The substitution of guanine (G) for adenine (A) at positioln 3243 of mitochondrial DNA, first demonstrated in MELAS patients, has been found in some diabetics, and this mutation seems to be one of genetic factors for diabetes mellitus. Because mutational mitochondria coexist with normal mitochondria (heteroplasmy), conventional PCR-RFLPm methods may not detect a mutation When a majority of the mitochondrial DNA in leukocytes is normal. We tested the efficacy of specific PCR amplification in detecting the 3243G mutation using a primer whonse 3' base was complementary to the mutational base. This mutation-specific PCR amplification method permitted detection of the mutation in 2 patients with MELAS and in a diabetic patient whose mutation was detected by the PCR-RFLP method in biopsied muscle but not in peripheral leukocytes, and in two other diabetic patients.Specific PCR amplification for detection of the 3243G mutation is a simple and senisitive method and is useful inevaluating this mutation in diabetes mellituls.
In order to assess glomerular endothelial permeability, we established an in vitro model using an immortalized bovine glomerular endothelial cell line. We examined the effect of high glucose concentration on albumin permeability across glomerular endothelial cell monolayers. Thirty millimolar D-glucose doubled albumin permeability (12.1±1.3%) compared with 10mM D-glucose (6.4±0.8%), but the albumin permeability of endothelial cells exposed to 10mM glucose+20mM mannitol was 6.8+0.5%. In addition, H-7 (25μM), a protein kinase C (PKC) inhibitor, suppressed the increased albumin permeability induced by 30mM glucose (7.8±0.9%). Lactate dehydrogenase release by endothelial cells incubated with 30mM glucose, on the other hand, was no different than when incubated with 10mM glucose, indicating that cytolysis was not associated with these events. These findings suggest that PKC activation is an important signal transduction pathway by which high glucose concentration increases glomerular endothelial albumin permeability.