Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells

Replication of mitochondrial DNA (mtDNA) is dependent on nuclear-encoded factors. It has been proposed that this reliance may exert spatial restrictions on the sites of mtDNA replication within the cytoplasm, as a previous study only detected mtDNA synthesis in perinuclear mitochondria. We have studied mtDNA replication in situ in a variety of human cell cultures labeled with 5-bromo-2'-deoxyuridine. In contrast to what has been reported, mtDNA synthesis was detected at multiple sites throughout the mitochondrial network following short pulses with bromodeoxyuridine. Although no bromodeoxyuridine incorporation was observed in anuclear platelets, incorporation into mtDNA of fibroblasts that had been enucleated 2 h prior to labeling was readily detectable. Blotting experiments indicated that the bromodeoxyuridine incorporation into mtDNA observed in situ represents replication of the entire mtDNA molecule. The studies also showed that replication of mtDNA occurred at any stage of the cell cycle in proliferating cells and continued in postmitotic cells, although at a lower level. These results demonstrate that mtDNA replication is not restricted to mitochondria in the proximity of the nucleus and imply that all components of the replication machinery are available at sufficient levels throughout the mitochondrial network to permit mtDNA replication throughout the cytoplasm.     

Neurotransmitter alterations in embryonic succinate semialdehyde dehydrogenase (SSADH) deficiency suggest a heightened excitatory state during development

Background  

SSADH (aldehyde dehydrogenase 5a1 (Aldh5a1); γ-hydroxybutyric (GHB) aciduria) deficiency is a defect of GABA degradation in which the neuromodulators GABA and GHB accumulate. The human phenotype is that of nonprogressive encephalopathy with prominent bilateral discoloration of the globi pallidi and variable seizures, the latter displayed prominently in Aldh5a1^-/- mice with lethal convulsions. Metabolic studies in murine neural tissue have revealed elevated GABA [and its derivatives succinate semialdehyde (SSA), homocarnosine (HC), 4,5-dihydroxyhexanoic acid (DHHA) and guanidinobutyrate (GB)] and GHB [and its analogue D-2-hydroxyglutarate (D-2-HG)] at birth. Because of early onset seizures and the neurostructural anomalies observed in patients, we examined metabolite features during Aldh5a1^-/- embryo development.     

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.     

Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme.

Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20 degrees C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolated in lauryl maltoside contains these same nine different polypeptides and three others, including homologues of subunits VIa and VIb of the mammalian enzyme.     

Nucleotide sequence of the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking regions.

A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted Alu dimer repeat was identified further downstream.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria

Background  

Colorectal cancer is the third most-common cancer and the second most-common cause of cancer related death in UK. Although chemotherapy plays significant role in the treatment of colorectal cancer, morbidity and mortality due to drug resistance and cancer metastasis are yet to be eliminated. Recently, doxycycline has been reported to have cytotoxic and anti-proliferating properties in various cancer cells. In this study, whether doxycycline was apoptosis threshold lowering agent in colorectal cancer cells by targeting mitochondria was answered.