Medizin - Open Access LMU - Teil 09/22 Archiv

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.

During storage of CPD-A1 preserved whole blood factors of the complement cascade become activated, as evidenced by a rapid increase in the concentrations of C3a-desArg and C4a-desArg. After 10 to 14 days of whole blood storage, the elevations of C3a and C4a levels were highly significant. This increase was paralleled by an increase in the concentration of the lysosomal proteinase elastase from polymorphonuclear (PMN) granulocytes. By contrast, the concentration of the C3 activator complex C4b2b remained unchanged even after 3 weeks of storage. The supplementation of the anticoagulant CPD-A1 with the polyvalent-proteinase-inhibitor aprotinin and the specific elastase- inhibitor eglin C failed to inhibit complement activation, whereas leukocyte depletion could partially abolish the increase of the concentration of C4a, but had no effect on C3a concentrations. These observations support the notion that cleavage of C4 during storage of whole blood is partially leukocyte dependent, whereas the activation of C3 is possibly caused by the activation of the alternate pathway of the complement system by contact of plasma with plastic surfaces.

The plasmid-encoded outer membrane protein YadA of enteropathogenic yersiniae is associated with pathogenicity. Recently, collagen binding of YadA-positive yersiniae was reported without detailed characterization (L. Emödy, J. Heesemann, H. Wolf-Watz, M. Skurnik, G. Kapperud, P. O'Toole, and T. Wadström, J. Bacteriol. 171:6674-6679, 1989). To elucidate the nature of collagen binding to YadA, we used a recombinant Yersinia strain expressing the cloned YadA gene. Direct binding of YadA-positive yersiniae to collagens was demonstrated in affinity blot experiments on nitrocellulose filters. A spectrum of collagen types in a wide concentration range were tested for their ability to block binding of 125I-labeled collagen type II to YadA-positive yersiniae. The results indicate a specific binding site(s) for YadA in collagen types I, II, III, IV, V, and XI. In contrast, collagen type VI did not bind to YadA. To characterize the binding site(s) more precisely, isolated collagen chains and cyanogen bromide fragments were investigated. These studies revealed that binding of YadA to collagen type I is confined to the alpha 1(I) chain, whereas the binding site within collagen type XI is localized in the alpha 3(XI) chain. alpha 2(I), alpha 1(XI), and alpha 2(XI) did not bind to YadA. Most interestingly, in the alpha 1(II) chain the specific binding site for YadA resides in the cyanogen bromide fragment CB10. The latter might indicate a binding site that does not depend on conformation. Based on these findings, further fragmentation and the synthesis of peptides may allow definition of the peptide sequence(s) relevant for YadA binding.

Subchondral bone density (by means of CT osteoabsorptiometry), and cartilage thickness (directly measured on photocopies of frozen sections), were examined in 30 human patellae, with an age range from 47 to 90 y. A surface demonstration of the distribution was prepared, and representative pictures produced by summation with a computer. Subchondral bone-density maxima were found in the proximal part of the lateral facet, and the density pattern interpreted as the expression of the long-term distribution of stress in the joint. It is reasonable to assume that cartilage thickness, of which two-thirds of the maximum values occupy a lateral position, is also dependent on the local stress. The 2 distributions show correlation coefficients > 0.5 in approximately a third of the cases we examined. Displacement of the higher values of cartilage thickness relative to the subchondral density maxima is attributed to incongruence in the medial part of the joint.

The relation of (multiple) organ failure (OF) to the release of inflammatory mediators and the incidence of infection and sepsis was studied prospectively in 100 patients with multiple trauma (injury severity score=37). Sixteen patients died of OF, 47 patients survived OF, and 37 patients had no OF. Fifteen (24%) of the patients with OF showed no signs of infection. In patients with early onset of OF (n=45), infection followed with a lag of 2 or more days. In 16 (44%) of these patients, infection led to a deterioration in organ function. With late onset of OF (n=18), infection preceded OF in nine patients. Polymorphonuclear leukocyte—elastase, neopterin, C-reactive protein, lactate, antithrombin III, and phospholipase A discriminated significantly among the three outcome groups. Of all factors, only polymorphonuclear leukocyte—elastase showed a difference between patients with and without infection or sepsis, respectively. These data indicate that infection might not play a crucial role in the pathogenesis of posttraumatic OF in a substantial portion of patients with trauma. Early OF, especially, seems to be mainly influenced by the direct sequelae of tissue damage and shock (eg, the release of inflammatory mediators). Since infection and sepsis did not lead to an augmented release of mediators in patients with trauma, the role of both entities remains unclear.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Antifolding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein. Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were cross-linked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOMS, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.