CXCR7 contributes to the aggressive phenotype of cholangiocarcinoma cells

Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with β-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with β-arrestin 2.     

Correlation between cardiac oxidative stress and myocardial pathology due to acute and chronic norepinephrine administration in rats

BACKGROUND: To investigate the cardiotoxic role of reactive oxygen species (ROS) and of products derived from catecholamines auto-oxidation, we studied: (1) the response of antioxidant cardiac cellular defence systems to oxidative stress induced by norepinephrine (NE) administration, (2) the effect of NE administration on cardiac beta1-adrenergic receptors by means of receptor binding assay, (3) the cellular morphological alterations related to the biologically cross-talk between the NE administration and cytokines [tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), interleukins IL6, IL8, IL10]. METHODS AND RESULTS: A total of 195 male rats was used in the experiment. All animals underwent electrocardiogram (EKG) before being sacrificed. The results obtained show that NE administration influences the antioxidant cellular defence system significantly increasing glutathione peroxidase (GPx) activity, glutathione reductase (GR) and superoxide dismutase (SOD). The oxidized glutathione (GSH/GSSG) ratio significantly decreases and malondialdehyde (MDA) levels increase showing a state of lipoperoxidation of cardiac tissue. We describe a significant apoptotic process randomly sparse in the damaged myocardium and the effect of ROS on the NE-mediated TNF-alpha, MCP-1, and IL6, IL8, IL10 production. CONCLUSIONS: Our results support the hypothesis that catecholamines may induce oxidative damage through reactive intermediates resulting from their auto-oxidation, irrespective of their interaction with adrenergic receptors, thus representing an important factor in the pathogenesis of catecholamines-induced cardiotoxicity. The rise of the cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium with respect to the cardiac dysfunction resulting from NE injection.     

Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells

The endothelium is a metabolically active organ that regulates the interaction between blood or lymph and the vessel or the surrounding tissue. Blood endothelium has been the object of many investigations whereas lymphatic endothelium biology is yet poorly understood. This report deals with a proteomic approach to the characterization and comparative analysis of lymphatic and blood vessel endothelial cells (ECs). By 2-DE we visualized the protein profiles of EC extracts from the thoracic aorta, inferior vena cava, and thoracic duct of Bos taurus. The three obtained electropherograms were then analyzed by specific software, and 113 quantitative and 25 qualitative differences were detected between the three endothelial gels. The cluster analysis of qualitative and quantitative differences evidenced the protein pattern of lymphatic ECs to be more similar to the venous than to the arterial one. Moreover, venous ECs were interestingly found showing a protein expression profile more similar to the lymphatic ECs than to the arterial ones. We also identified 64 protein spots by MALDI-TOF MS and ESI-IT MS/MS and three reference maps of bovine endothelium were obtained. The functional implications of the identified proteins in vascular endothelial biology are discussed.     

Phytotoxic activity of caulerpenyne from the Mediterranean invasive variety of Caulerpa racemosa: a potential allelochemical

Allelopathy has been postulated as a factor in the colonisation success of Caulerpa racemosa var. cylindracea, an introduced chlorophyte alga which has become invasive in the Mediterranean Sea. In order to reveal their possible phytotoxic activity, secondary metabolites were isolated from a population growing in the Gulf of Naples (Italy) and tested on leaf tissue of the native seagrass Cymodocea nodosa, which often co-occurs with Caulerpa in nature. The seaweed metabolites were identified by ¹H and ¹³C NMR analysis and leaf portions of C. nodosa were exposed to crude diethyl ether extract and purified compounds in the laboratory, under controlled conditions of irradiance and temperature for a period of 144 h. Eventual changes in the photosynthetic performance of the seagrass tissue following the treatment were tested by monitoring its optimal quantum yield with a pulse amplitude modulated (PAM) fluorometer. One of the identified compounds, caulerpenyne, showed a phytotoxic effect, while the other purified metabolites did not reveal any effect at the assayed concentrations. Thus, our results seem to suggest a possible allelopathic activity of caulerpenyne, which may play a role in the successful competition of the invasive C. racemosa var. cylindracea with native macrophytes, such as seagrasses.     

Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway

The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.     

Neuropilin-1 is upregulated in Sj?gren’s syndrome and contributes to pathological neovascularization

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sj?gren’s syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sj?gren’s syndrome.     

Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1

Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.     

Effects of the antimicrobial peptide BMAP-27 in a mouse model of obstructive jaundice stimulated by lipopolysaccharide

An experimental study was designed to investigate the efficacy of BMAP-27, a compound of the cathelicidin family, in neutralizing Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct-ligated mice. Main outcome measures were: endotoxin and TNF-alpha concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. Adult male BALB/c mice were injected intraperitoneally with 2 mg/kg E. coli 0111:B4 LPS 1 week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam-piperacillin (TZP), sham with 1 mg/kg BMAP-27, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1mg/kg BMAP-27. After LPS, TNF-alpha plasma levels were significantly higher in BDL mice compared to sham-operated animals. BMAP-27 achieved a significant reduction of plasma endotoxin and TNF-alpha concentration when compared with placebo- and TZP-treated groups. On the other hand, both TZP and BMAP-27 significantly reduced the bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo- and TZP-treated treated mice and no lethality in sham-operated mice, while only BMAP-27 significantly reduced the lethality to 10%. In light of its dual antimicrobial and anti-endotoxin properties, BMAP-27 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.     

A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.