Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell‐free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. Apocytochrome c synthesized in vitro was found in the post‐ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labelling kinetics do not suggest a precursor role of microsomal apocytochrome c or holocytochrome c. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The probles of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.

Megakaryocytes are difficult to isolate because of their fragility, their tendency to aggregate, and their varying sizes. For purification of cells at different stages of maturation and of different sizes (ploidy classes) we developed an immunomagnetic cell sorting method (MACS) to enrich the whole spectrum of the megakaryocytic cell lineage. The use of small magnetic beads coupled to various antibodies and labelling with fluorescent antibodies allowed direct analysis of enrichment and evaluation of the isolated fraction without further staining or detachment procedures. CD 61 (Y2/51), a monoclonal antibody directed against platelet glycoprotein IIIa, was employed to perform the separation procedure. An enrichment up to 47% of CD 61‐positive cells with an average of 37% and a recovery rate of 37% was obtained by using the MACS technique. Pre‐enrichment by Percoll density centrifugation, followed by MACS separation, resulted in an enrichment of 65% and a recovery rate of 67%. The relative amount of small megakaryocytic cells in only MACS‐enriched cell populations, however, was higher than in Percoll/MACS fractions. As a parameter of vitality we tested cytokine secretion of the enriched megakaryocytes in reverse haemolytic plaque assays. Secretion of IL‐1, IL‐6, GM‐CSF, and PDGF with and without stimulation by phorbol myristate acetate was demonstrable at the single cell level.

We have undertaken a randomized study on 20 patients undergoing coronary artery bypass surgery in order to determine the influence of cardiopulmonary pump prime solutions on colloid osmotic pressure and extravascular lung water. Crystalloid priming with Ringer lactate was compared with an albumin solution of nearly physiological colloid osmotic composition (4%). Measurements of extravascular lung water were performed by a modified, highly sensitive thermal dye technique, with additional detection of tracer signals in the pulmonary artery. In the Ringer lactate group, a significantly greater decrease in colloid osmotic pressure occurred immediately after onset of cardiopulmonary bypass. The more pronounced decrease in colloid osmotic pressure and in transcapillary gradient (difference between colloid osmotic pressure and pulmonary capillary wedge pressure) in the Ringer lactate group was associated with a significant increase in extravascular lung water (by 60%) in the postoperative period; the human albumin group, however, showed only a slight tendency to increased lung water. There were no differences in haemodynamic or respiratory states after operation.

Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crussa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells.Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria.The transfer of immunoprecipitablc mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c.Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiales between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins.In the cell-free homogenate membrane-bound ribosonies are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles.The results suggest that the transport of cytoplasniically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation ; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.In the preceding paper we studied the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurosporu crassu [l]. la whole cells, the processes of synthesis and transport are not easily separated. Furthermore, post-labelling fractionation artifacts may obscure the intracellular location of mitochondrial proteins which are on their path from the site of synthesis to the site of function.In considering systems in vitro a number of possibilities exist, ranging Crom cell-free homogenates to reconstituted systems involving isolated and purified protein and organelle components. In the present study we have investigated synthesis and transport in a cell-free homogenate. We. have employed this system in an attempt lo decide between two mechanisms currently proposed for the transport of mitochondria] proteins. The first mechanism is based on observations with whole IC'emrospora cells [l]. This mechanism proposes the existence of extramitochondrial pools of mitochondrial proteins from which they are imported into the mitochondria. The second mechanism is that proposed by Butow and his colleagues [3 -61. This latter mechanism proposes a special class of cytoplasmic ribosomes which are attached to the mitochondria at specific sites. The nascent polypeptides from these ribosomes are discharged in a vectorial manner into the mitochondria.The results we present here support our earli...

BackgroundEpidemiological studies have shown that ambient particulate matter (PM) and changes in air temperature are associated with increased cardiopulmonary events.ObjectiveWe hypothesized that patients with previous myocardial infarction (MI) experience changes in heart rate (HR) and repolarization parameters, such as Bazett-corrected QT interval (QTc), and T-wave amplitude (Tamp), in association with increases in air pollution and temperature changes.MethodsBetween May 2003 and February 2004, 67 MI survivors from the Augsburg KORA-MI registry repeatedly sent 16 sec electrocardiograms (ECGs) with a personal transmitter (Viapac) via telephone to the Philips Monitoring Center, where ECG parameters were immediately analyzed. Meteorological data and air pollutants were acquired from fixed monitoring sites on an hourly basis. Additive mixed models were used for analysis. Effect modification by patient characteristics was investigated.ResultsThe analysis of the 1,745 ECGs revealed an increased HR associated with interquartile range (IQR) increases in PM levels among participants not using beta-adrenergic receptor blockers and among those with body mass index ≥ 30 kg/m2. We observed a 24- to 47-hr lagged QTc prolongation [0.5% change (95% confidence interval, 0.0–1.0%)] in association with IQR increases in levels of PM ≤ 2.5 μm in aerodynamic diameter, especially in patients with one [0.6% (0.1–1.0%)] or two [1.2% (0.4–2.1%)] minor alleles of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) single-nucleotide polymorphism rs2364725. Positive immediate (0–23 hr) and inverse delayed (48–71 hr up to 96–119 hr) associations were evident between PM and Tamp. We detected an inverse U-shaped association between temperature and Tamp, with a maximum Tamp at 5°C.ConclusionsIncreased air pollution levels and temperature changes may lead to changes in HR and repolarization parameters that may be precursors of cardiac problems.