs 6 mm Hg) but not in the Trendelenburg (2.1 vs 6 mm Hg) and reverse Trendelenburg positions (3.4 vs 6 mm Hg). Rectal and gastric pressures were also position dependent and technically less reliable. CONCLUSIONS: Increased IAP has a major influence on pulmonary compliance (50% decrease at 16 mm Hg). Measurements of IAP by intraorgan manometry are position dependent and may not accurately reflect the intraperitoneal pressure.

Abdomen↗
PubMed1995

Mas37p, a novel receptor subunit for protein import into mitochondria.

By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria. Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions. Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration-driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p. Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p. Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other. We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.

Amino Acid Sequence↗
PubMed1994

Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane.

The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.

Adenosine Triphosphate↗
PubMed1994

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C-terminal hexa-histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel-derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.

Adenosine Triphosphate↗
PubMed1993

Synthesis of phosphorylated oligosaccharides in lysozyme is enhanced by fusion to cathepsin D.

Chinese hamster ovary cells transfected with human lysozyme cDNA encoding Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme, with about 60% of the molecules bearing carbohydrate. This carbohydrate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation of [Asn22] lysozyme fused to human cathepsin D is altered relative to [Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa precursor that is cleaved to enzymatically active and antigenically positive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the lysozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the precursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intracellularly released lysozyme portion of the fusion protein contains trimmed oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enriched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate processing in [Asn22]lysozyme, including the synthesis of mannose 6-phosphate residues and of lactosamine repeats, is altered by the attached cathepsin D. The phosphorylation of the carbohydrate on the lysozyme portion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.

Animals↗
PubMed1993

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.

Amino Acid Sequence↗