A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly.

UNLABELLED: We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION: Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. WHAT IS KNOWN: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

Protein translocation into mitochondria: the role of TIM complexes.

Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins. Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane. This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.

False-positive reactions of some monoclonal anti-c reagents.

Cross-reactivities of monoclonal reagents for red blood cell (RBC) typing are seen very rarely, e.g. in some reagents for testing of ABO or Lewis antigens. We report on two patients in whom we observed weak reactions using a monoclonal anti-c reagent containing clone MS35, but no reactions with anti-c reagents containing other monoclonal or polyclonal antibodies. To resolve this discrepancy, we studied RHCE exon 1 (Ex1) and 2 from genomic DNA of one patient and performed quality controls of the false-positive reacting anti-c reagent. RHCE Ex1 showed no abnormalities as well as RH Ex2 examined with primers specific for Ex2 D/C. No amplification product of RH Ex2 was received with primers specific for c. A titration study with untreated, papain-treated and sialidase-treated adult and newborn RBCs showed anti-i reactivity of the false-positive reacting reagent. This reactivity could not be removed by absorption with c-negative newborn RBCs without reducing the anti-c titre in the same way, indicating a cross-reactivity. Some manufacturers give a remark on a possible cold agglutinin activity of their monoclonal anti-c reagents in the instruction leaflet, but in order to avoid such irritation, we recommend to remove this cross-reactivity by dilution during the manufacturing process.

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.

Yeast mitochondrial F1F0-ATPase: the novel subunit e is identical to Tim11.

We report here the identification of the novel subunit of the mitochondrial F1F0-ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled-coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co-immunoprecipitated with a subunit of the F1-ATPase, demonstrate that subunit e is a subunit of the F1F0-ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0-ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.

The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom.

The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.

Torsades de pointes arrhythmia in a patient with left ventricular myxoma.

A 38-year-old woman suffered from syncopes due to torsades de pointes arrhythmias. Echocardiography showed a left ventricular mass that was resected. By immunohistochemical investigations, an organized thrombus was excluded and the diagnosis of a rare ventricular myxoma was confirmed. Postoperatively the arrhythmias resolved without recurrence. We conclude that left ventricular myxoma may cause life-threatening arrhythmias, possibly by irritation of cardiac mechanoreceptors. To our knowledge, this is the first case of a left ventricular myxoma associated with a torsades de pointes arrhythmia.

[Change in congener analysis caused by percutaneous absorption of propanol-containing antiseptics]. / Veränderung von Begleitstoffanalysen durch percutane Resorption propanolhaltiger Antiseptika.

According to the forensic literature, an alteration of blood alcohol concentration through alcohol disinfection before venepuncture is unlikely, however, percutaneous resorption of alcohol containing antiseptics is well documented. There are no investigations available, elucidating to what extent this is also correct for congener alcohols, particularly propanol-1/-2, which have serum levels in range of mg/l. In experiments with surgical hand and local skin disinfection, carried out with and without oral alcohol, relevant levels of propanol-1/-2 between 0.2 and 2 mg/l were measured. We conclude, that the routine clinical use of antiseptics is able to alterate and to falsify congener levels and can lead to incorrect interpretation of congener analysis.

Acute myopathy in a type 2 diabetic patient on combination therapy with metformin, fenofibrate and rosiglitazone.

AIMS/HYPOTHESIS: This report describes the case of a 75-year-old male type 2 diabetic Caucasian who was admitted to the clinical ward because of acute pain and cramps in both calf muscles. MATERIALS AND METHODS: Neuromuscular function was assessed by electromyography and electroneurography of the right leg. An open biopsy was taken from the left vastus lateralis muscle for histological and histochemical analyses. Southern blotting was performed to detect defects in mitochondrial DNA and tRNA. Cytochrome P450 (CYP-P450) polymorphisms were analysed in blood cells. RESULTS: Fifteen weeks before admission, the patient's lipid-lowering medication was switched from simvastatin to fenofibrate because of predominant hypertriglyceridaemia; this did not affect creatine kinase levels. Three weeks before admission, rosiglitazone was added to his existing metformin therapy because of worsening metabolic control. Upon admission, serum enzymes indicating myopathy were elevated (creatine kinase 6897 U/l, myoglobin 902 ng/ml) and kidney function was impaired (creatinine 0.116 mmol/l, blood urea nitrogen 2.3 mmol/l). Electrophysiology revealed myopathy and sensory polyneuropathy. Histology showed multiple damage of the myofibrillar architecture. There was no evidence of defects in mitochondrial DNA or tRNA. Furthermore, no functional limitations in CYP2C9, CYP2C19 and CYP2D6 were detected. Following withdrawal of the oral medication and intravenous hydration, clinical symptoms and laboratory parameters gradually decreased. CONCLUSIONS/INTERPRETATION: Until more data from controlled trials are available, we recommend that combination therapy with fibrates and thiazolidinediones should be monitored frequently by measurements of serum creatine kinase and creatinine, specifically in patients with pre-existing nephropathy and polyneuropathy.

Import of proteins into mitochondria: a novel pathomechanism for progressive neurodegeneration.

The vast majority of mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is therefore the transfer of nuclear-encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes. During the past years the use of simple model organisms such as the yeasts S. cerevisiae and N. crassa has helped considerably to identify and unravel the structure and function of a substantial number of components involved in targeting of nuclear-encoded preproteins to mitochondria. Several pathways and a number of components were characterized that are involved in guiding mitochondrial preproteins to their specific sites of function. In particular, import of nuclear-encoded precursor proteins into and across the mitochondrial inner membrane is mediated by two distinct translocases, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the general preprotein translocase of the outer membrane, TOM complex. The TIM complexes differ in the their substrate specificity. While the TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal, the TIM22 complex facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane. Most recently the rapid progress of research has allowed elucidation of a new mitochondrial disease on the molecular level. This rare X-linked progressive neurodegenerative disorder, named Mohr-Tranebjaerg (MT syndrome), is caused by mutations in the DDP1 gene and includes sensorineural deafness, blindness, mental retardation and a complex movement disorder. The analysis of the novel pathomechanism is based on the homology of the affected DDP1 protein to a family of conserved yeast components acting along the TIM22 pathway. This contribution briefly summarizes the current knowledge of the pathways of protein import and proposes a mechanism to explain how defective import leads to neurodegeneration.

Mini organ culture of thyroid tissue: a new technique for maintaining the structural and functional integrity of thyroid tissue in vitro.

We have developed a culture technique by which the normal configuration of follicles and most other structural characteristics of the thyroid gland are well preserved. Small tissue fragments (diameter 0.5 to 0.9 mm) from various mammalian species were prepared and kept in hydrophobic culture dishes to prevent their attachment. It was essential to raise the oxygen concentration in the incubator to 50% and the D-glucose concentration to 5.6 mM. Under these conditions, mini organs were formed by the outgrowth of epithelial cells from follicles located on the fragment surface and opened during tissue preparation. After 2 to 3 days, the mini organs were enclosed by a confluent and tight monolayer of follicle cells and [3H]thymidine incorporation returned to background levels. The structural integrity of follicles underneath the monolayer and in the center of the mini organs could be maintained for at least several weeks. Light microscope autoradiographs of mini organs incubated with iodide, sulfate, or phosphate revealed that the vectorial transport and the posttranslational modifications of thyroglobulin were maintained. An assay for the precise quantitation of biosynthetic activities in individual mini organs was developed. The observations indicate that thyroid-specific functions in mini organs can be preserved for prolonged periods of time.

Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria.

Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior. Dimer formation is promoted by the membrane potential delta psi. Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex. Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space. This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation. We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane. The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein.

[Acute aortic insufficiency following endocarditis due to infection with Campylobacter fetus subspecies fetus]. / Akute Aorteninsuffizienz nach Endokarditis durch Infektion mit Campylobacter fetus subspecies fetus.

A 50-year-old male alcoholic addict, examined because of diarrhoea with fever was found to have Campylobacter jejuni in blood and stool cultures. After administration of broad-spectrum penicillin all acute symptoms disappeared but he lost 8 kg within 3 months and his general state health gradually deteriorated. After 3 months he suddenly developed leg oedema, dyspnoea and bouts of fever up to 38.8 degrees C. A loud cardiac murmur was now heard. Echocardiography demonstrated vegetations on the regurgitant aortic valve. Endocarditis being suspected he was at first treated with penicillin G (15 mega IU/d) and gentamycin (160 mg/d). The fever regressed, but after 8 days the blood culture grew Campylobacter fetus subspecies fetus. Antibiotic treatment was switched to imipenem, twice daily 500 mg, in accordance with sensitivity test results. Further blood cultures were sterile. Despite this the cardiac status deteriorated, the aortic regurgitation reaching grade IV. The valve was replaced with a bioprosthesis and the patient quickly improved postoperatively. Antibiotic treatment was stopped and the cardiovascular status became normal. The patient has now been free of symptoms and recurrence for 7 months.

The blood saving potential of vortex versus roller pump with and without aprotinin.

To evaluate the potential of centrifugal blood pumps for saving blood, 120 patients scheduled for elective coronary artery bypass grafting were entered into a prospective randomized trial. A standard roller pump (group I) was compared with a centrifugal blood pump (group II) and roller pump plus aprotinin (group III). There was no significant difference between groups I and II with respect to free haemoglobin, lactic dehydrogenase, serum bilirubin, platelet surface glycoprotein IIb-IIIa and granule membrane protein 140, chest-tube drainage, use of blood products, length of stay in intensive care, time on ventilator and postoperative mortality. Aprotinin reduced chest-tube drainage and use of blood products significantly. Three cases of graft occlusions were noted in group III. Centrifugal blood pumps offer no advantage in routine heart surgery over conventional roller pumps. Aprotinin reduces blood loss, but does not influence GP IIb-IIIa and GMP 140 expression on blood platelets.

Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22.

Translocation of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery. Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import. Tim23 and Tim17 are constituents of a translocation channel across the inner membrane. Tim44 is associated with this channel at the matrix side, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mgel, which drive protein translocation into the matrix using ATP as an energy source. Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17. Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals. Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23-Tim17 complex. Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22.

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins.

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Mitochondrial disorders. A diagnostic challenge in clinical chemistry.

Mitochondria play a pivotal role in cellular metabolism and in energy production in particular. Defects in structure or function of mitochondria, mainly involving the oxidative phosphorylation (OXPHOS), mitochondrial biogenesis and other metabolic pathways, have been shown to be associated with a wide spectrum of clinical phenotypes. The ubiquitous nature of mitochondria and their unique genetic features contribute to the clinical, biochemical and genetic heterogeneity of mitochondrial diseases. We will focus on the recent advances in the field of mitochondrial disorders and their consequences for an advanced clinical and genetic diagnostics. In addition, an overview on recently identified genetic defects and their pathogenic molecular mechanisms will be given.

The role of the TIM8-13 complex in the import of Tim23 into mitochondria.

Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.

The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system.

We have identified a complex in mitochondria that functions as a part of the preprotein import machinery of the inner membrane (MIM complex). Two known components, MIM23 and MIM17, and two novel components, MIM33 and MIM14, were found as constituents of this complex. In the presence of a translocating chain, the outer membrane import machinery (MOM complex) and the MIM complex form translocation contact sites. On the matrix side, the MIM complex is associated with the mt-Hsp70-MIM44 system. We propose a structure of the import machinery in which the MIM complex constitutes a proteinaceous channel that accepts preproteins from the MOM complex, facilitates their reversible transmembrane movement, and mediates unidirectional transport by linkage to the ATP-dependent mt-Hsp70-MIM44 system.