Are the 'core' proteins of the mitochondrial bc1 complex evolutionary relics of a processing protease?

The so-called 'core' proteins of the respiratory cytochrome bc1 complex and the two subunits of the mitochondrial processing peptidase (MPP) are structurally similar but their evolutionary relationship remains a mystery. Here, we present a model suggesting that the core proteins originated from an ancient proteolytic enzyme that was integrated into the bc1 complex during early stages of endosymbiosis.

The transport of hydrophobic ions across lipid bilayers.

The three-capacitor model for hydrophobic ion adsorption in lipid membranes (Andersen, O.S., Feldberg, S., Nakadomari, H., Levy, S. and McLaughlin, S. (1978) Biophys. J. 21, 35-70) is extended to ion transport whereby electrostatic effects from the interfacially adsorbed hydrophobic space charge have been encountered. The phenomena of current saturation with increasing concentrations of hydrophobic ions in the bulk electrolyte and the associated increase of the time constant of current relaxation can be quantitatively understood on the basis of space charge-limited currents as well as the nonexponential current decay.

Molecular structure of the 8.0 kDa subunit of cytochrome-c reductase from potato and its delta psi-dependent import into isolated mitochondria.

The cytochrome-c reductase (EC 1.10.2.2) of the mitochondrial respiratory chain couples electron transport from ubiquinol to cytochrome c with proton translocation across the inner mitochondrial membrane. The enzyme from potato was shown to be composed of 10 subunits. Isolation and characterization of cDNA clones for the second smallest subunit reveal an open reading frame of 216 bp encoding a protein of 8.0 kDa. The protein exhibits similarities to a 7.2/7.3 kDa subunit of cytochrome-c reductase from bovine and yeast, that is localized on the intermembrane space side of the enzyme complex. It also shows similarity to a previously unidentified 7.8 kDa protein of cytochrome-c reductase from Euglena. The potato 8.0 kDa protein has a segmental structure, as its sequence can be divided into four parts, each comprising a central Arg-(Xaa)5-Val motif. N-terminal sequencing of the mature 8.0 kDa proteins indicates the absence of a cleavable mitochondrial targeting sequence. Import of the in vitro synthesized 8.0 kDa protein into isolated potato mitochondria confirms the lack of a presequence and reveals a dependence of the transport on the membrane potential delta psi across the inner mitochondrial membrane. These features are unique among the intermembrane space proteins known so far.

Wave-guide spectroscopy on planar lipid bilayers doped with hydrophobic ions.

Wave-guide spectroscopy exploits the light pipe properties of planar lipid bilayers by propagating a light wave along the plane of the bilayer. Applying this technique to the optical absorption of chromophore in the membrane, results in an enhanced sensitivity when compared to normal incidence spectroscopy. This gain factor is of the order of 100 per mm optical path along the bilayer, thus transforming the weak absorbances in lipid membranes into easily measurable quantities. Wave-guide spectroscopy has been used to measure the adsorption isotherm of hydrophobic dipicrylamine ions in a phosphatidylcholine membrane. The adsorption isotherm is linear for low aqueous concentrations, in the micromolar range however, it changes into a sublinear dependence. The addition of an inert alkali salt to the electrolyte favours the adsorption of hydrophobic ions. Current saturation is observed with the transition to the sublinear isotherm. When using the time constant for current relaxation as an indicator of changes in the magnitude of the surface potential, it does not seem to vary with the additional dipicrylamine which adsorbs in the presence of high concentrations of alkali salt in the electrolyte. A compensation of hydrophobic charge by the alkali ions from the inert electrolyte is proposed.

Primary structure, cell-free synthesis and mitochondrial targeting of the 8.2 kDa protein of cytochrome c reductase from potato.

Cytochrome c reductase from potato comprises ten subunits with apparent molecular sizes between 55 and < 10 kDa. The subunit with the highest electrophoretic mobility on SDS-polyacrylamide gels was isolated and analysed by cyclic Edman degradation. Mixtures of degenerative oligonucleotides were derived from the obtained sequence data and used for the isolation of corresponding cDNA clones. The clones encode a protein of 72 amino acids which exhibits significant sequence identity with a 9.5 kDa subunit of cytochrome c reductase from bovine and a 11 kDa subunit of the enzyme complex from yeast. Comparison between the deduced amino acid sequence of the open reading frame and the sequence of the mature protein reveals that only the initiator methionine is absent in the functional subunit. Hence the protein has a calculated molecular mass of 8.2 kDa. Transcripts of the potato 8.2 kDa protein were not translated in reticulocyte lysates but in vitro translation worked efficiently with wheat germ lysate. Import of the radiolabelled protein into isolated mitochondria from potato seems to depend on a potential across the inner membrane and confirms the absence of a cleavable mitochondrial presequence.

Primary structure and expression of a gene encoding the cytosolic ribosomal protein S4 from potato.

The primary structure of a cDNA clone encoding the S4 protein from the small subunit of 80S ribosomes from potato was determined. Cytosolic ribosomal protein S4 is hydrophilic and has a prevalence for positively charged residues. In potato it is 264 amino acids long and contains a putative nuclear targeting signal close to the N-terminus. Having 65-69% identical amino acids cytosolic ribosomal protein S4 from mammals, fungi and plants belongs to the highly conserved proteins. The S4 gene is transcribed in all potato tissues analysed and has a relatively high expression level in comparison to nuclear genes encoding mitochondrial proteins.

The mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which plays an essential role in mitochondrial protein import. It cleaves off the N-terminal targeting signals of nuclear encoded mitochondrial proteins upon their transport into the organelle. In mammals and yeast the enzyme is localized in the mitochondrial matrix while in plants it is integrated into a protein complex of the respiratory chain. As the activity of MPP is essential for the viability of eukaryotic cells it is conceivable that inhibitors of MPP which are specific for the soluble enzyme only present in fungi and animals may work as fungicides or insecticides.

Purification and sequencing of cytochrome b from potato reveals methionine cleavage of a mitochondrially encoded protein.

Several mitochondrial genes from a large number of different fungi, mammals and plants have been sequenced but little is known about the corresponding translation products. We have affinity purified cytochrome c reductase from potato mitochondria and isolated the mitochondrially encoded cytochrome b protein. Amino-terminal sequencing reveals that the polypeptide does not start with a methionine. Comparison of the amino acid sequence with the recently published sequence of the gene encoding the cytochrome b apoprotein suggests that the N-formylmethionine is removed. This result provides the first evidence for the presence of a deformylase and a methionine aminopeptidase in mitochondria.

The 'Hinge' protein of cytochrome c reductase from potato lacks the acidic domain and has no cleavable presequence.

The 'Hinge' protein of cytochrome c reductase from fungi and mammals is thought to support electron transport from cytochrome c1 to cytochrome c and was reported to be one of the most acidic proteins known. Isolation and analysis of cDNA clones of the first 'Hinge' protein from a plant source reveals that it has a surplus of basic residues in potato. While the overall identity between the deduced amino acid sequence of the potato 'Hinge' protein and the proteins from yeast and bovine is in the range of 40%, the characteristic acidic domain is lacking. Therefore the numerous theories on the function of the mitochondrial 'Hinge' protein seem not to apply for the protein from potato. Also the atypical acidic presequence of the 'Hinge' protein from fungi and mammals is absent as revealed by N-terminal sequencing of the isolated potato 'Hinge' protein. Functional implications of these results for the 'Hinge' proteins from other organisms are discussed.

[Multilocular cystic nephroma]. / Multilokuläres zystisches Nephrom.

We report on a case of the rare entity of multilocular cystic nephroma. The clinical symptoms, the problems with differential diagnosis and the therapy are described. The histological findings, origin and classification are discussed.

[Report of 2 cases of isolated, primary amyloidosis of the male urethra and urinary bladder of a female]. / Bericht über zwei Fälle einer isolierten, primären Amyloidose der männlichen Harnröhre und der Harnblase einer Frau.

Primary localized amyloidosis of the bladder and urethra is a rare condition. Two cases of isolated primary amyloidosis, of the urethra in a man and of the bladder in a woman, are reported. The clinical specificity of the location and the specific therapy are discussed. We draw attention to the extended, palette of diagnostic investigations now available and the new classification made possible by the introduction of immunohistochemistry and immunoelectron microscopy.

[Primary paraurethral malignant mixed tumor (carcinosarcoma) of the vagina. A case report]. / Primärer paraurethraler maligner Mischtumor (Karzinosarkom) der Vagina. Ein Fallbericht.

In total malignant mixed tumors (carcinosarcomas) represent a very rare gynecological neoplasm. In accordance with the embryological genesis primary localizations are with descending probability the uterus, the ovarians and finally the tubes. We report on a 55 year old female patient, with a paraurethral mass, which was primarily diagnosed in 1994. Histopathological evaluation of a transvaginal biopsy did not demonstrate evidence of malignancy at that time. After painful enlargement the paraurethral mass was resected surgically and histopathological evaluation revealed a primary paraurethral malignant mixed tumor (carcinosarcoma). Postoperatively, the patient underwent percutaneous (46.4 Gy) and intracavitary (2 x 6 Gy) radiation. Twelve months postoperatively there is no evidence of disease.

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels.

Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.

[Rates of prostate-specific antigen testing for early detection of prostate cancer: a first comparison of German results with current international data]. / PSA-basierte Früherkennung des Prostatakarzinoms durch den Hausarzt: Erste deutsche Ergebnisse im internationalen Vergleich.

OBJECTIVE: Measurement of prostate-specific antigen (PSA) is not only used as a screening instrument by urologists, but also by general practitioners and internal specialists (GP-IS). Until now, there are neither data on the approach of German GP-IS in practicing this nor have data been classified in the context of available international literature on this topic. MATERIALS AND METHODS: Between May and December 2012, a questionnaire containing 16 items was sent to 600 GP-IS in Brandenburg and Berlin. The response rate was 65% (392/600). Six indicator questions (IQ1-6) were selected and results were set in the context of available international data. The quality of present studies was evaluated by the Harden criteria. RESULTS: Of the 392 responding physicians, 317 (81%) declared that they would use PSA testing for early detection of PCA (IQ1) and, thus, formed the study group. Of these GP-IS, 38% consider an age between 41 and 50 years as suitable for testing begin (IQ2), while 53% and 14% of the GP-IS perform early detection until the age of 80 and 90 years, respectively (IQ3). A rigid PSA cut-off of 4 ng/ml is considered to be reasonable by 47% of the involved GP-IS, whereas 16% prefer an age-adjusted PSA cut-off (IQ4). Patients with pathological PSA levels were immediately referred to a board-certified urologist by 69% of the GP-IS. On the other hand, 10% first would independently control elevated PSA levels themselves after 3-12 months (IQ5). Furthermore, 14% of the interviewed physicians consider a decrease of PCA-specific mortality by PSA screening as being proven (IQ6). Knowledge regarding PCA diagnostics is mainly based on continuous medical education for GP-IS (33%), personal contact with urologists (6%), and guideline studies (4%). While 53% indicated more than one education source, 4% did not obtain any PCA-specific training. The results provided by this questionnaire evaluating response of German GP-IS to six selected indicator questions fit well into the international context; however, further studies with sufficient methodical quality are required. CONCLUSIONS: Despite current findings and controversial recommendations of the two large PCA screening studies on this issue, German GP-IS still frequently use PCA screening by PSA measurement. Primary strategies of early detection as well as follow-up after assessment of pathologically elevated PSA levels poorly follow international recommendations. Thus, an intensification of specific education is justified.

Engineering photorespiration: current state and future possibilities.

Reduction of flux through photorespiration has been viewed as a major way to improve crop carbon fixation and yield since the energy-consuming reactions associated with this pathway were discovered. This view has been supported by the biomasses increases observed in model species that expressed artificial bypass reactions to photorespiration. Here, we present an overview about the major current attempts to reduce photorespiratory losses in crop species and provide suggestions for future research priorities.

Identification of novel homologues of three low molecular weight subunits of the mitochondrial bc1 complex.

Large-scale random cDNA sequencing projects have been started for several organisms and are a valuable tool for the analysis of quantitative and qualitative aspects of gene expression. However, the reliability of the obtained data is limited as most of the clones are only partially analysed on one strand. As a consequence the sequence entries derived from random cDNA sequencing projects usually comprise incomplete open reading frames. They nevertheless define complete and reliable coding sequences, if two prerequisites are fulfilled: (i) the clones encode very small proteins, and (ii) the clones have a high frequency in the cDNA-banks. The present study describes the use of cDNA databases for the identification of homologues of three low-molecular-weight subunits of the mitochondrial bc1 complex, termed the QCR6, QCR9 and QCR10 proteins. These polypeptides are only characterized for a small number of organisms, have a scarcely defined function and exhibit a low degree of structural conservation if compared between different species. Several clones were identified for each polypeptide by searches with TBLASTN using the known sequences as probes. Most of the database entries contain complete open reading frames and sequencing queries could be excluded due to the abundancy of the clones. Multiple sequence alignments are presented for all three polypeptides and consensus sequences are given which may provide a basis for the investigation of the proteins by site-directed mutagenesis.