Ectopic expression of an Old Yellow Enzyme (OYE3) gene from Saccharomyces cerevisiae increases the tolerance and phytoremediation of 2-nitroaniline in rice.

2-nitroaniline (2-NA) is an environmental pollutant and has been extensively used as intermediates in organic synthesis. The presence of 2-NA in the environment is not only harmful for aquatic life but also mutagenic for human beings. In this study, we constructed transgenic rice expressing an Old Yellow Enzyme gene, ScOYE3, from Saccharomyces cerevisiae. The ScOYE3 transgenic plants were comprehensively investigated for their biochemical responses to 2-NA treatment and their 2-NA phytoremediation capabilities. Our results showed that the rice seedlings exposed to 2-NA stress, showed growth inhibition and biomass reduction. However, the transgenic plants exhibited strong tolerance to 2-NA stress compared to wild-type plants. Ectopic expression of ScOYE3 could effectively protect transgenic plants against 2-NA damage, which resulted in less reactive oxygen species accumulation in transgenic plants than that in wild-type plants. Our phytoremediation assay revealed that transgenic plants could eliminate more 2-NA from the medium than wild-type plants. Moreover, omics analysis was performed in order to get a deeper insight into the mechanism of ScOYE3-mediated 2-NA transformation in rice. Altogether, the function of ScOYE3 during 2-NA detoxification was characterized for the first time, which serves as strong theoretical support for the phytoremediation potential of 2-NA by Old Yellow Enzyme genes.

N-terminal truncation (N-) and directional proton transfer in an old yellow enzyme enables tunable efficient producing (R)- or (S)-citronellal.

(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of (-)-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production. The activity of OYE from Corynebacterium glutamicum (CgOYE) is increased, as well as superior thermal stability and pH tolerance via truncating the different lengths of regions at N-terminal of CgOYE. Next, we converted the truncation mutant N31-CgOYE, a protein involved in proton transfer for the asymmetric hydrogenation of CC bonds, into highly (R)- and (S)-stereoselective enzymes using only three mutations. The mixture of racemic (E/Z)-citral is reduced into the (R)-citronellal with ee and conversion up to 99 % by the mutant of CgOYE, overcoming the problem of the reduction for the mixtures of (E/Z)-citral in biocatalytic reaction. The present work provides a general and effective strategy for improving the activity of OYE, in which the partially conserved histidine residues provide "tunable gating" for the enantioselectivity for both the (R)- and (S)-isomerases.

Novel insights into the whole-blood DNA methylome of asthma in ethnically diverse children and youth.

BACKGROUND: The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. METHODS: DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. RESULTS: 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. CONCLUSIONS: We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.

Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket.

The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.

Autosomal recessive Leber hereditary optic neuropathy, a new neuro-ophthalmo-genetic paradigm.

Leber hereditary optic neuropathy (LHON) is a primary inherited neurodegenerative disorder of the optic nerve. It has been ascribed to variants in the mitochondrial genome, mainly the m.3460G>A, m.11778G>A and m.14484T>C mutations in ND1, ND4 and ND6, respectively. Nonetheless, inconclusive molecular diagnosis is not uncommon. Recently, biallelic mutations in the NDUFS2, DNAJC30, MCAT and NDUFA12 nuclear genes have been identified in unresolved LHON cases, identifying an autosomal recessive LHON (arLHON, OMIM:619382). The clinical presentation of arLHON copies that of typical LHON due to mtDNA mutations (mtLHON), with an acute phase of sudden and severe vision loss, telangiectatic and tortuous vessels around the optic nerve and swelling of the retinal nerve fibre layer. This is followed by a chronic phase of retinal nerve fibre layer loss, but eventually affected individuals recover partial or full visual acuity. Idebenone treatment significantly improved vision recovery in DNAJC30-associated patients. As for mtLHON, arLHON predominantly affected male compared with female carriers. The discovery of arLHON cases breaks with the dogma of exclusive maternal inheritance. It defines a new neuro-ophthalmo-genetic paradigm, which should be considered in individuals manifesting a LHON phenotype but with an inconclusive molecular diagnosis. NDUFS2, DNAJC30, MCAT and NDUFA12 should be investigated in these individuals, knowing that other arLHON genes might exist.

NamiRNA-enhancer network of miR-492 activates the NR2C1-TGF-ß/Smad3 pathway to promote epithelial-mesenchymal transition of pancreatic cancer.

Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-ß/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-ß, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-ß/Smad3 pathway.

Ascorbate peroxidase postcold regulation of chloroplast NADPH dehydrogenase activity controls cold memory.

Exposure of Arabidopsis (Arabidopsis thaliana) to 4°C imprints a cold memory that modulates gene expression in response to a second (triggering) stress stimulus applied several days later. Comparison of plastid transcriptomes of cold-primed and control plants directly before they were exposed to the triggering stimulus showed downregulation of several subunits of chloroplast NADPH dehydrogenase (NDH) and regulatory subunits of ATP synthase. NDH is, like proton gradient 5 (PGR5)-PGR5-like1 (PGRL1), a thylakoid-embedded, ferredoxin-dependent plastoquinone reductase that protects photosystem I and stabilizes ATP synthesis by cyclic electron transport (CET). Like PGRL1A and PGRL1B transcript levels, ndhA and ndhD transcript levels decreased during the 24-h long priming cold treatment. PGRL1 transcript levels were quickly reset in the postcold phase, but expression of ndhA remained low. The transcript abundances of other ndh genes decreased within the next days. Comparison of thylakoid-bound ascorbate peroxidase (tAPX)-free and transiently tAPX-overexpressing or tAPX-downregulating Arabidopsis lines demonstrated that ndh expression is suppressed by postcold induction of tAPX. Four days after cold priming, when tAPX protein accumulation was maximal, NDH activity was almost fully lost. Lack of the NdhH-folding chaperonin Crr27 (Cpn60ß4), but not lack of the NDH activity modulating subunits NdhM, NdhO, or photosynthetic NDH subcomplex B2 (PnsB2), strengthened priming regulation of zinc finger of A. thaliana 10, which is a nuclear-localized target gene of the tAPX-dependent cold-priming pathway. We conclude that cold-priming modifies chloroplast-to-nucleus stress signaling by tAPX-mediated suppression of NDH-dependent CET and that plastid-encoded NdhH, which controls subcomplex A assembly, is of special importance for memory stabilization.

Molecular Analysis Uncovers the Mechanism of Fertility Restoration in Temperature-Sensitive Polima Cytoplasmic Male-Sterile Brassica napus.

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

Multi-Omics Approach to Mitochondrial DNA Damage in Human Muscle Fibers.

Mitochondrial DNA deletions affect energy metabolism at tissue-specific and cell-specific threshold levels, but the pathophysiological mechanisms determining cell fate remain poorly understood. Chronic progressive external ophthalmoplegia (CPEO) is caused by mtDNA deletions and characterized by a mosaic distribution of muscle fibers with defective cytochrome oxidase (COX) activity, interspersed among fibers with retained functional respiratory chain. We used diagnostic histochemistry to distinguish COX-negative from COX-positive fibers in nine muscle biopsies from CPEO patients and performed laser capture microdissection (LCM) coupled to genome-wide gene expression analysis. To gain molecular insight into the pathogenesis, we applied network and pathway analysis to highlight molecular differences of the COX-positive and COX-negative fiber transcriptome. We then integrated our results with proteomics data that we previously obtained comparing COX-positive and COX-negative fiber sections from three other patients. By virtue of the combination of LCM and a multi-omics approach, we here provide a comprehensive resource to tackle the pathogenic changes leading to progressive respiratory chain deficiency and disease in mitochondrial deletion syndromes. Our data show that COX-negative fibers upregulate transcripts involved in translational elongation and protein synthesis. Furthermore, based on functional annotation analysis, we find that mitochondrial transcripts are the most enriched among those with significantly different expression between COX-positive and COX-negative fibers, indicating that our unbiased large-scale approach resolves the core of the pathogenic changes. Further enrichments include transcripts encoding LIM domain proteins, ubiquitin ligases, proteins involved in RNA turnover, and, interestingly, cell cycle arrest and cell death. These pathways may thus have a functional association to the molecular pathogenesis of the disease. Overall, the transcriptome and proteome show a low degree of correlation in CPEO patients, suggesting a relevant contribution of post-transcriptional mechanisms in shaping this disease phenotype.

NADP+-Dependent Dehydrogenase SCO3486 and Cycloisomerase SCO3480: Key Enzymes for 3,6-Anhydro-L-Galactose Catabolism in Streptomyces coelicolor A3(2).

Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-L-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-L-galactonate, and SCO3480 is a 3,6-anhydro-L-galactonate cycloisomerase that converts 3,6-anhydro-L-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50°C, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.

Novel NDUFA12 variants are associated with isolated complex I defect and variable clinical manifestation.

Isolated biochemical deficiency of mitochondrial complex I is the most frequent signature among mitochondrial diseases and is associated with a wide variety of clinical symptoms. Leigh syndrome represents the most frequent neuroradiological finding in patients with complex I defect and more than 80 monogenic causes have been involved in the disease. In this report, we describe seven patients from four unrelated families harboring novel NDUFA12 variants, with six of them presenting with Leigh syndrome. Molecular genetic characterization was performed using next-generation sequencing combined with the Sanger method. Biochemical and protein studies were achieved by enzymatic activities, blue native gel electrophoresis, and western blot analysis. All patients displayed novel homozygous mutations in the NDUFA12 gene, leading to the virtual absence of the corresponding protein. Surprisingly, despite the fact that in none of the analyzed patients, NDUFA12 protein was detected, they present a different onset and clinical course of the disease. Our report expands the array of genetic alterations in NDUFA12 and underlines phenotype variability associated with NDUFA12 defect.

Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

The complete plastomes of two flowering epiparasites (Phacellaria glomerata and P. compressa): Gene content, organization, and plastome degradation.

A plant parasite obligately parasitizing another plant parasite is referred to as epiparasite, which is extremely rare in angiosperms, and their complete plastome sequences have not been characterized to date. In this study, the complete plastomes of two flowering epiparasites: Phacellaria compressa and P. glomerata (Amphorogynaceae, Santalales) were sequenced. The plastomes of both species are of similar size, structure, gene content, and arrangement of genes to other hemiparasites in Santalales. Their plastomes were characterized by the functional loss of plastid-encoded NAD(P)H-dehydrogenase and infA genes, which strongly coincides with the general pattern of plastome degradation observed in Santalales hemiparasites. Our study demonstrates that the relatively higher level of nutritional reliance on the host plants and the reduced vegetative bodies of P. compressa and P. glomerata do not appear to cause any unique plastome degradation compared with their closely related hemiparasites.

Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment.

Bone marrow engraftment of the hematopoietic stem and progenitor cells (HSPCs) involves homing to the vasculatures and lodgment to their niches. How HSPCs transmigrate from the vasculature to the niches is unclear. Here, we show that loss of diaphanous-related formin mDia2 leads to impaired engraftment of long-term hematopoietic stem cells and loss of competitive HSPC repopulation. These defects are likely due to the compromised trans-endothelial migration of HSPCs since their homing to the bone marrow vasculatures remained intact. Mechanistically, loss of mDia2 disrupts HSPC polarization and induced cytoplasmic accumulation of MAL, which deregulates the activity of serum response factor (SRF). We further reveal that beta2 integrins are transcriptional targets of SRF. Knockout of beta2 integrins in HSPCs phenocopies mDia2 deficient mice. Overexpression of SRF or beta2 integrins rescues HSPC engraftment defects associated with mDia2 deficiency. Our findings show that mDia2-SRF-beta2 integrin signaling is critical for HSPC lodgment to the niches.

Cyclic electron flow modulate the linear electron flow and reactive oxygen species in tomato leaves under high temperature.

The cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in photosynthesis and also functions in plant tolerance of abiotic environmental stress. However, the role of PGR5/PGRL1- and NDH-dependent CEF in tomato under hightemperature (HT) is poorly understood. Here, we assessed the photoprotective effect of these pathways in tomato leaves under HT by using antimycin A (AA) and rotenone (R), which are chemical inhibitors of PGR5/PGRL1- and NDH-dependent CEF, respectively. The results showed that AA treatment caused significantly greater inhibition of CEF under HT compared to R treatment. Moreover, AA treatment caused a greater decrease in maximal photochemistry efficiency (Fv/Fm) and increased damage to the donor and acceptor side of photosystem II (PSII); however, the limitation of the acceptor side in PSI [Y(NA)] was significantly increased. In addition, thylakoid membrane integrity was compromised and reactive oxygen species, proton gradient (ΔpH), antioxidant enzyme activity, and the expression of photosystem core subunit genes were significantly decreased under AA treatment. These findings indicate that PGR5/PGRL1-dependent CEF protects PSII and PSI from photooxidative damage through the formation of ΔpH while maintaining thylakoid membrane integrity and normal gene expression levels of core photosystem components. This study demonstrates that PGR5/PGRL1-dependent CEF plays a major role in HT response in tomato.

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE.

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

Photochemical regeneration of flavoenzymes - An Old Yellow Enzyme case-study.

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells.

Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Arabidopsis thaliana alternative dehydrogenases: a potential therapy for mitochondrial complex I deficiency? Perspectives and pitfalls.

BACKGROUND: Complex I (CI or NADH:ubiquinone oxidoreductase) deficiency is the most frequent cause of mitochondrial respiratory chain defect. Successful attempts to rescue CI function by introducing an exogenous NADH dehydrogenase, such as the NDI1 from Saccharomyces cerevisiae (ScNDI1), have been reported although with drawbacks related to competition with CI. In contrast to ScNDI1, which is permanently active in yeast naturally devoid of CI, plant alternative NADH dehydrogenases (NDH-2) support the oxidation of NADH only when the CI is metabolically inactive and conceivably when the concentration of matrix NADH exceeds a certain threshold. We therefore explored the feasibility of CI rescue by NDH-2 from Arabidopsis thaliana (At) in human CI defective fibroblasts. RESULTS: We showed that, other than ScNDI1, two different NDH-2 (AtNDA2 and AtNDB4) targeted to the mitochondria were able to rescue CI deficiency and decrease oxidative stress as indicated by a normalization of SOD activity in human CI-defective fibroblasts. We further demonstrated that when expressed in human control fibroblasts, AtNDA2 shows an affinity for NADH oxidation similar to that of CI, thus competing with CI for the oxidation of NADH as opposed to our initial hypothesis. This competition reduced the amount of ATP produced per oxygen atom reduced to water by half in control cells. CONCLUSIONS: In conclusion, despite their promising potential to rescue CI defects, due to a possible competition with remaining CI activity, plant NDH-2 should be regarded with caution as potential therapeutic tools for human mitochondrial diseases.

Reaction of Mast Cells in the Zone of Polypropylene Mesh Implantation.

Reaction of mast cells of adult male Wistar rats (n=15) in the zone of polypropylene mesh fixation was studied by histochemical, immunohistochemical, and traditional morphological methods on days 1, 5, 10, and 30 after implantation. Immediately after the intervention, mast cells stimulated the processes aimed at wound healing. Secretion of mast cells was clearly regulatory. These cells migrated to the zone of injury for subsequent activation of their function. The number of cNOS+ mast cells near the polypropylene mesh was maximum on day 1 and the number of iNOS+ mast cells peaked on day 5 of the experiment, which probably represented a compensatory reaction. Presumably, stimulation of fibrillogenesis was largely due to the activatory effect of mast cells on the fibroblast function, but not to collagen production by these mast cells.