Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Development and characterization of 33 polymorphic microsatellite markers in Trachurus japonicus by SLAF-seq technology

Trachurus japonicus is an economically important fish in the northwestern Pacific Ocean. However, its resources have declined seriously and there is an urgent need for a wide-range of investigations of the existing genetic resources. This requires a large number of diverse molecular markers with high discriminating power. In this study, we identified 43,264 perfect SSRs in T. japonicus genome using SLAF-seq technology. Of these, we randomly selected 106 SSRs (tri-nucleotide to hexa-nucleotide) to test for polymorphism. Eventually, we successfully developed a total of 33 loci including 8 tri-nucleotide and 25 long repeat motifs (tetra-nucleotide to hexa-nucleotide). The number of alleles (Na) of these loci ranged from 4 to 24 (mean 12.6). The observed heterozygosity (Ho) and expected heterozygosity (He) varied from 0.258 to 0.969 (mean 0.723) and from 0.452 to 0.962 (mean 0.827), respectively. All loci except TJ6-7 were highly informative (PIC > 0.5). These results showed that the shortlisted 33 loci exhibited moderate to relatively high genetic diversity, of which 18 were regarded as highly polymorphic and well-resolved. In summary, these diverse and potential microsatellites detected in our study provide substantial genetic basis for the screening of polymorphic SSR markers of T. japonicus and also provide a powerful tool to perform further studies on the genetic resource assessment and conservation of T. japonicus.     

GDF11 prevents the formation of thoracic aortic dissection in mice: Promotion of contractile transition of aortic SMCs

Thoracic aortic dissection (TAD) is an aortic disease associated with dysregulated extracellular matrix composition and de-differentiation of vascular smooth muscle cells (SMCs). Growth Differentiation Factor 11 (GDF11) is a member of transforming growth factor β (TGF-β) superfamily associated with cardiovascular diseases. The present study attempted to investigate the expression of GDF11 in TAD and its effects on aortic SMC phenotype transition. GDF11 level was found lower in the ascending thoracic aortas of TAD patients than healthy aortas. The mouse model of TAD was established by β-aminopropionitrile monofumarate (BAPN) combined with angiotensin II (Ang II). The expression of GDF11 was also decreased in thoracic aortic tissues accompanied with increased inflammation, arteriectasis and elastin degradation in TAD mice. Administration of GDF11 mitigated these aortic lesions and improved the survival rate of mice. Exogenous GDF11 and adeno-associated virus type 2 (AAV-2)-mediated GDF11 overexpression increased the expression of contractile proteins including ACTA2, SM22α and myosin heavy chain 11 (MYH11) and decreased synthetic markers including osteopontin and fibronectin 1 (FN1), indicating that GDF11 might inhibit SMC phenotype transition and maintain its contractile state. Moreover, GDF11 inhibited the production of matrix metalloproteinase (MMP)-2, 3, 9 in aortic SMCs. The canonical TGF-β (Smad2/3) signalling was enhanced by GDF11, while its inhibition suppressed the inhibitory effects of GDF11 on SMC de-differentiation and MMP production in vitro. Therefore, we demonstrate that GDF11 may contribute to TAD alleviation via inhibiting inflammation and MMP activity, and promoting the transition of aortic SMCs towards a contractile phenotype, which provides a therapeutic target for TAD.     

Plasma Neutrophil Elastase and Elafin Imbalance Is Associated with Acute Respiratory Distress Syndrome (ARDS) Development

Background

We conducted an exploratory study of genome-wide gene expression in whole blood and found that the expression of neutrophil elastase inhibitor (PI3, elafin) was down-regulated during the early phase of ARDS. Further analyses of plasma PI3 levels revealed a rapid decrease during early ARDS development. PI3 and secretory leukocyte proteinase inhibitor (SLPI) are important low-molecular-weight proteinase inhibitors produced locally at neutrophil infiltration site in the lung. In this study, we tested the hypothesis that an imbalance between neutrophil elastase (HNE) and its inhibitors in blood is related to the development of ARDS.

Methodology/Principal Findings

PI3, SLPI, and HNE were measured in plasma samples collected from 148 ARDS patients and 63 critical ill patients at risk for ARDS (controls). Compared with the controls, the ARDS patients had higher HNE, but lower PI3, at the onset of ARDS, resulting in increased HNE/PI3 ratio (mean = 14.5; 95% CI, 10.9–19.4, P<0.0001), whereas plasma SLPI was not associated with the risk of ARDS development. Although the controls had elevated plasma PI3 and HNE, their HNE/PI3 ratio (mean = 6.5; 95% CI, 4.9–8.8) was not significantly different from the healthy individuals (mean = 3.9; 95% CI, 2.7–5.9). Before the onset (7-days period prior to ARDS diagnosis), we only observed significantly elevated HNE, but the HNE-PI3 balance remained normal. With the progress from prior to the onset of ARDS, the plasma level of PI3 declined, whereas HNE was maintained at a higher level, tilting the balance toward more HNE in the circulation as characterized by an increased HNE/PI3 ratio. In contrast, three days after ICU admission, there was a significant drop of HNE/PI3 ratio in the at-risk controls.

Conclusions/Significance

Plasma profiles of PI3, HNE, and HNE/PI3 may be useful clinical biomarkers in monitoring the development of ARDS.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cytoplasm-localized SIRT1 enhances apoptosis

In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.     

Dauricine induces apoptosis,inhibits proliferation and invasion through inhibiting NF‐κB signaling pathway in colon cancer cells

Dauricine, a bioactive component of Asiatic Moonseed Rhizome, has been widely used to treat a large number of inflammatory diseases in traditional Chinese medicine. In our study, we demonstrated that dauricine inhibited colon cancer cell proliferation and invasion, and induced apoptosis by suppressing nuclear factor‐kappaB (NF‐κB) activation in a dose‐ and time‐dependent manner. Addition of dauricine inhibited the phosphorylation and degradation of IκBα, and the phosphorylation and translocation of p65. Moreover, dauricine down‐regulated the expression of various NF‐κB‐regulated genes, including genes involved cell proliferation (cyclinD1, COX2, and c‐Myc), anti‐apoptosis (survivin, Bcl‐2, XIAP, and IAP1), invasion (MMP‐9 and ICAM‐1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that dauricine significantly suppressed colonic tumor growth. Taken together, our results demonstrated that dauricine inhibited colon cancer cell proliferation, invasion, and induced cell apoptosis by suppressing NF‐κB activity and the expression profile of its downstream genes. These findings provide evidence for a novel role of dauricine in preventing or treating colon cancer through modulation of NF‐κB singling pathway. J. Cell. Physiol. 225: 266–275, 2010. © 2010 Wiley‐Liss, Inc.     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Selective inhibition of protein kinase C β2 attenuates the adaptor P66Shc-mediated intestinal ischemia–reperfusion injury

Apoptosis is a major mode of cell death occurring during ischemia–reperfusion (I/R) induced injury. The p66^Shc adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ₂/p66^Shc pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia–reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ₂ were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ₂ suppressed p66^Shc overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ₂ via small interfering RNA (siRNA) inhibited H/R-induced p66^Shc overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66^Shc phosphorylation and this was inhibited by ruboxistaurin and PKCβ₂ siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ₂ inhibition protects mice from gut I/R injury by suppressing the adaptor p66^Shc-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.