Guanylin and related peptides.

Guanylin and uroguanylin are short peptides homologous to heat-stable enterotoxins of Escherichia coli and other enteric bacteria. Guanylin and uroguanylin are synthetized from the respective prepropeptides mainly in gastrointestinal mucosa and are secreted both into intestinal lumen and into the blood. Luminally secreted peptides stimulate chloride and bicarbonate secretion in the intestine through the mechanism involving guanylate cyclase C receptor, cyclic GMP, protein kinase G and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Bacterial enterotoxins, which have greater potency than endogenous peptides, induce excessive fluid secretion into intestinal lumen leading to secretory diarhea. Uroguanylin is expressed mainly in enterochromaffin cells of duodenum and proximal small intestine whereas guanylin is abundant in goblet cells of colonic epithelium. Uroguanylin and guanylin increase urinary sodium and potassium excretion both as circulating hormones and as paracrine mediators produced within the kidney. Uroguanylin functions as "intestinal natriuretic hormone" which is secreted in response to oral sodium loading and maintains sodium balance during postprandial period. Plasma and urinary concentrations of guanylin and uroguanylin increase in renal failure and heart failure. Guanylin peptides possess antiproliferative activity in intestinal cells culture and their expression decreases in colonic carcinoma indicating that their deficiency may contribute to the pathogenesis of this disease.     

Swimming stress-induced histochemical and morphological changes in various regions of the rat hippocampus

In rats, swimming stress enhanced functional activity of the hippocampus' pyramidal neurones as manifested by diminishing of the glycogen contents, increasing amount of nucleic acids, augmented nucleus/cytoplasm ratio.     

Differences in surface-dwelling beetles of grasslands invaded and non-invaded by goldenrods (Solidago canadensis,S. gigantea) with special reference to Carabidae

We evaluated surface-dwelling Coleoptera with special reference to ground beetles (Coleoptera: Carabidae) using pitfall traps across fourteen stands of grasslands invaded and non-invaded by invasive goldenrods (Solidago canadensis L. and S. gigantea Ait.) over a 3 year period. We analysed differences in assemblages of invaded and non-invaded grasslands and tested responses of surface-dwelling beetles and carabids to invasion of goldenrods. We identified 29 Coleoptera families and 91 Carabidae species. Solidago invaded grasslands showed significantly higher activity-abundance of rove and carrion beetles and supported greater diversity and significantly higher evenness of surface-dwelling Coleoptera and the number of sampled families and individuals was higher too. We found lower taxonomic richness and significantly lower activity-abundance of carabids across goldenrods stands. Several less common Carabidae species and significantly higher representation of stenotopic brachypterous habitat specialists were also observed within invaded stands. We confirmed that differences in plant cover connected with invasion of goldenrods, soil moisture and abandonment of invaded habitats are the driving mechanisms of changes in surface-dwelling Coleoptera and ground beetles assemblages composition across Solidago invaded grasslands. Overall, changes of grassland biotopes connected with invasion of goldenrods significantly alter Coleoptera families and Carabidae assemblages, but not necessarily reduce diversity.     

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain

Background

Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown.

Results

Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.

Conclusion

Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

     

Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

Introduction

Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures.

Methods

In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals.

Results

EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3⁺ and CD8⁺ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction).

Conclusions

Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.     

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.     

Hpz1 modulates the g1-s transition in fission yeast

Here we characterize a novel protein in S. pombe. It has a high degree of homology with the Zn-finger domain of the human Poly(ADP-ribose) polymerase (PARP). Surprisingly, the gene for this protein is, in many fungi, fused with and in the same reading frame as that encoding Rad3, the homologue of the human ATR checkpoint protein. We name the protein Hpz1 (Homologue of PARP-type Zn-finger). Hpz1 does not possess PARP activity, but is important for resistance to ultraviolet light in the G1 phase and to treatment with hydroxyurea, a drug that arrests DNA replication forks in the S phase. However, we find no evidence of a checkpoint function of Hpz1. Furthermore, absence of Hpz1 results in an advancement of S-phase entry after a G1 arrest as well as earlier recovery from a hydroxyurea block. The hpz1 gene is expressed mainly in the G1 phase and Hpz1 is localized to the nucleus. We conclude that Hpz1 regulates the initiation of the S phase and may cooperate with Rad3 in this function.     

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6–8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.     

Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris

Papillomaviruses are known to cause benign or malignant lesions in various animals. In cattle, bovine papillomavirus (BPV) is the etiologic agent of papillomatosis and neoplasia of the upper gastrointestinal tract and urinary bladder. Currently, there are no standard diagnostic tests or prophylactic vaccines. Protection against papillomavirus infection is conferred by neutralizing antibodies directed towards the major structural protein L1. These antibodies can be efficiently induced by immunization with virus-like particles that are formed spontaneously after L1 gene expression in recombinant systems. The yeast Pichia pastoris is known to provide an efficient system for expression of proteins due to reduced cost and high levels of protein production. We evaluated P. pastoris for expression of the L1 gene from BPV1, BPV2 and BPV4. After methanol induction, the recombinants were able to produce L1 proteins of the three different BPV types. To increase heterologous L1 protein levels, a codon optimization strategy was used for production under bioreactor conditions. The BPV1 L1 protein was identified by monoclonal antibody anti-6xHis. This is the first report of BPV L1 expression in yeast.     

Longitudinal Analysis of the Eco‐Design Management Standardization Process in Furniture Companies

This article discusses how eco‐design management standards have been adopted and the environmental and economic results that have been obtained by the Spanish furniture manufacturers. This is precisely the industry sector in Spain where the dissemination of eco‐design standards has been most important. Using multiple case‐study methodology, the research has shown that, in three companies, more than 90% of the environmental impact of the companies’ products occurs within the manufacturing phase. Companies have implemented tools for life cycle assessment with eco‐indicators values that allow them to assess complex products and evaluate their significant environmental impacts at each stage. The environmental strategies of these companies are based on the continuous improvement of the internal processes and the review and monitoring of their activities. In this approach, the proper choice of materials and the environmental management of the supply chain are the main problems for companies. The outcomes achieved by the companies included some improvements, such as a greater control of product management and a reduction in operating costs, that have allowed them to obtain competitive advantages. Moreover, the adoption of standard management has enabled the companies to drive innovation of products, improve the image of companies and their products, significantly reduce the environmental impact of their products, and adapt to new, more demanding environmental laws and regulations.