Expression Procedure Optimization of <Emphasis Type="Italic">Carassius aurantus</Emphasis> CYP1A in <Emphasis Type="Italic">Shewanella</Emphasis> Using Plasmid Construction Strategy

Cytochrome P4501A (CYP1A) has attracted an increasing interest due to its important role in metabolism of contaminants in aquatic environment and kinds of biomarkers to monitor the pollutants. CYPs are reported to express in E. coli, yeast and insect cells, while expression levels in these systems are too low to continue further study, such as functional and structural research. In this study, we construct an expression system using Shewanella oneidensis to produce goldfish CYP1A. RBS sequence that can elevate expression level by initiating the translation was added. A leading signal peptide which will direct the goal protein into periplasm of the host was introduced. Moreover, large-size plasmid construction strategy was applied during the successful construction process of expression system. At the position of ~60 kDa, a single band was seen clearly after expression; furthermore the amount of expressed CYP1A was as high as 0.02 micromoles per liter in the culture. Heme test was also performed, the result showed the typical P450 hemoprotein spectra. All these data suggest the possible suitable expression system for fish P4501A system was constructed.     

Comparative analysis of conventional and biological treatment in healing of bone disease

The healing of Bone tissue consists of a complex process. Hence, we designed our study to evaluate chondrial diseases, which are as they have a very low healing capacity. Seventy two elderly osteoarthritis (OA) and 54-paediatric juvenile idiopathic arthritis (JIA) patients were included. The group was divided as 24 OA patients and 18 JIA patients in each group. Group I received Hyualuronic acid and glucocorticoides. Group II received platelet rich plasma and fibrin glue. Group III received PRP, fibrin glue, and MSC. 40 control patients received only PRP treatment. Out of 72 OA patients 35 (48.6%) male and 37 (51.4%) female with mean age of 48 ± 6.5 years. 64 (88.9%) Patients had pain and swelling. 52 (72.2%) lacked flexibility. 42 (58.3%) had hypertrophy. 28 (38.9%) had less cartilage thickness. 34 (47.2%) were in grade 3, grade 2 has 28 (38.9%) and grade 1 has 10 (13.9%) patients respectively. Among 54 JIA patients 28 (51.9%) male and 26 (48.1%) female patients with mean, age 4.6 ± 3.8 years. 39 (72.2%) had pain and swelling. 32 (59.3%) lacked flexibility. 29 (53.7%) children’s had functional disability. Group I patients showed 30% improvement with no statistical significance (P < 0.21). Group II showed 45% improvement with statistical significance (P < 0.01). In Group III 80%, improvement was observed with statistical significance (P < 0.001). In 40 control patients, 60% improvement was observed. In conclusion, use of these MSC, PRP, and PPP are safe and less cost effective for treating OA and JIA.     

不同基因型苦荞幼苗对低磷胁迫的响应

采用沙培法,以4个不同耐低磷苦荞(Fagopyrum tataricum(L.) Gaertn)品种为材料,设正常磷处理(P1,2 mmol/L对照)、低磷胁迫(P2,1 mmol/L)和极低磷胁迫(P3,0.2 mmol/L) 3个处理,研究低磷胁迫对苦荞苗期农艺性状、生理生化指标以及植株磷利用的影响。结果显示:(1)低磷胁迫下,苦荞苗期株高、茎粗、叶面积、地上部干重、根系干重、根系平均直径、根系表面积、根系体积等指标均有所下降;主根伸长、根冠比有所升高,但不同品种的升降幅度有所不同。(2)低磷胁迫使苦荞叶绿素含量、可溶性蛋白含量和根系活力均有所下降,根系的SOD活性、POD活性、酸性磷酸酶活性、可溶性糖含量、游离脯氨酸含量显著增加,且表现为耐低磷苦荞品种的增幅大于不耐低磷苦荞。(3)低磷胁迫使苦荞植株全磷含量和单株磷积累量下降,却使磷利用效率升高。研究结果表明耐低磷品种通过主根伸长下扎以及分泌较多的酸性磷酸酶,合理吸收与利用土壤磷素,通过保持叶片较高的叶绿素含量维持较强的光合能力,通过保持较高的抗氧化酶活性降低膜脂过氧化伤害,最大程度的适应低磷环境。     

Structured illumination microscopy with interleaved reconstruction (SIMILR)

Structured illumination microscopy (SIM) is the commonly used super‐resolution (SR) technique for imaging subcellular dynamics. However, due to its need for multiple illumination patterns, the frame rate is just a fraction of that of conventional microscopy and is thus too slow for fast dynamic studies. A new SR image reconstruction method that maximizes the use of each subframe of the acquisition series is proposed for improving the super‐resolved frame rate by N times for N illumination directions. The method requires no changes in raw data and is appropriate for many versions of SIM setup, including those implementing fast illumination pattern generation mechanism based on spatial light modulator or digital micromirror device. The performance of the proposed method is demonstrated through imaging the highly dynamic endoplasmic reticulum where continuous rapid growths or shape changes of tiny structures are observed.      

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation

Uncoupled endothelial nitric oxide synthase (eNOS) produces O₂^? instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O₂^? production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O₂^? burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O₂^? releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.     

PKM2 knockdown influences SREBP activation and lipid synthesis in bovine mammary-gland epithelial MAC-T cells

Objective

The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.

Results

MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.

Conclusions

The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.

     

Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.     

Effects of 1-Methylcyclopropene on Rice Growth Characteristics and Superior and Inferior Spikelet Development Under Salt Stress

Journal of Plant Growth Regulation - Salinity stress hampers rice growth and development due to its osmotic, ionic, and hormonal (ethylene) stresses. High ethylene production affects inferior and...     

Influence of DNA sequence identity on efficiency of targeted gene replacement.

We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.     

Localization of the major ethidium bromide binding site on tRNA.

Binding of ethidium bromide to Escherichia coli tRNAVal and an RNA minihelix based on the acceptor stem and T-arm of tRNAVal was investigated by 19F and 1H NMR spectroscopy of RNAs labeled with fluorine by incorporation of 5-fluorouracil. Ethidium bromide selectively intercalates into the acceptor stem of the tRNAVal. More than one ethidium bromide binding site is found in the acceptor stem, the strongest between base pairs A6:U67 and U7:A66. 19F and 1H spectra of the 5-fluorouracil-substituted minihelix RNA indicate that the molecule exists in solution as a 12 base-paired stem and a single-stranded loop. Ethidium bromide no longer intercalates between base pairs corresponding to the tRNAVal acceptor stem in this molecule. Instead, it intercalates between base pairs at the bottom of the long stem-loop structure. These observations suggest that ethidium bromide has a preferred intercalation site close to the base of an RNA helical stem.