Proteoglycans support proper granule formation in pancreatic acinar cells

ArticleCopyright © Springer-Verlag Berlin Heidelberg 2015Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.German Research FoundationPortuguese Foundation for Science and Technology (FCT

Walnut (Juglans regia L.) leaves: phenolic compounds, antimicrobial activity ans antioxidant potential of different cultivars

Different cultivars of walnut (Juglans regia L.) leaves (Cv. Lara, Franquette, Mayette, Marbot, Mellanaise and Parisienne) grown in Portugal, were investigated in what concerns phenolic compounds and antimicrobial and antioxidant properties. Phenolics analysis was performed by reversed-phase HPLC/DAD and 10 compounds were identified and quantified: 3- and 5-caffeoylquinic acids, 3- and 4-pcoumaroylquinic acids, p-coumaric acid, quercetin 3-galactoside, quercetin 3-pentoside derivative, quercetin 3-arabinoside, quercetin 3-xyloside and quercetin 3-rhamnoside. The antimicrobial capacity was screened against Gram positive (Bacillus cereus, B. subtilis, Staphylococcus aureus) and Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans). Walnut leaves selectively inhibited the growth of Gram positive bacteria, being B. cereus the most susceptible one (MIC 0.1 mg/mL). Gram negative bacteria and fungi were resistant to the extracts at 100 mg/mL. Lara walnut leaves were also submitted to antibacterial assays using 18 clinical isolates of Staphylococcus sp. Antioxidant activity was accessed by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and b-carotene linoleate model system. In a general way, all of the studied walnut leaves cultivars presented high antioxidant activity (EC50 values lower than 1 mg/mL), being Cv. Lara the most effective one

Inhibition of nitric oxide biosynthesis by anthocyanin fraction of blackberry extract.

Anthocyanins are natural colorant belonging to the flavonoid family, widely distributed among flowers, fruits, and vegetables. Some flavonoids have been found to possess anticarcinogenic, cytotoxic, cytostatic, antioxidant, and anti-inflammatory properties. Since increased nitric oxide (NO) plays a role in inflammation, we have investigated whether the pharmacological activity of the anthocyanin fraction of a blackberry extract (cyanidin-3-O-glucoside representing about 88% of the total anthocyanin content) was due to the suppression of NO synthesis. The markedly increased production of nitrites by stimulation of J774 cells with lipopolysaccharide (LPS) for 24 h was concentration-dependently inhibited by the anthocyanin fraction (11, 22, 45, and 90 μg/ml) of the extract. Moreover, this inhibition was dependent on a dual mechanism, since the extract attenuated iNOS protein expression and decreased the iNOS activity in lungs from LPS-stimulated rats. Inhibition of iNOS protein expression appeared to be at the transcriptional level, since the extract and similarly cyanidin-3-O-glucoside (10, 20, 40, and 80 μg/ml, amounts corresponding to the concentrations present in the extract) decreased LPS-induced NF-κB activation, through inhibition of IκBα degradation, and reduced ERK-1/2 phosphorylation in a concentration-dependent manner. In conclusion, our study demonstrates that at least some part of the anti-inflammatory activity of blackberry extract is due to the suppression of NO production by cyanidin-3-O-glucoside, which is the main anthocyanin present in the extract. The mechanism of this inhibition seems to be due to an action on the expression/activity of the enzyme. In particular, the protein expression was inhibited through the attenuation of NF-κB and/or MAPK activatio

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans

Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-ß1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factor-ß1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-ß1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-ß1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease

Anthocyanins and Flavonoids of Vaccinium L.

Vaccinium L., comprising approximately 450 species primarily in the Northern Hemisphere, is a genus of shrubs or lianas in the family Ericaceae. The berries of many species are harvested for household consumption and commercial sale. The genus produces a wide range of compounds such as anthocyanins, flavonoids, chromones, coumarins, lignans, benzoic acids, iridoids, sterols, and triterpenoids, but is best known for the production of anthocyanins and flavonoids. Extracts and isolates of anthocyanins and flavonoids from Vaccinium fruits or leaves showed antioxidative, anti-inflammatory, antitumor, antiviral, vasoprotective, and antifungal activities. To data, more than 116 anthocyanins and flavonoids compounds have been isolated and identified primarily from the fruits or leaves of Vaccinium. This article reviews phytochemistry and pharmaceutical properties of these compounds

Smad and p38 MAP kinase-mediated signaling of proteoglycan synthesis in vascular smooth muscle

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the "response to retention" hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor β (TGF-β) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-β stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-β increased [ 35S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-β-mediated [ 35S]sulfate incorporation with 92% inhibition at 3 μM. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration dependently blocked TGF-β-mediated [ 35S]sulfate incorporation. TGF-β increased [ 3H]glucosamine incorporation into glycosaminoglycans by 180% and [ 35S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-β to increase proteoglycan to LDL binding. TGF-β mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease