Effects of mutant TDP-43 on the Nrf2/ARE pathway and protein expression of MafK and JDP2 in NSC-34 cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and lacks an effective treatment. The disease pathogenesis has not been clarified at present. Pathological transactive response DNA-binding protein 43 (TDP-43) plays an important role in the pathogenesis of ALS. Nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is found in a mutant TDP-43 transgenic cell model, but its downstream antioxidant enzyme expression is decreased. To elucidate the specific mechanism of Nrf2/ARE (antioxidant responsive element) signaling dysfunction, we constructed an ALS cell model with human mutant TDP-43 using the NSC-34 cell line to evaluate the impact of the TDP-43 mutation on the Nrf2/ARE pathway. We found the nuclear translocation of Nrf2, but the expression of total Nrf2, cytoplasmic Nrf2, and downstream phase II detoxifying enzyme (NQO1) was decreased in NSC-34 cells transfected with the TDP-43-M337V plasmid. Besides, TDP-43-M337V plasmid-transfected NSC-34 cells were rounded with reduced neurites, shortened axons, increased levels of intracellular lipid peroxidation products, and decreased viability, which suggests that the TDP-43-M337V plasmid weakened the antioxidant capacity of NSC-34 cells and increased their susceptibility to oxidative damage. We further showed that expression of the MafK protein and the Jun dimerization protein 2 (JDP2) was reduced in TDP-43-M337V plasmid-transfected NSC-34 cells, which might cause accumulation of Nrf2 in nuclei but a decrease in NQO1 expression. Taken together, our results confirmed that TDP-43-M337V impaired the Nrf2/ARE pathway by reducing the expression of MafK and JDP2 proteins, and provided information for further research on the molecular mechanisms of TDP-43-M337V in ALS.

First Report of Bacterial Leaf Streak of Strelitzia reginae Caused by Burkholderia cepacia.

In 2011, the bacterial leaf streak disease of the monocotyledonous flowering plant, commonly known as bird of paradise (Strelitzia reginae), occurred in a nursery in Guangzhou, Guangdong Province, China. Lesions on diseased leaves began as water-soaked leaf spots or streaks near the central and secondary veins, eventually expanded along veins and became brown necrotic streaks. Occasionally, during wet conditions, seedlings were completely blighted. The disease incidence was about 12% in the nursery. Bacteria were consistently isolated on nutrient agar (NA) (4) from surface-sterilized symptomatic lesions and purified on NA. Three bacterial strains were tested for pathogenicity on S. reginae plants. Three plants were inoculated per bacterial strain (bacterial suspensions 107 CFU/ml in nutrient broth [NB] [4]) by wounding three young, fully expanded leaves (four wounds per leaf) with needle. Plants were placed in polyethylene bags 1 day before inoculation and maintained for 7 days after inoculation. Three control plants were inoculated with NB. Water-soaked areas on leaves were observed on all inoculated plants 7 days after inoculation. Within 10 days, brown streaks were observed. All strains induced similar symptoms as those observed on the plants in the nursery. Control plants showed no symptoms. For molecular identification, a near full-length sequence of the 16S rRNA gene was amplified from strain TNT1-1 (GenBank Accession No. JX901049.1) with primers 27F and 1492R (3), obtaining a PCR product of ~1,500 bp. A BLAST search in GenBank revealed the highest similarity (99.5%) to sequences of Burkholderia cepacia (FN178432.1 and FN178432.1). BIOLOG identification showed that TTN1-1 had the highest probability index of 0.85 and highest similarity index of 0.85 to B. cepacia. For biochemical characteristics, the strain was gram negative, anaerobic growth test negative, oxidase negative, catalase positive, did not produce fluorescent pigment on KB (4), did not grow on DIM agar (4), arginine dihydrolysis negative, nitrate reduction negative, starch hydrolysis negative, gelatin liquefaction negative, citrate, D-arabinose, L-fructose, trehalose, and maltose utilization positive, didn't produce acid from glucose, and grew on Tween 80 medium at 41°C. The above characteristics were identical to that of reference isolate B. cepacia ATCC 25416. Additionally, bacteria isolated on NA from the leading edge of lesions of inoculated plants with the strain were identical to the inoculated strain based on 16S rDNA sequence analysis, but no bacteria were recovered from the wounded sites on the control plants. Therefore, bacterial leaf streak of bird of paradise is caused by B. cepacia based on Koch's postulates. In contrast, two bacterial diseases on S. reginae were previously reported to be caused by Xanthomonas campestris (1) and B. gladioli (2) in the United States and Italy, respectively. A similar leaf streak disease on S. nicolai was caused by Acidovorax avenae subsp. avenae in the United States (5). To our knowledge, this is the first report of a leaf streak disease on S. reginae caused by B. cepacia. References: (1) A. R. Chase and J. B. Jones. Plant Dis. 71:845, 1987. (2) G. Cirvilleri et al. Plant Dis. 90:1553, 2006. (3) I. M. Lee et al. Appl. Environ. Microbiol. 63:2631, 1997. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (5) T. E. Seijo and N. A. Peres. Plant Dis. 95:1474, 2011.

Microsatellite analysis of different ploidy offspring of artificial gynogenesis in Cyprinus carpio.

Gynogenesis was induced by using UV-irradiated spermatozoa of blunt snout bream Megalobrama amblycephala to activate eggs of common carp Cyprinus carpio. The maternal genome was then duplicated by cold shock in 0 to 4° C cold water to retain the second polar body. Two kinds of fry, normal fry and abnormal tortuous fry, were hatched. Their DNA content was measured by flow cytometry. The normal fry were identified as diploid, representing the successful gynogenesis in C. carpio whereas the abnormal tortuous fry were haploid. Ten microsatellite loci were used to study the genetic diversity among C. carpio, diploid gynogenetic C. carpio and unduplicated haploid tortuous fry. The results indicated that the genetic homozygosity of gynogenetic C. carpio was significantly higher than that of C. carpio. The genetic homozygosity of the haploid C. carpio was intermediate between that of gynogenetic C. carpio and C. carpio. It might be easier for the allogenetic DNA fragments to be integrated into the haploid genome than into diploid gynogenetic genome.

[Effects of sIL-13Rα2 on the nasal mucosa goblet cell apoptosis of allergic rhinitis of rats].

Objective: To investigate the effects of sIL-13Rα2 on the apoptosis of goblet cell in nasal mucosa of allergic rhinitis rats. Methods: Forty healthy male Wistar rats were randomly divided into 4 groups (10 rats per group): control group (group A), AR group (group B), sIL-13Rα2 group (group C) and triamcinolone acetonide group (group D). Ovalbumin (OVA) and aluminum hydroxide were used to establish the AR rat model. After the establishment of AR rat models, 50 µl PBS, 100 µg/50 µl IL-13Rα2 and 3.5 µg/50 µl triamcinolone acetonide were respectively dropped into each nasal cavity of every rat two times a week from 4 to 10 week in group B, group C and group D. Group A was operated with saline instead of OVA. The nasal mucosa tissues were collected at 24 h after the final administration. AB-PAS staining method was used to detect the quantity and secretion of goblet cells in the nasal mucosa tissue of all groups. Immunohistochemistry method was used to detect the expression of Bax proteins.Apoptosis was detected by TUNEL method.ANOVA analysis was used to compare multiple groups, and LSD-t test was used to compare the two groups.Pearson correlation analysis was used to analyze the correlation between the Bax positive cell rate of goblet cells and the rate of apoptotic cells. The difference was statistically significant with P<0.05. Results: Compared with group A, there were more goblet cells and hypersecretion of mucus in the nasal mucosa tissue of rats in group B while fewer in group C. The goblet cells in group C and group D were significantly fewer than that in group B (0.639 00±0.831 vs 0.956 7±0.980, 0.661 90±0.657 vs 0.956 7±0.980, t value was 2.748, 2.767, respectively, all P<0.05). The immunohistochemistry results showed that the positive expression rates of Bax protein in goblet cells of group C and group D were significantly higher than that in group B (0.880 2±0.125 vs 0.568 7±0.953, 0.938 4±0.200 vs 0.568 7±0.953, t value was -2.292, -2.685, respectively, all P<0.05). The apoptosis rates of goblet cell in nasal mucosa of group C and group D were also significantly higher than that in group B (0.516 0±0.079 vs 0.274 0±0.056, 0.535 4±0.829 vs 0.274 0±0.056, t value was -17.671, -2.225, respectively, all P<0.05). The expression of Bax protein and apoptosis of goblet cells were positively correlated (r=0.859, P<0.01). Conclusion: sIL-13Rα2 can induce apoptosis of the goblet cells in nasal mucosa of allergic rhinitis rats, by inhibiting IL-13 and up regulating Bax.

[Effects of inhibiting the phosphorylation of JNK by absorbed INF-γon the remodeling of nasal mucosa in allergic rhinitis rats].

Objective:To study the role of phosphorylated JNK(c-Jun N-terminal kinase) on nasal mucosa remodeling in allergic rhinitis(AR) rats and the influence of IFN-γon IL-1ß,JNK and nasal mucosa remodeling.Method:According to random number table,48 Wistar rats were divided into control group(A group),AR group(B group),IFN-γgroup(C group) and triamcinolone acetonide group(D group).The rats in group B,C and D were sensitized and provocated for inducing AR by intraperitoneal injection of ovalbumin(OVA) and Al(OH)3.Thirty minutes before intranasally challenged,rats in three groups were administrated by instillation of PBS,IFN-γand triamcinolone acetonide into nasal cavities,while the group A rats were administrated by saline solution.Ten rats in each group were selected to enter the final experiment.The density of IL-1ßin serum and nasal lavage fluid were tested by ELISA.The mean absorbance (mA) of phosphorylated JNK and c-Jun were tested by immunohistochemistry.Western Blot detected the P-JNK level in nasal tissue homogenate.Result:The density of IL-1ßin serum and nasal lavage fluid in group C and group D were significantly lower than that of group B (P<0.01).Immunohistochemistry study showed that the protein expression level of phosphorylated JNK and c-Jun of nasal mucosa were significantly increased in group B,but significantly reduced in group C and group D .The mA of phosphorylated JNK and c-Jun in group B were significantly higher than those in the group C and group D(P<0.01).The Western blot showed that the P-JNK of nasal tissue homogenate in group B was higher than that of group C and group D (P<0.01).Conclusion: The phosphorylation of JNK played an important role in nasal mucosa remodeling.IFN-γcould inhibit the phosphorylation of JNK and reduce the nasal mucosa remodeling.The mechanisms may be achieved through down-regulation of IL-1ß.

Animal mitochondria: evolution, function, and disease.

Mitochondria are sub-cellular organelles responsible for producing the majority of cellular energy through the process of oxidative phosphorylation (OXPHOS), and are found in nearly all eukaryotic cells. Mitochondria have a unique genetic system, mitochondrial DNA (mtDNA), which is a small, self-replicating and diverse genome. In the past 30 years, mtDNA has made significant contribution to molecular ecology and phylogeography. Mitochondria also represent a unique system of mitochondrial-nuclear genomic cooperation. Additionally, mitochondrial dysfunction can be fatal. In this paper, we review several aspects of mitochondria, including evolution and the origin of mitochondria, energy supply and the central role of mitochondria in apoptosis, and mitochondrial dysfunction. It is shown that mitochondria play a critical role in many aspects of life.

Analysis of mitochondrial respiratory-related genes reveals nuclear and mitochondrial genome cooperation in allotetraploid hybrid.

An allotetraploid hybrid lineage derived from the distant hybridization of red crucian carp (Carassius auratus red var., ♀, 2n =100) × common carp (Cyprinus carpio L., ♂, 2n =100) was investigated for its mitochondrial and nuclear genome inheritance patterns. Based on liver transcriptomic data for this hybrid, red crucian carp, and common carp, we identified 94, 136, and 86 contigs corresponding to 41, 46, and 37 mitochondrial respiratory chain nuclear genes, respectively. Mitochondrial respiratory chain nuclear gene sequences from red crucian carp and common carp were both detected in the allotetraploid hybrid, indicating that both parental nuclear genomes were participated in the synthesis of mitochondrial respiratory protein complexes in the hybrid. For mitochondrial respiratory related genes, high sequence similarity (>90%) and a low nucleotide divergence rate (<0.2) between red crucian carp and common carp could be a critical factor allowing cooperation of the three genomes (red crucian carp mitochondrial genome, red crucian and common carp nuclear genomes) in the allotetraploid hybrid lineage. Interestingly, gene duplication events were identified in the allotetraploid hybrid, red crucian and common carp, as confirmed by analysis of orthologous gene trees for these fish. Our findings provide valuable information with which to study cooperation between the nuclear and mitochondrial genomes of other hybrids, and will provide basic genetic information of relevance to mitochondrial-related diseases in humans and animals.

Minifish mtDNA has abundance of repeat sequences and inefficient replication in vitro.

Paedocypris is a newly described minifish genus endemic to Southeast Asia. Besides a tiny adult size of ~8 mm in length, minifish feature fragmentary habitats of acidic peat blackwater swamps, an unusual reproduction mode, truncated development and one of the smallest known genomes. A complete sequence is absent for the minifish mitochondrial DNA (mtDNA). Here we report the complete mtDNA sequence and its unusual feature in the minifish P. progenetica (Pp). We show that the Pp mtDNA is a circular molecule of 17,382 bp in length and has the same number of similarly oriented genes as in other vertebrates. Specifically, it comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 D-loop. Surprisingly, the D-loop is elusive for amplification by standard PCR conditions. The D-loop possesses a 28-bp dinucleotide TA repeat and more intriguingly, up to 25 copies of a 34-bp tandem repeat sequence. These tandem repeats predict the formation of paired regions. Hence, besides a generally conserved mtDNA with other vertebrates, the Pp mtDNA features an unusual D-loop and compromised DNA replication in vitro.

Inheritance of the complete mitochondrial genomes in three different ploidy fishes.

Diploid natural gynogenetic goldfish (2nGRCG), triploid hybrids (3nRB) and tetraploid hybrids (4nRB) are generated by distant hybridization of red common goldfish (RCG, Carassius auratus red var.) and blunt snout bream (BSB, Megalobrama amblycephala). In the present study, we obtained the complete mitochondrial DNA (mtDNA) sequences of the hybrid offspring and compared them with the homologous sequences of RCG and BSB. All mtDNA sequences of these hybrids were 16,580bp in length, and the genes number, size, and order were quite similar to that of RCG. Genetic analysis revealed that the mtDNA sequences of these hybrids had high similarity (>99%) and low divergence (<2%) to their maternal RCG, yet lower similarities (84%) and higher divergences (16%) to their paternal BSB. The phylogenetic analysis also showed that the sequences of 2nGRCG, 3nRB and 4nRB were clustered with RCG rather than with BSB. These results indicate that the mitochondrial genomes of 2nGRCG, 3nRB and 4nRB remain maternally inherited after hybridization and polyploidization. Moreover, clade separation of hybrid offspring from their paternal BSB in the phylogenetic tree implies that phylogenetic analysis of mtDNA is incomplete for elucidating the true relationships between different species, particularly when they have undergone hybridization or allopolyploidization. Our study provides significant information for both evolution and genetic studies of mtDNA for hybrid species and allopolyploidization species.