A Commensal Strain of Staphylococcus epidermidis Overexpresses Membrane Proteins Associated with Pathogenesis When Grown in Biofilms.

Staphylococcus epidermidis has emerged as one of the major nosocomial pathogens associated with infections of implanted medical devices. The most important factor in the pathogenesis of these infections is the formation of bacterial biofilms. Bacteria grown in biofilms are more resistant to antibiotics and to the immune defence system than planktonic bacteria. In these infections, the antimicrobial therapy usually fails and the removal of the biofilm-coated implanted device is the only effective solution. In this study, three proteomic approaches were performed to investigate membrane proteins associated to biofilm formation: (i) sample fractionation by gel electrophoresis, followed by isotopic labelling and LC-MS/MS analysis, (ii) in-solution sample preparation, followed by isotopic labelling and LC-MS/MS analysis and (iii) in-solution sample preparation and label-free LC-MS/MS analysis. We found that the commensal strain S. epidermidis CECT 231 grown in biofilms expressed higher levels of five membrane and membrane-associated proteins involved in pathogenesis: accumulation-associated protein, staphylococcal secretory antigen, signal transduction protein TRAP, ribonuclease Y and phenol soluble modulin beta 1 when compared with bacteria grown under planktonic conditions. These results indicate that a commensal strain can acquire a pathogenic phenotype depending on the mode of growth.

Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Actin and myosin contribute to mammalian mitochondrial DNA maintenance.

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

Clinical spectrum of gram-positive infections in lung transplantation.

PURPOSE: Gram-positive (GP) organisms are among the most common cause of infections in early postsurgical and immunocompromised populations. Patients recovering from lung transplantation (LT) are particularly susceptible owing to the physiologic stress imposed by surgery and induction with intense immunosuppression. Sites, types, and timing of GP infections following LT are not well documented. This report describes the clinical spectrum of GP infections and their effects on surgical airway complications (SAC) and bronchiolitis obliterans syndrome (BOS) following LT. METHODS AND MATERIALS: Data were collected from 202 patients undergoing 208 LT procedures at a single institution between November 1990 and November 2005. Data were retrospectively analyzed according to timing, location, and causative pathogen. RESULTS: In the median follow-up period of 2.7 years (range, 0-13.6 years), 137 GP infections were confirmed in 72 patients. Sites of infection included respiratory tract (42%), blood (27%), skin, wound and catheter (21%), and other (10%). GP pathogens identified were Staphylococcus species (77%), Enterococcus species (12%), Streptococcus species (6%), Pneumococcus (4%), and Eubacterium lentum (1%). The likelihood of SAC and BOS was increased in lung allograft recipients with GP pneumonia as compared with those without (hazard ratio 2.1; 95% confidence interval=1.5-3.1). CONCLUSIONS: GP organisms were responsible for infections in 40% of lung allograft recipients and most commonly isolated from the respiratory tract and blood stream. Staphylococcal species were most frequently identified, 42% of which were methicillin-resistant Staphylococcus aureus (MRSA). Given the strong association of respiratory tract infections with the development of SAC and BOS, empiric antimicrobial strategies after LT should include agents directed against GP organisms, especially MRSA.

Inhibitors of the catalytic domain of mitochondrial ATP synthase.

An understanding of the mechanism of ATP synthase requires an explanation of how inhibitors act. The catalytic F1-ATPase domain of the enzyme has been studied extensively by X-ray crystallography in a variety of inhibited states. Four independent inhibitory sites have been identified by high-resolution structural studies. They are the catalytic site, and the binding sites for the antibiotics aurovertin and efrapeptin and for the natural inhibitor protein, IF1.

Assessment of subchondral bone mineral density in equine metacarpophalangeal and stifle joints.

Functional relationships between articular cartilage and underlying subchondral bone have been shown to be associated with the progression of osteoarthritis (OA). However, quantifiable factors substantiating this relationship do not exist. Therefore, the study objective involved quantifying subchondral bone mineral density (BMD) of the equine metacarpophalangeal (MCP) and stifle joints as a step in determining if regional subchondral BMD may be associated with OA changes. BMD was bilaterally quantified using dual energy xray absorptiometry (DEXA) for four subchondral regions: palmer and dorsal aspect of the medial 3rd metacarpal (MC3P, MC3D), medial femoral condyle (MFC), and the medial trochlear ridge (MTR). BMD (g/cm2) was measured using a 5.2 x 5.2 mm region of interest in the subchondral bone area. To account for the differences in thickness, BMD (g/cm2) was divided by sample thickness providing volumetric BMD (g/cm3). No significant effect of side on BMD values was found (p > 0.72). However, there were significant differences in BMD found between all regions (p < 0.05). The volumetric BMD of the MC3P region was significantly greater than the MC3D and the MC3D was greater than the MFC, while the MFC was greater than the MTR (MC3P > MC3D > MFC > MTR). The MCP regions had a higher volumetric BMD than the stifle regions possibly due to higher weight distribution, smaller articular surface area, and joint geometry. The adaptive bone structural variations between the regions studied were shown to be significant. Mechanical properties of the overlying cartilage are currently being analyzed to correlate with these results and provide a possible diagnostic method to investigate OA progression.

Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

How should the chest wall be opened at necropsy?

AIMS: To compare several different instruments used to open the chest wall during necropsy and to assess whether any one type reduced the production of sharp rib ends and thus the potential for receiving an injury. METHODS: During the necropsy the pathologist opened the chest wall using two randomly assigned instruments from a selection of hand saw, electric saw, rib shears, and bread knife. The age, weight, sex, and height of the deceased were recorded, in addition to the textures of the resultant exposed rib ends. During the procedure, the speed, length, production of spray, and site of incision were also noted. The thoracic cavity was inspected and any details of tumours, adhesions, fluid, or organ damage were noted. RESULTS: Twenty four necropsies were carried out on an equal number of men and women. The total number of ribs that were incised was 422, with 206 through the bony aspect (49%). Sixty seven per cent of the bony rib ends were rough, and this was found to be instrument dependent. The rib shears produced the highest number of rough bony and cartilage rib ends. The electric saw produced the smoothest contoured rib ends. Spray occurred in 29% of cases, exclusively with the use of the electric saw. Organ damage was most frequently associated with the use of the bread knife. CONCLUSION: Rib shears, the instrument most frequently used to open the chest wall, appear to cause the highest frequency of rough, potentially dangerous rib ends. The electric saw produced the smoothest rib ends, both in cartilage and bone, and thus seems to offer the most efficacious method of reducing the potential hazard associated with ragged, spiky bone ends during the opening of the thoracic cavity. Although each of the procedures detailed in this study was shown to have its own advantages and disadvantages, personal preference and operator experience are perhaps the most important factors in ultimately determining the method used.

The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.

Pirfenidone for chronic progressive multiple sclerosis.

Current treatment of secondary progressive multiple sclerosis is unsatisfactory in stabilizing or reversing the disabilities associated with the disease. Pirfenidone is a new non-peptide drug which has been shown in vitro and in vivo to decrease synthesis of Tumor Necrosis Factor-alpha (TNF-alpha) and block receptors for TNF-alpha. Since TNF-alpha seems to be a key cytokine in demyelination, a pilot study of oral pirfenidone was undertaken in an open-label baseline vs treatment protocol over a 2-year period in 20 patients. Fourteen (14120) patients (70%) remained in the study for 2 years. Three (3/20) patients dropped out early because of gastrointestinal adverse reactions, and another three patients dropped out for personal reasons after 1 year (not because of adverse reactions). The remaining patients did not manifest any other drug-related adverse reactions and complications. Improvement or stabilization occurred in most patients at about 3 months, and it was sustained at 6, 12 and 24 months as evaluated by both primary and secondary outcome measures. Magnetic resonance imaging foiled to reveal any new lesions. Thus, pirfenidone appears to offer protection against the usual slow progression of the disease. Most patients experienced a distinct decrease in their neurological disability. These findings indicate that an extensive multi-center double blind and placebo controlled trial is warranted.

Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis.

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half-closed" conformation. This structure probably represents the posthydrolysis, pre-product release step on the catalytic pathway. A catalytic scheme for hydrolysis (and synthesis) at physiological rates and a mechanism for the ATP-driven rotation of the gamma subunit are proposed based on the crystal structures of the bovine enzyme.

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase.

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

The structure and nucleotide occupancy of bovine mitochondrial F(1)-ATPase are not influenced by crystallisation at high concentrations of nucleotide.

Analysis of tryptophan mutants of F(1)-ATPase from Escherichia coli [Löbau et al. (1997) FEBS Lett. 404, 15-18] suggested that nucleotide concentrations used to grow crystals for the determination of the structure of bovine F(1)-ATPase [Abrahams et al. (1994) Nature 370, 621-628] would be sufficient to occupy only two catalytic sites, and that higher concentrations of nucleotide would result in all three sites being occupied. We have determined the structure of bovine F(1)-ATPase at 2.9 A resolution with crystals grown in the presence of 5 mM AMPPNP and 5 microM ADP. Similar to previous structures of bovine F(1)-ATPase determined with crystals grown in the presence of lower nucleotide concentrations, only two beta-subunits have bound nucleotide and the third subunit remains empty.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals.

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate.

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.