Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.     

Integration of cell attachment,cytoskeletal localization,and signaling by integrin-linked kinase (ILK), CH-ILKBP,and the tumor suppressor PTEN

Cell attachment and the assembly of cytoskeletal and signaling complexes downstream of integrins are intimately linked and coordinated. Although many intracellular proteins have been implicated in these processes, a new paradigm is emerging from biochemical and genetic studies that implicates integrin-linked kinase (ILK) and its interacting proteins, such as CH-ILKBP (alpha-parvin), paxillin, and PINCH in coupling integrins to the actin cytoskeleton and signaling complexes. Genetic studies in Drosophila, Caenorhabditis elegans, and mice point to an essential role of ILK as an adaptor protein in mediating integrin-dependent cell attachment and cytoskeletal organization. Here we demonstrate, using several different approaches, that inhibiting ILK kinase activity, or expression, results in the inhibition of cell attachment, cell migration, F-actin organization, and the specific cytoskeletal localization of CH-ILKBP and paxillin in human cells. We also demonstrate that the kinase activity of ILK is elevated in the cytoskeletal fraction and that the interaction of CH-ILKBP with ILK within the cytoskeleton stimulates ILK activity and downstream signaling to PKB/Akt and GSK-3. Interestingly, the interaction of CH-ILKBP with ILK is regulated by the Pi3 kinase pathway, because inhibition of Pi3 kinase activity by pharmacological inhibitors, or by the tumor suppressor PTEN, inhibits this interaction as well as cell attachment and signaling. These data demonstrate that the kinase and adaptor properties of ILK function together, in a Pi3 kinase-dependent manner, to regulate integrin-mediated cell attachment and signal transduction.     

A Metagenomic Study Highlights Phylogenetic Proximity of Quorum-Quenching and Xenobiotic-Degrading Amidases of the AS-Family

Quorum-sensing (QS) signals of the N-acylhomoserine lactone (NAHL) class are cleaved by quorum-quenching enzymes, collectively named NAHLases. Here, functional metagenomics allowed the discovery of a novel bacterial NAHLase in a rhizosphere that was treated with γ-caprolactone. As revealed by rrs-DGGE and rrs-pyrosequencing, this treatment increased the percentage of the NAHL-degrading bacteria and strongly biased the structure of the bacterial community, among which Azospirillum dominated. Among the 29 760 fosmids of the metagenomic library, a single one was detected that expressed the qsdB gene conferring NAHL-degradation upon E. coli and decreased QS-regulated virulence in Pectobacterium. Phylogenetic analysis of the 34 orfs of the fosmid suggested that it would belong to an unknown Proteobacterium - probably a γ-proteobacterium. qPCR quantification of the NAHLase-encoding genes attM, qsdA, and qsdB revealed their higher abundance in the γ-caprolactone-treated rhizosphere as compared to an untreated control. The purified QsdB enzyme exhibited amidase activity. QsdB is the first amidase signature (AS) family member exhibiting NAHLase-activity. Point mutations in the AS-family catalytic triad K-S-S abolished the NAHLase activity of QsdB. This study extends the diversity of NAHLases and highlights a common phylogenic origin of AS-family enzymes involved in the degradation of natural compounds, such as NAHLs, and xenobiotics, such as nylon and linuron.     

MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the lpa proteins, and their dedicated secretion apparatus, the Mxi—Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored lpa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation ( mxiG *), which had no effect on the stability of the protein, did not affect lpa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocon are involved not only in entry but also in spread of shigella between epithelial cells.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Nouvelles données micropaléontologiques(Foraminifères planctoniques et petits Foraminifères benthiques) sur le stratotype de l'Aquitanien

The authors study the microfauna of small benthonic and planktonic Foraminifera from the Aquitanian stratotype and of a few exposures in the stratotype vicinity.Benthonic microfauna is rich: more than 150species have been collected; among them Miliolidae mainly belonging to the genera Quinqueloculina and Miliola.Planktonic Foraminifera are much less abundant:only 20 species. They authorize to place the stratotype at the boundary of N4–N5 Blow's zones.The figurations of many species are made withthe scanning electronic microscope.     

Nouvelles données micropaléontologiques(Foraminifères planctoniques et petits Foraminifères benthiques) sur le stratotype bordelais du Burdigalien

The present work deals with the microfauna of small benthonic and planktonic Foraminifera from the Burdigalian stratotype of Aquitaine basin (France) and from a few outcrops in the stratotype vicinity showing the upper or lower boundary of the stratotype.Benthonic fauna is rich: more than 250 species divided in 92 genera have been collected. The most frequent are hyaline Foraminifera (Pararotalia, Elphidium, Asterigerina, Florilus, Virgulopsis, Bolivina). The miocene feature is more pronounced than in the Aquitanian stratotype.Planktonic Foraminifera are much less abundant:only 20 species. However, some of them authorize to place the stratotype (or its equivalent l. s.) in N5 Blow's zone and perhaps in the basel N6 zone.The figurations of many species are made withthe scanning electronic microscope.     

Dietary protein restriction during lactation in primiparous sows with different live weights at farrowing: I. Consequences on sow metabolic status and litter growth

The hypothesis that the restriction of dietary protein during lactation has different impacts on sow metabolic status and milk production according to body weight was evaluated. From 5-months of age until farrowing, the gilts were fed to achieve body weights of 180 or 240 kg at farrowing. At this time, 38 sows were assigned to one of three groups: " 180 kg" sows not restricted in dietary protein during lactation (180CP); "180 kg" restricted in protein (180LP), or "240 kg" sows restricted in protein (240LP). Catheters were fitted in the jugular vein of 24 sows and serial blood samples were collected 1 d before and 1 d after weaning. Amongst the protein-restricted animals, heavy sows (240LP) had a smaller appetite than light sows in early lactation, resulting in lower energy and protein intakes in the 240LP than in the 180LP sows. Body protein losses were 8, 11 and 13.5% of calculated body protein mass at farrowing in the 180CP, 180LP and 240LP sows, respectively. At the end of lactation, IGF-I concentrations were lower in the 180LP than in the sows from the other groups, probably because of the uncoupling between GH and IGF-I secretions. Low IGF-I concentrations likely promote lean tissue mobilization. Glucose and insulin profiles suggested an insulin resistance state in the 240LP sows compared with the 180LP sows, which may explain, at least in part, the lower feed intake and body reserve mobilization in these sows. Plasma pre- and post-prandial concentrations of amino acids in late lactation differed among the three treatment groups. Throughout lactation, litters from the 180LP and 240LP sows had a slower growth rate than litters from sows which were not restricted, suggesting that endogenous protein mobilization throughout lactation does not completely compensate for a low protein intake.     

Dietary protein restriction during lactation in primiparous sows with different live weights at farrowing: II. Consequences on reproductive performance and interactions with metabolic status

The hypothesis that the restriction of dietary protein during lactation has different impacts on reproductive performance in light and heavy sows at farrowing was investigated, as well as the relationships between reproductive parameters and sow metabolic data. At farrowing, 38 primiparous sows were assigned to one of three groups: sows weighing 180 kg not restricted in dietary protein during lactation (180CP); sows weighing 180 or 240 kg restricted in protein (180LP and 240LP). Twenty-four sows were catheterized and serial blood samples were collected 1 d before and 1 d after weaning. The sows were inseminated at the first estrus after weaning and slaughtered at d 30 of gestation. Protein restriction reduced the proportion of sows that returned to estrus within 8 d after weaning in the 180LP sows (P < 0.03), but not in the 240LP sows. It also induced a reduction in ovulation rate in the 180LP sows (P < 0.05) and, to a lesser extent, in the 240LP sows (P = 0.12). When the sows were categorized according to return to estrus (WOI < or = 8 or > 8 d), basal and mean concentrations of LH increased after weaning only in sows with a short WOI. Sows with a delayed estrus exhibited a higher ratio of plasma tyrosine to large neutral amino acids (AA, P < 0.01). In conclusion, large body reserves at farrowing buffer, at least in part, the detrimental effect of a strongly negative nitrogen balance on reproduction. We suggest that the alteration of AA profiles induced by dietary protein restriction and body protein loss alters LH secretion via modifications of the neurotransmitters involved in GnRH secretion.