Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.     

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by subtractive genotyping, to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.     

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by “subtractive genotyping”, to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.     

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.     

Contribution of NADH dehydrogenase subunit I and cytochrome C oxidase subunit I sequences toward identifying a case of human coenuriasis in France

Coenuriasis is a parasitic disease induced by larval taeniid tapeworms that is rarely observed in humans. In December 2005, a case was diagnosed in Nancy, France, after surgical excision of a cyst on a 24-yr-old woman returning from the C?te d'Ivoire. Morphological and epidemiological criteria suggested that the infection was due to Taenia serialis. Molecular analysis of NADH dehydrogenase subunit I (NDI) and cytochrome c oxidase subunit I (COI) sequences was also in favor of T. serialis identification, but the absence of available genetic data on T. brauni and T. glomeratus and the small number of published sequences for T. serialis and T. multiceps must be considered with caution. The NDI partial sequences presented more variations within species of Taenia than the COI sequences, which make them more useful targets for species identification and analysis of intraspecific polymorphisms. The present study points to the usefulness of molecular biology tools to help make up for the shortcomings of the commonplace parasitological diagnosis for coenuriasis.     

Intraspecific variability among NADH dehydrogenase subunit 1 sequences of Taenia hydatigena.

This paper describes intraspecific variability of the partial sequences of the mitochondrial ND1 gene among isolates of Taenia hydatigena from pigs in Poland, Ukraine and Wales. The differences between studied isolates ranged from 0.4 to 5.5%, which exceeds the variability within the same fragment between the different genetic variants of Echinococcus multilocularis and is comparable with the variability between the most closely related strains (G5/G6/G7) of E. granulosus. The biggest difference (5.5%) was found between the geographically most distant Ukrainian and Welsh samples of T. hydatigena while the samples collected from the neighbouring locations in Poland, were most similar to each other.     

Liver alcohol dehydrogenase subunit equivalence studied by rapid sampling of alcohol product formed from sequentially bound [4alpha-3H]NADH.

Proteoglycans of calf and steer articular cartilage were studied with a view of assessing structure and changes occurring as a result of the aging process. The average reduction in hydrodynamic size noted in steer was associated with a diminution in size of the chondroitin sulfate-rich region of the core protein as well as the chondroitin sulfate chains themselves. By contrast the keratan sulfate-rich region was hydrodynamically larger in steer although the keratan sulfate chains were only slightly longer than in calf. The proteoglycans showed a maturation-related decrease in chondroitin sulfate content (shorter chains, fewer chains, smaller chondroitin sulfate-rich region) and an enrichment in keratan sulfate chains in both the chondroitin sulfate-rich and keratan sulfate-rich regions. Proteoglycans from both age groups contained an oligosaccharide which was recovered mainly from outside of the keratan sulfate-rich region. There were no significant differences in size between keratan sulfate chains recovered from the keratan sulfate-rich region and the chondroitin sulfate-rich region.     

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, ψnad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this ψnad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.     

Periplasmic cold expression and one-step purification of human dihydrolipoamide dehydrogenase

Dihydrolipoamide dehydrogenase (LADH) is a FAD-linked subunit of alpha-ketoglutarate, pyruvate and branched-chain amino acid dehydrogenases and the glycine cleavage system. As an oxidoreductase it transfers electrons from the dihydrolipoic acid prosthetic group to the NAD(+) cofactor via its FAD center. Besides its physiological function it is capable of generating harmful reactive oxygen species (ROS) in pathological settings therefore it is implicated in neurodegeneration, ischemia-reperfusion, cancer and several other disorders. Pathological mutants of the enzyme cause severe, sometimes lethal syndromes like hypotonia, metabolic acidosis or inefficiency in development. Recently it has been revealed that LADH is a moonlighting protease when specific mutations in the dimerization surface destabilize the functional homodimer and expose a serine-protease-like catalytic dyad. As the basis of versatile functions of LADH is far from elucidation, there is a constant need for a pure and functional enzyme product for investigations. Several studies used recombinant human LADH before, however, it was generated by more complicated and/or physiologically less compatible protocols than reported here; most papers on functional and structural studies do not even report detailed protocols and characteristics (most importantly the purity) of their protein products. Here we describe the details of an optimized, easy-to-use periplasmic expression and one-step purification protocol for obtaining a highly pure, active and authentic (tag-cleaved) enzyme with the characterization of the protein product. The purified LADH can be used in biophysical and structural studies while the published protocol is easily convertible to a protein labeling procedure.     

A new subunit of horse liver alcohol dehydrogenase and subunit composition of the polymorphic form.

The most cathodal (on starch-gel electrophoresis), steroid-active band of horse liver alcohol dehydrogenase, whose catalytic properties were shown to be dependent on the livers used as a starting material [Pietruszko (1974) Biochem. Biophys. Res. Commun. 60, 687-694], has been prepared from A-type and S-type horse livers by identical methods. Results presented here show that different isoenzymes are present in these preparations.