Development of pharmacological strategies for mitochondrial disorders.

Mitochondrial diseases are an unusually genetically and phenotypically heterogeneous group of disorders, which are extremely challenging to treat. Currently, apart from supportive therapy, there are no effective treatments for the vast majority of mitochondrial diseases. Huge scientific effort, however, is being put into understanding the mechanisms underlying mitochondrial disease pathology and developing potential treatments. To date, a variety of treatments have been evaluated by randomized clinical trials, but unfortunately, none of these has delivered breakthrough results. Increased understanding of mitochondrial pathways and the development of many animal models, some of which are accurate phenocopies of human diseases, are facilitating the discovery and evaluation of novel prospective treatments. Targeting reactive oxygen species has been a treatment of interest for many years; however, only in recent years has it been possible to direct antioxidant delivery specifically into the mitochondria. Increasing mitochondrial biogenesis, whether by pharmacological approaches, dietary manipulation or exercise therapy, is also currently an active area of research. Modulating mitochondrial dynamics and mitophagy and the mitochondrial membrane lipid milieu have also emerged as possible treatment strategies. Recent technological advances in gene therapy, including allotopic and transkingdom gene expression and mitochondrially targeted transcription activator-like nucleases, have led to promising results in cell and animal models of mitochondrial diseases, but most of these techniques are still far from clinical application.

Deficits of non-verbal auditory perception in postlingually deaf humans using cochlear implants.

Temporal integration in the time domain of a few seconds was investigated with a subjective accentuation paradigm in 11 monochannel cochlear implant users, who showed auditory comprehension deficits. While listening to metronome beats generated at various frequencies, patients were asked to accentuate mentally every n-th beat and create an individual rhythmic pattern. The extent of temporal integration was defined as the duration of perceptual units consisting of subjectively grouped beats at particular metronome frequencies. The results indicate that there is reduced capacity for temporal integration in implant recipients, particularly for lower metronome frequencies, in comparison to normally hearing. These observations point to the coincidence of specific temporal processing disorders and deficits in auditory comprehension after cochlear implantation.

The antimutagenic effect of a truncated epsilon subunit of DNA polymerase III in Escherichia coli cells irradiated with UV light.

It has previously been suggested that inhibition of the proofreading 3'-5' exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations in Escherichia coli. Our previous work showing that overproduction of epsilon, the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response of E. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of epsilon we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of epsilon--ExoI and ExoII; the third segment, ExoIII, which is essential for 3'-5' exonuclease activity, is deleted. We show that at 40 degrees C, overproduction of the truncated epsilon subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in a trpE65 strain growing at 40 degrees C causes a 10-fold decrease in the frequency of UV-induced Trp+ mutations. This antimutagenic effect of the truncated epsilon is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains the dnaQ49 allele encoding an epsilon subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in the trpE65 strain. We propose that the DNA binding ability of free epsilon, rather than its 3'-5' exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD'-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis.

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli.

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective epsilon subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122::Tn5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC+ plasmid. Interestingly, while the frequency of UV-induced GC-->AT transitions is greatly reduced, the frequency of AT-->TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC+; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.

Evolutionary conservation of the transcribed spacer sequences of the rDNA repeat unit in three species of the genus Aspergillus.

We have cloned and sequenced the two intervening transcribed spacers in the rDNA repeat unit of three Aspergillus species--A. nidulans, A. awamori and A. wentii. The A. wentii and A. awamori spacers are almost identical and share a high degree of homology with the A. nidulans spacers. All spacers have a high G-C content (66%-76%) and the potential of forming complex secondary structures, which may indicate that they play a role in the maturation of pre-rRNA molecules.