Studies on the role of goat heart galectin-1 as a tool for detecting post-malignant changes in glycosylation pattern

Galectins are mammalian lectins established to play a crucial role in the progression of various cancer types by the virtue of their differential expression in normal and cancerous cells. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its potential role in the detection of post-malignant changes in glycosylation pattern. When exposed to superoxide radicals generated from a pyrogallol auto-oxidation system, GHG-1 treated erythrocyte suspension released higher amount of oxyhemoglobin than the unagglutinated erythrocytes. The extent of erythrocyte hemolysis was found to be directly proportional to concentrations of hypochlorous acid. GHG-1 was used to detect the change in the β-galactoside expression pattern in erythrocyte membrane from human donors suffering from prostate and breast cancer. No significant change was observed in the hemolysis of lectin agglutinated erythrocytes collected from pre-operated breast cancer patients, whereas significant increase was observed in normal healthy control and post-operated samples. Findings of this study proclaim GHG-1 as an important tool for the detection of post-malignant changes in glycosylation pattern.Abbreviations: Gal-1, galectin-1; GHG-1, goat heart galectin-1; HOCl, hypochlorous acid; OxyHb, oxyhemoglobin     

Detection of Olive‐infecting Viruses in Tunisia

During a virus survey in autumn 2007 and spring 2008 of two Tunisian olive mother blocks, 175 olive samples were collected from 19 different cultivars and tested by RT‐PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV‐1), Olive latent virus 2 (OLV‐2), Olive leaf yellowing‐associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV), using specific sets of primers. The PCR‐negative samples were also subjected to dsRNA and mechanical transmission tests. PCR results indicated that c. 86% of the trees were infected with at least one virus, whereas visible bands were shown by 3 of 24 PCR‐negative samples in dsRNA analysis. OLYaV was the most prevalent virus (49.1%), followed by OLV‐1 (34.3%), CMV (25.7%), OLRSV (16.6%), CLRV (13.1%), SLRSV (7.4%) and OLV‐2 (6.9%), whereas ArMV was not detected. Very high infection rates were found in the two main oil cvs. Chemlali (84.6%) and Chétoui (86.9%).     

A multiwell platform for studying stiffness-dependent cell biology

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.     

Prenylated anthronoid antioxidants from the stem bark of Harungana madagascariensis

Two new prenylated anthronoids, harunmadagascarins A and B, were isolated from the stem bark of Harungana madagascariensis along with six known compounds including two anthronoids: harunganol B and harungin anthrone, one benzophenone: methyl 3-formyl-2,4-dihydroxy-6-methyl benzoate and three pentacyclic triterpenes: friedelin, lupeol and betulinic acid. Harunmadagascarins A and B were characterized as 8,9-dihydroxy-4,4-bis-(3,3-dimethylallyl)-6-methyl-2,3-(2,2-dimethylpyrano)anthrone and 8,9-dihydroxy-4,4,5-tris-(3,3-dimethylallyl)-6-methyl-2,3-(2,2-dimethylpyrano)anthrone, respectively. The structures of these secondary metabolites were determined by spectroscopic means and comparison with the published data. Methyl 3-formyl-2,4-dihydroxy-6-methyl benzoate was isolated for the first time from a plant. Harunmadagascarins A and B, harunganol B and harungin anthrone exhibited significant antioxidant activity.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K _cat/K _m, 4.3×10⁴, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.     

Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant

The present investigation dealt with the use of previously isolated and studied gamma-ray mutant strain Pseudomonas aeruginosa EBN-8 for the production of biosurfactant by using different hydrocarbon substrates viz. n-hexadecane, paraffin oil and kerosene oil, provided in minimal medium, as the sole carbon and energy sources. The batch experiments were conducted in 250 mL Erlenmeyer flasks, containing 50 mL minimal salt media supplemented with 1% (w/v) hydrocarbon substrate, inoculated by EBN-8 and incubated at 37 degrees C and 100 rpm in an orbital shaker. The sampling was done on 24 h basis for 10 d. The surface tension of cell-free culture broth decreased from 53 to 29 mN/m after 3 and 4 d of incubation when the carbon sources were paraffin oil and n-hexadecane, respectively. The largest reduction in interfacial tension from 26 to 0.4 mN/m was observed with n-hexadecane, while critical micelle dilution was obtained as 50 x CMC for paraffin oil as carbon source. When grown on n-hexadecane and paraffin oil, the EBN-8 mutant strain gave 4.1 and 6.3 g of the rhamnolipids/L, respectively. These surface-active substances subsequently allowed the hydrocarbon substrates to disperse readily as emulsion in aqueous phase.     

BDNF and DYRK1A Are Variable and Inversely Correlated in Lymphoblastoid Cell Lines from Down Syndrome Patients

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.