Search for mitochondrial DNA mutation at position 3243 in German patients with a positive family history of maternal diabetes mellitus.

Mitochondrial diabetes is one of the most common monogenetic forms of diabetes mellitus. However, variable prevalences have been reported by different investigators. Therefore, the aim of our study was to investigate the prevalence of the most prevalent mitochondrial DNA mutation at position 3243 (A --> G) in german patients with a positive family history of maternally inherited diabetes mellitus and/or hearing loss. We screened 1460 patients with diabetes mellitus by a questionnaire and identified 122 patients with a positive family history of maternal diabetes mellitus. Seven of the 122 patients suffered from hearing loss in addition. An EDTA blood sample of each patient was examined by polymerase chain reaction followed by a digestion with Bsp120I. In addition all samples were further examined by denaturing gradient gel electrophoresis to increase the detection limit for heteroplasmy. Only one mt DNA mutation at position 3243 could be detected in the 122 patients. The detection limit of denaturing gradient gel electrophoresis (DGGE) for heteroplasmy was 3%. We detected one new polymorphism at position 3333 (C --> T) of the mitochondrial genome (0.8% of the patients), and a known polymorphism at position 3197 (T --> C) in 10.6% of the patients. We therefore conclude that the frequency of the A3243G mutation is much lower in the investigated study population, mostly originating from Saxonia, than in asian populations.

Detection of thyroid-stimulating hormone receptor and Gsalpha mutations: in 75 toxic thyroid nodules by denaturing gradient gel electrophoresis.

The actual frequency of constitutively activating thyrotropin receptor or Gsalpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7-9 of the Gsalpha protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser-->Leu) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or Gsalpha mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsalpha play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or Gsalpha genes as the molecular cause of these hot nodules.

Phosphorothioate oligonucleotides reduce melanoma growth in a SCID-hu mouse model by a nonantisense mechanism.

In our efforts to investigate the biologic role of Ha-ras oncogenes in human melanoma by Ha-ras phosphorothioate antisense oligonucleotides, we observed that antisense, sense, and scrambled control oligonucleotides at a concentration of 10 microM all similarly and strongly inhibited growth of our human melanoma target cell line SK-2 in vitro but without specific decrease of the target protein. Cell numbers with respect to the untreated control were reduced by 84% +/- 4.2% (ISD), 82.9% +/- 3.6%, and 84% +/- 3%, respectively. In vivo studies in a SCID-hu mouse model confirmed these findings. Both antisense and sense control oligonucleotides administered through osmotic pumps significantly (p < 0.006) reduced the mean tumor weight (1.5 g +/- 0.4 g and 1.8 g +/- 0.8 g, respectively) in comparison with saline-treated (5.7 g +/- 0.7 g) or untreated control animals (5.8 g +/- 1.0 g). The vascularity of oligonucleotide-treated tumors was greatly reduced. Clinical signs of oligonucleotide-related toxicity were not observed, and there was no evidence of histopathologic alterations in a variety of mouse tissues. We could demonstrate that the antimelanoma effects can be abrogated in vitro by adding basic fibroblast growth factor (bFGF). In the context of the importance of bFGF in melanocyte biology and angiogenesis, we argue in favor of an interaction between polyanionic phosphorothioate oligonucleotides and bFGF in our melanoma system. These findings stress the notion that phosphorothioate oligonucleotides may be promising antineoplastic lead compounds capable of employing antitumor effects by mechanisms other than specific inhibition of gene expression.

Targeting of the master receptor MOM19 to mitochondria.

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP.