Sanguinarine inhibits VEGF-induced angiogenesis in a fibrin gel matrix

BACKGROUND: The identification of possible ways to block blood vessels formation has become a major scientific objective of the last decade and several phytochemicals are currently being exploited to target tumour angiogenesis. AIM: The effects of Sanguinarine (SA), an alkaloid from the root of Sanguinaria Canadensis, were evaluated in an in vitro angiogenesis model; moreover the effects on Akt phosphorylation in porcine aortic endothelial cell line (AOC) were also examined. METHODS: SA (300 nM) was tested in the presence or absence of VEGF (100 ng/ml) in a three dimensional angiogenesis bioassay obtained pipetting a suspension of AOC on microcarrier beads in a fibrinogen solution before the addition of thrombine. Endothelial cell proliferation was measured at 48, 96, 144, 192 h. The phosphorylation of Akt was measured by ELISA in 2 x 10(5) AOC treated as described above. RESULTS: The addition of SA abolished (p< 0.001) VEGF stimulatory effect on AOC growth at all the examined times. In addition, the stimulatory effect induced by VEGF on Akt phosphorylation was significantly (p< 0.001) inhibited by SA. CONCLUSION: SA appear to be an antiangiogenic natural product by directly suppressing the proliferative effect of VEGF on endothelial cell line: this effect could be mediated by blocking the VEGF-induced Akt activation.     

Regulation of progastricsin mRNA levels in sea bass (Dicentrarchus labrax) in response to fluctuations in food availability

In this study the sea bass (Dicentrarchus labrax) pepsinogen C gene was isolated. The nucleotide sequences of all exons are presented. The organization of the gene is compatible with that of other aspartic proteinases. The predicted 388-residue amino acid (aa) sequence of sea bass pepsinogen C consists of a signal sequence of 16 amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two characteristic active-site aspartic acids. We also analyzed fasting-induced changes in the expression of progastricsin mRNA, using real-time RT-PCR absolute quantification. Progastricsin mRNA copy number was downregulated under conditions of negative energy balance, such as starvation, and upregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass progastricsin gene and support a role of this gastric digestive enzyme in the regulation of food intake in sea bass.     

EVA regulates thymic stromal organisation and early thymocyte development

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.     

2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida

The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second molecule of GSH with much lower affinity (Kd approximately 2 to 11 microM). The enzyme is unstable in the absence of GSH. The turnover number in the forward direction (47 s(-1) at 25 degrees C) greatly exceeds off rates for GSH (koff approximately 10(-3) to 10(-2) s(-1) at 10 degrees C), suggesting that GSH acts as a tightly bound cofactor in the reaction. The crystal structure of the enzyme at 1.7 A resolution reveals that the isomerase is closely related to class kappa GSH transferases. Diffraction quality crystals could only be obtained in the presence of GSH and HCCA/tHBPA. Clear electron density is seen for GSH. Electron density for the organic substrates is located near the GSH and is best modeled to include both HCCA and tHBPA at occupancies of 0.5 for each. Although there is no electron density connecting the sulfur of GSH to the organic substrates, the sulfur is located very close (2.78 A) to C7 of HCCA. Taken together, the results suggest that the isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.     

Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.     

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.