Peroxisome Biogenesis Disorders.

Peroxisomes are presented in all eukaryotic cells and play essential roles in many of lipid metabolic pathways, including ß-oxidation of fatty acids and synthesis of ether-linked glycerophospholipids, such as plasmalogens. Impaired peroxisome biogenesis, including defects of membrane assembly, import of peroxisomal matrix proteins, and division of peroxisome, causes peroxisome biogenesis disorders (PBDs). Fourteen complementation groups of PBDs are found, and their complementing genes termed PEXs are isolated. Several new mutations in peroxins from patients with mild PBD phenotype or patients with phenotypes unrelated to the commonly observed impairments of PBD patients are found by next-generation sequencing. Exploring a dysfunctional step(s) caused by the mutation is important for unveiling the pathogenesis of novel mutation by means of cellular and biochemical analyses.

Ultrasound processing of liquid system(s) and its antimicrobial mechanism of action.

Ultrasound creates cavitation phenomena, resulting in the formation of several free radicals, namely OH˙ and H˙, due to the breakdown of the H2 O molecule. These radicals affect the cellular integrity of the bacteria, causing the inactivation of several processes, and thus it is important to unravel the mechanism of action of this technology. This research looks into the application and mechanism of action of ultrasound technology as a means of disinfection by acoustic cavitation. Sterile water and synthetic waste water were inoculated with different mutants of Escherichia coli K12 strains containing deletions in genes affecting specific functional properties of E. coli. These were: dnak soxR, soxS, oxyR, rpoS, gadA/gadB, gadC and yneL. Escherichia coli K-12 ΔoxyR appeared to be more resistant to the treatment together with gadW, gadX, gabT and gabD, whereas the mutant K-12 ΔdnaK was more sensitive with c. 2·5 log (CFU per ml) reduction in comparison to their isogenic wild-type E. coli K-12. This indicates that the dnaK gene participates in general stress response and more specifically to hyperosmotic stress. The other E. coli deleted genes tested (soxS, rpoS, gadB, gadC, yneL) did not appear to be involved in protection of microbial cells against ultrasound. SIGNIFICANCE AND IMPACT OF THE STUDY: This study looks at the mechanism of action of ultrasound technology for the disinfection of wastewater. Different mutants with deleted genes were used to study the respective sensitivity or resistance to this treatment. This is essential to characterize changes at the molecular level, which might be occurring during treatment, resulting in bacterial adaptation.

Enrichment of cells with TALEN-induced mutations using surrogate reporters.

Targeted gene knockout using engineered nucleases such as transcription activator like-effector nucleases (TALENs) is a gold standard for investigating the functions of a gene of interest. Although most TALENs can cleave chromosomal DNA efficiently, the activities of designed TALENs are not always high enough to allow the efficient derivation of cells containing TALEN-driven mutations. Thus, simple methods to enrich cells containing TALEN-directed mutations would facilitate the use of TALENs. Here we describe the enrichment of such cells using surrogate episomal reporters coupled with flow cytometric sorting, magnetic separation, or hygromycin selection.

Intracranial self-stimulation-reward or immobilization-aversion had different effects on neurite extension and the ERK pathway in neurotransmitter-sensitive mutant PC12 cells.

Various actions trigger pleasure (reward) or aversion (punishment) as emotional responses. Emotional factors that negatively affect brain neural control processes for long periods of time might cause various mental diseases by inducing neuronal changes. In the present study, newly developed PC12m12 cells which are　highly sensitivity to neurotransmitters such as acetylcholine (ACh), were used. Exposing the cells to plasma from rats that had been subjected to intracranial self-stimulation (ICSS) markedly upregulated neurite outgrowth. In addition, voluntary running in a wheel or forced on a rotating rod was used to induce behavioral excitation in rats, and examinations of their plasma confirmed that the ICSS-induced neurite outgrowth was not associated with the ICSS behavior itself. Furthermore, immunoblotting and treatment with U0126, an ERK (extracellular signal-regulated kinase) antagonist, showed that the ICSS-induced neurite outgrowth was related to neuronal ERK activity. Exposing the same cells to plasma from rats that had been subjected to immobilization (IMM) also increased neurite outgrowth. Although the degree of enhancement was not as great as that seen after the ICSS rat plasma treatment, it was less than that observed after treatment with ACh as a positive control. These results indicate that ICSS or IMM lead to varying degrees of morphological changes, such as enhanced neurite outgrowth, in PC12m12 cells, but the neuronal signal transduction pathways underlying these effects differ; i.e.,the former morphological change might involve the activation of the ERK pathway, whereas the latter changes might not. Using PC12m12 cells which exhibit sensitivity to neurotransmitters, it might be possible to clarify the pathogeneses of mental diseases at the neuronal level and search for therapeutic drugs.

Electrical stimulation induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway.

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in electrical stimulation-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to electrical stimulation at 100 mA for 30 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The frequency of neurite outgrowth induced by electrical stimulation was approximately 10-fold greater than that of neurite outgrowth induced by NGF alone. The neurite outgrowth induced by electrical stimulation was inhibited by a specific p38 MAPK inhibitor, SB203580. The activation of p38 MAPK induces activation of the transcription factor CREB. The activation of CREB induced by electrical stimulation was inhibited by SB203580. Longer electrical stimulation of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that electrical stimulation-induced activation of the p38 MAPK/CREB pathway is responsible for the neurite outgrowth and survival of PC12m3 cells.

Transcriptome sequencing and metabolic pathways of astaxanthin accumulated in Haematococcus pluvialis mutant under 15% CO2.

Transcriptome sequencing and annotation was performed on Haematococcus pluvialis mutant red cells induced with high light under 15% CO2 to demonstrate why astaxanthin yield of the mutant was 1.7 times higher than that of a wild strain. It was found that 56% of 1947 differentially expressed genes were upregulated in mutant cells. Most significant differences were found in unigenes related to photosynthesis, carotenoid biosynthesis and fatty acid biosynthesis pathways. The pyruvate kinase increased by 3.5-fold in mutant cells. Thus, more pyruvate, which was beneficial to carotenoids and fatty acid biosynthesis, was generated. Phytoene synthase, zeta-carotene desaturase, lycopene beta-cyclase involved in ß-carotene biosynthesis in mutant cells were upregulated by 10.4-, 4.4-, and 5.8-fold, respectively. Beta-carotene 3-hydroxylase catalyzing conversion of ß-carotene into astaxanthin was upregulated by 18.4-fold. The fatty acid biosynthesis was promoted because of the upregulation of acetyl-CoA synthetase and acetyl-CoA carboxylase, thus increasing astaxanthin esterification and accumulation in mutant cells.

Transcriptome-based analysis on carbon metabolism of Haematococcus pluvialis mutant under 15% CO2.

To elucidate the mechanism underlying the enhanced growth rate in the Haematococcus pluvialis mutated with 60Co-γ rays and domesticated with 15% CO2, transcriptome sequencing was conducted to clarify the carbon metabolic pathways of mutant cells. The CO2 fixation rate of mutant cells increased to 2.57gL-1d-1 under 15% CO2 due to the enhanced photosynthesis, carbon fixation, glycolysis pathways. The upregulation of PetH, ATPF0A and PetJ related to photosynthetic electron transport, ATP synthase and NADPH generation promoted the photosynthesis. The upregulation of genes related to Calvin cycle and ppdK promoted carbon fixation in both C3 and C4 photosynthetic pathways. The reallocation of carbon was also enhanced under 15% CO2. The 19-, 14- and 3.5-fold upregulation of FBA, TPI and PK genes, respectively, remarkably promoted the glycolysis pathways. This accelerated the conversion of photosynthetic carbon to pyruvate, which was an essential precursor for astaxanthin and lipids biosynthesis.

Blue Native PAGE: Applications to Study Peroxisome Biogenesis.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.

Generation of Peroxisome-Deficient Somatic Animal Cell Mutants.

Cell mutants with a genetic defect affecting various cellular phenotypes are widely utilized as a powerful tool in genetic, biochemical, and cell biological research. More than a dozen complementation groups of animal somatic mutant cells defective in peroxisome biogenesis have been successfully isolated in Chinese hamster ovary (CHO) cells and used as a model system reflecting fatal human severe genetic disorders named peroxisome biogenesis disorders (PBD). Isolation and characterization of peroxisome-deficient CHO cell mutants has allowed the identification of PEX genes and the gene products peroxins, which directly leads to the accomplishment of isolation of pathogenic genes responsible for human PBDs, as well as elucidation of their functional roles in peroxisome biogenesis. Here, we describe the procedure to isolate peroxisome-deficient mammalian cell mutants from CHO cells, by making use of an effective, photo-sensitized selection method.