Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-β Signaling Pathway

Post-operative adhesions are a critical problem in pelvic and abdominal surgery despite a multitude of studies dedicated to finding modalities to prevent their occurrence. Ghrelin administration promotes an anti-fibrotic response in a surgical mouse model of adhesion-induction, but the mechanisms mediating this effect have not been established. In the current study, the molecular mechanisms that underlie the anti-adhesion effect of ghrelin were investigated. Post-surgical adhesions were experimentally created in C57BL/6 wild-type mice via a combination of ischemic peritoneal buttons and cecal multiple abrasions. Ghrelin or saline intraperitoneal injections were given twice daily from two days before surgery to selected time points post-surgically to assess the phenotypic and molecular effects of treatment (1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) after surgery). Endpoints included the scoring of adhesions and gene and protein expression analysis of pro-fibrogenic factors conducted on peritoneal ischemic tissue by quantitative PCR and Western blot. Ghrelin administration significantly reduced post-surgical adhesions and down-regulated pro-inflammatory gene and protein expression, including Tgfb3 and Tgfbr2. The up-regulation of inhibitory proteins Smad6 and Smad7 confirmed the ghrelin-induced blockage of TGF-β signaling. Ghrelin is a candidate therapeutic drug for post-operative adhesion prevention, inhibiting inflammatory responses via blockage of the TGF-β signaling pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.     

Cooperative 3-epimerization of chenodeoxycholic acid byClostridium innocuum andEubacterium lentum

In the course of an in vitro screening of intestinal microorganisms from human feces to assess their ability to perform 3-hydroxy epimerization of chenodeoxycholic acid, we isolated aClostridium innocuum strain with 3-hydroxysteroid dehydrogenating activity; this, when cocultured with a strain ofEubacterium lentum able to produced small amounts of the 3-epimer from chenodeoxycholic acid, was able to markedly strengthen the transformation. The activity of both strains has been tested on 3-oxo-CDCA and on 3-hydroxy-CDCA in an effort to clarify the cooperative epimeric conversion.     

Ultrastructure of long-range transport carriers moving from the trans Golgi network to peripheral endosomes

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60- to 100-nm clathrin-coated vesicles to larger, convoluted tubular-vesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.     

Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.