Production of peroxidases by hairy roots ofBrassica napus

Hairy roots cultures derived from leaf explants ofBrassica napus L. produced and secreted peroxidases. The enzyme activity in the medium increased with growth but it remained nearly constant in the tissue. The changes in extracellular peroxidase activity seemed to be correlated with the increase in a basic peroxidase of pI: 9.6. Four isoenzymes with pI in the range 8.5–9.6 and a neutral peroxidase of pI 6.3 were the most important peroxidases detected in cell extracts. Ca²⁺ addition at the beginning of the culture stimulated both the excretion of peroxidase to the medium and the enzyme activity in hairy roots but the isoenzyme profiles did not show qualitative changes during the growth cycle for both culture conditions.     

Genetic and Immunohistochemical Expression of Integrins ITGAV,ITGA6, and ITGA3 As Prognostic Factor for Colorectal Cancer: Models for Global and Disease-Free Survival

Objective

To evaluate the relationship between the expression profiles of 84 extracellular matrix (ECM) genes and the prognosis of patients with colorectal cancer (CRC).

Methods

This retrospective study included 114 patients with stage I–IV CRC who underwent primary tumour resection. Quantitative real-time PCR and immunohistochemistry assays were conducted using primary tumour samples. Kaplan-Meier survival curves were also generated to identify differences in global survival (GS) and disease-free survival (DFS) for the hypo- or hyperexpression status of each marker. The log-rank test was used to verify whether the differences were significant. Stepwise Cox regression models were also used to identify the risk factors associated with GS and DFS in a multivariate mode, and then were used to score the risk of death associated with each marker, either independently or in association.

Results

In the univariate analyses, significant differences in GS in relation to the expression profiles of ITGAV (p = 0.001), ITGA3 (p = 0.002), ITGA6 (p = 0.001), SPARC (p = 0.036), MMP9 (p = 0.034), and MMP16 (p = 0.038) were observed. For DFS, significant differences were observed in associated with ITGAV (p = 0.004) and ITGA3 (p = 0.001). However, only the ITGAV and ITGA6 gene markers for GS (hazard ratio (HR) = 3.209, 95% confidence interval (CI) = 1.412–7.293, p = 0.005 and HR = 3.105, 95% CI = 1.367–7.055, p = 0.007, respectively), and ITGA3 for DFS (HR = 3.806, 95% CI = 1.573–9.209, p = 0.003), remained in the final Cox regression models. A scoring system was developed to evaluate the risk of patient death based on the number of markers for the components of the final GS model. Scores of 0, 1, or 2 were associated with the following mean survival rates [CI]: 47.162 [44.613–49.711], 39.717 [35.471–43.964], 30.197 [24.030–36.327], respectively.

Conclusions

Multivariate mathematical models demonstrated an association between hyperexpression of the ITGAV and ITGA6 integrins and GS, and also between the ITGA3 integrin and DFS, in patients with colorectal tumours. A risk scoring system based on detected hyperexpression of 0, 1, or 2 markers (e.g., ITGAV and/or ITGA6) was also found to accurately correlate with the GS curves generated for the present cohort.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

Ricerche Sul Movimento Degli Stami in Alcune Berberidacee

Riassunto

L'A. studia i movimenti degli stami di Berberis in natura: trova che nel bocciolo i movimenti sono dovuti a fatti di diversità di accrescimento e di evoluzione dei singoli tessuti: determina il momento e le condizioni fisiologiche in cui si iniziano e la sensibilità e le proprietà contrattili (movimento rapido): mediante osservazioni di tipo sperimentale prova che il movimento rapido di contrazione e quello lento di estensione non dipende da relazioni umorali o di altro genere tra il filamento e gli altri organi del fiore ma è unicamente proprietà intrinseca della cellula contrattile: basandosi su questo fatto e sui dati ottenuti negli altri lavori riporta ad un unico schema i vari movimenti che lo stame compie da quando ha acquistato la proprietà contrattile fino al suo distacco dall'asse fiorale.     

Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock‐down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as ‘enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.     

Development of Central Nervous System Autoimmunity Is Impaired in the Absence of Wiskott-Aldrich Syndrome Protein

Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in Was^−/− mice. We describe here that Was^−/− mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in Was^−/− mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in Was^−/− mice. However, adoptive transfer of MOG-activated T cells from Was^−/− mice in WT mice failed to induce EAE. Was^−/− mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. Was^+/− heterozygous mice developed an intermediate clinical phenotype between WT and Was^−/− mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.     

Functional analysis of a fatty acid binding protein produced by Aphidius ervi teratocytes

Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of various aphid species, including Acyrthosiphon pisum (Homoptera, Aphididae), the model host used in the present study. Parasitized hosts show a marked increase of their nutritional suitability for the developing parasitoid larvae. This alteration of the biochemical and metabolic profile is due to a castration process mediated by the combined action of the venom, injected at the oviposition, and of the teratocytes, cells deriving from the dissociation of the embryonic membrane. Teratocytes produce and release in the host haemocoel two parasitism-specific proteins, which are of crucial importance for the development of their sister larvae. One of the proteins is a fatty acid binding protein (Ae-FABP), which shows a high affinity for C14-C18 saturated fatty acids (FAs) and for oleic and arachidonic acids. To better define the possible nutritional role of this protein, we have studied its immunolocalization profile in vivo and the impact on FA uptake by the epidermal and midgut epithelia of A. ervi larvae. During the exponential growth of A. ervi larvae, Ae-FABP is distributed around discrete lipid particles, which are abundantly present in the haemocoel of parasitized host aphids and in the midgut lumen of parasitoid larvae. Moreover, a strong immunodetection signal is evident on the surface of the two larval epithelia involved in nutrient absorption: the parasitoid midgut epithelium and the external epidermal layer. These two epithelia can effectively absorb radiolabelled myristic acid, but the FA transport rates are not affected by the presence in the medium of Ae-FABP. The protein appears to act essentially as a vector in the host haemolymph, transferring FAs from the digestion sites of host lipids to the growing parasitoid larvae. These data indicate that the proteins produced by A. ervi teratocytes may play complementary roles in the nutritional exploitation of the host.     

Expression,surface immobilization,and characterization of functional recombinant cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.