Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

Climate,copepods and seabirds in the boreal Northeast Atlantic – current state and future outlook

The boreal Northeast Atlantic is strongly affected by current climate change, and large shifts in abundance and distribution of many organisms have been observed, including the dominant copepod Calanus finmarchicus, which supports the grazing food web and thus many fish populations. At the same time, large‐scale declines have been observed in many piscivorous seabirds, which depend on abundant small pelagic fish. Here, we combine predictions from a niche model of C. finmarchicus with long‐term data on seabird breeding success to link trophic levels. The niche model shows that environmental suitability for C. finmarchicus has declined in southern areas with large breeding seabird populations (e.g. the North Sea), and predicts that this decline is likely to spread northwards during the 21st century to affect populations in Iceland and the Faroes. In a North Sea colony, breeding success of three common piscivorous seabird species [black‐legged kittiwake (Rissa tridactyla), common guillemot (Uria aalge) and Atlantic puffin (Fratercula arctica)] was strongly positively correlated with local environmental suitability for C. finmarchicus, whereas this was not the case at a more northerly colony in west Norway. Large seabird populations seem only to occur where C. finmarchicus is abundant, and northward distributional shifts of common boreal seabirds are therefore expected over the coming decades. Whether or not population size can be maintained depends on the dispersal ability and inclination of these colonial breeders, and on the carrying capacity of more northerly areas in a warmer climate.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Identification and Functional Validation of a 5′ Upstream Regulatory Sequence in the Human Tyrosinase Gene Homologous to the Locus Control Region of the Mouse Tyrosinase Gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non‐coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5′ upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at ?15 kb. We detected several stretches of homology within the first 30 kb 5′ tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5′ upstream regulatory sequence found between ?8 and ?10 kb of the human tyrosinase locus (termed h5′URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at ?9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue‐specific enhancer in the h5′URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5′URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.     

miRNA-20a促进卵巢癌细胞OVCAR3的转移

目的检测miRNA．20a对卵巢癌细胞系OVCAR3转移能力的影响。方法通过实时定量RT-PCR验证反义寡核苷酸与小干扰RNA封闭与过表达的效果,然后利用MTF、软琼脂集落形成和transwell侵袭实验检测封闭和过表达miRNA．20a对OVCAR3细胞增殖及转移能力的影响。结果封闭内源性miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显降低。过表达miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显升高。结论miRNA-20a可能参与了卵巢癌细胞OVCAR3的转移。     

Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.     

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to th...