Myringoplasty: is it worth performing in children?

OBJECTIVE: To evaluate the results of myringoplasty in children 4 to 14 years old at the time of surgery. DESIGN: Retrospective analysis of case notes for 100 consecutive children who had myringoplasty in a teaching hospital serving as a primary care and referral center. METHODS: Between March 1994 and March 1999, patients 14 years or younger at the time of surgery were identified by the computer database. There were 118 procedures performed in 100 patients (18 had a second procedure performed in the contralateral ear at a later date). Twenty-three patients were excluded because they underwent concurrent mastoid exploration, and 6 others because of inadequate follow-up, leaving 89 cases for analysis. Data from revision procedures were not included. MAIN OUTCOME MEASURES: Graft success was defined as an intact eardrum at 12 months postoperatively and middle ear effusion signaled graft failure. Success in terms of hearing was defined as an improvement in perception of pure-tone thresholds of 10 dB or greater over 2 consecutive frequencies compared with the results of the preoperative audiogram. RESULTS: Closure of perforation was achieved in 90% (80) of patients, but dropped to 88% (78) as 2 patients developed glue ear. Hearing improved in 64 patients (72%), deteriorated in 7 (8%), and remained unchanged in 18 (20%). There was no case of profound hearing loss. CONCLUSIONS: The success rate of myringoplasty in children is comparable to that reported for adults. The incidence of middle ear effusion in grafted ears is not higher than that reported for nongrafted ears, and children who have had myringoplasty can be treated as safely with ventilation tubes as any other children.

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

The preprotein translocase of the mitochondrial inner membrane: function and evolution.

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane.

We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria. The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner. Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria. Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria. The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria. The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria. These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi.

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Tom5 functionally links mitochondrial preprotein receptors to the general import pore.

Most mitochondrial proteins are synthesized as preproteins on cytosolic polysomes and are subsequently imported into the organelle. The mitochondrial outer membrane contains a multisubunit preprotein translocase (Tom) which has receptors on the cytosolic side and a general import pore (GIP) in the membrane. Tom20-Tom22 and Tom70-Tom37 function as import receptors with a preference for preproteins that have amino-terminal presequences or internal targeting information, respectively. Tom40 is an essential constituent of the GIP, whereas Tom6 and Tom7 modulate the assembly and dissociation of the Tom machinery. Here we report the identification of Tom5, a small subunit that has a crucial role importing preproteins destined for all four mitochondrial subcompartments. Tom5 has a single membrane anchor and a cytosolic segment with a negative net charge, and accepts preproteins from the receptors and mediates their insertion into the GIP. We conclude that Tom5 represents a functional link between surface receptors and GIP, and is part of an 'acid chain' that guides the stepwise transport of positively charged mitochondrial targeting sequences.

Role of mitochondrial GrpE and phosphate in the ATPase cycle of matrix Hsp70.

The yeast mitochondrial GrpE homologue, Mge1, assists matrix Hsp70 in both protein translocation across the mitochondrial membranes and subsequent protein folding. We expressed mtHsp70 and Mge1 in Escherichia coli and analyzed their function in the ATP hydrolysis cycle. Mge1 stimulates ATP hydrolysis by mtHsp70 about twofold. Addition of inorganic phosphate inhibits ATP hydrolysis by preventing ADP release from mtHsp70. Mge1 has no direct effect on gamma-phosphate release from mtHsp70, yet indirectly relieves the phosphate inhibition by stimulating ADP release. We conclude that Mge1 promotes the ATPase cycle of mtHsp70 by increasing the rate of ADP release. ATP then rapidly binds to mtHsp70 such that the total amount of mtHsp70-bound nucleotide is not changed by Mge1.

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane.

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex. (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.

Characterization of the preprotein translocase of the outer mitochondrial membrane by blue native electrophoresis.

The mitochondrial outer membrane contains import receptors for nuclear-encoded preproteins and a general import pore responsible for membrane translocation of preproteins. Receptors and the general import pore have been suggested to assemble into a loose complex. However, biochemical characterization of the complex has been limited so far. We report that blue native electrophoresis separates two complexes. One complex of approximately 400 kDa contains the receptor Tom22 and the general import pore component Tom40, the other complex of approximately 120 kDa contains the receptor Tom70. A preprotein accumulated at the general import pore apparently co-migrates with the larger complex, suggesting the functionality of the complex. We conclude that the translocase of the outer membrane consists of at least two subcomplexes and that blue native electrophoresis will be a powerful tool for biochemical analysis of the complexes.

Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins.

Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.

Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM).

The essential mitochondrial inner-membrane protein, Mim44, is involved in the translocation of preproteins across the mitochondrial inner membrane. Two other putative components of this protein-translocation system are the integral inner-membrane proteins, Mim23 and Mim17. Here, we present genetic evidence for functional co-operation of all three proteins. Furthermore, we show that Mim23 and Mim17 are associated in a protein complex that also contains two proteins of 55 kDa and 20 kDa. We speculate that this subcomplex forms the proteinaceous import channel of the inner-membrane which transiently interacts with a less abundant peripheral complex of Mim44 and mitochondrial heat-shock protein Hsp70.

The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site.

Three proteins of the mitochondrial inner membrane are known that are essential for the viability of yeast and seem to be involved in import of preproteins; the integral membrane proteins MIM17 and MIM23 and the peripheral membrane protein MIM44, MIM17 and MIM23 are homologous to each other in their hydrophobic domain, expose their termini to the intermembrane space, and span the inner membrane up to four times, each. A preprotein in transit across the mitochondrial membrane is specifically cross-linked to MIM17, MIM23, MIM44, and matrix hsp70. We conclude that MIM17 and MIM23 are integral parts of a preprotein translocation channel and cooperate with MIM44 and hsp70 at the same protein import site.

Neurilemmoma of the larynx.

Neurogenous tumours of the larynx are extremely uncommon. We describe a case of a neurilemmoma which presented clinically as a discrete nodule on the vocal cord. Simple endoscopic removal under direct vision was performed.

Papillary carcinoma in a thyroglossal cyst.

A thyroglossal duct cyst is a congenital anomaly that arises from remnants of the thyroglossal duct, which in the embryo connects the thyroid gland with the floor of the pharynx. Primary carcinomas of thyroglossal duct tissue are rare, and are usually diagnosed after surgery. Management of such tumours is not clearcut. A case of a papillary carcinoma of thyroglossal duct tissue is presented that has been followed up for 10 years following Sistrunk's operation, and the management of these uncommon tumours is discussed.

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

Yeast mitochondrial NAD(+)-dependent isocitrate dehydrogenase is an RNA-binding protein.

We have previously described the characterisation of an abundant mitochondrial protein (p40) that binds specifically to 5'-untranslated leaders of mitochondrial mRNAs in yeast. p40 consists of two polypeptides with M(r) of 40 and 39 kDa. Limited sequence analysis of p40 identifies it as the Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh). Both enzyme and RNA-binding activities are specifically lost in cells containing disruptions in either IDH1 or IDH2, the nuclear genes encoding the two subunits of the enzyme, thus conclusively identifying p40 as Idh and showing that both activities are dependent on the simultaneous presence of both subunits. Although we still must ascertain whether and how either function of Idh is regulated and whether the two functions are compatible or mutually exclusive, this combination of dehydrogenase activity and RNA-binding in a single protein may be part of a general regulatory circuit linking the need for mitochondrial function to mitochondrial biogenesis.

Wegener's granulomatosis: otological aspects.

Otological presentations are uncommon in Wegener's granulomatosis. We report a series of cases in which ear disease was a major manifestation of the disease and discuss the otogenic and neurological manifestations of Wegener's granulomatosis.

Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane.

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPII and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MP13) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.