A bacteriological survey of an oyster-growing area: The Oualidia Lagoon,Morocco

A 3-year bacteriological survey of an oyster-growing area in Morocco, where the Japanese oyster (Crassostrea gigas) is grown, showed that the contamination of this lagunar ecosystem was not continuous but intermittent and that animal manure and human recreational activities were important sources of pollution. The major source of contamination was of animal origin, except during the summer, when human contamination prevailed.     

cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.

When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.     

A finite element model of stress-mediated vascular adaptation: application to abdominal aortic aneurysms

Despite rapid expansion of our knowledge of vascular adaptation, developing patient-specific models of diseased arteries is still an open problem. In this study, we extend existing finite element models of stress-mediated growth and remodelling of arteries to incorporate a medical image-based geometry of a healthy aorta and, then, simulate abdominal aortic aneurysm. Degradation of elastin initiates a local dilatation of the aorta while stress-mediated turnover of collagen and smooth muscle compensates the loss of elastin. Stress distributions and expansion rates during the aneurysm growth are studied for multiple spatial distribution functions of elastin degradation and kinetic parameters. Temporal variations of the degradation function are also investigated with either direct time-dependent degradation or stretch-induced degradation as possible biochemical and biomechanical mechanisms for elastin degradation. The results show that this computational model has the capability to capture the complexities of aneurysm progression due to variations of geometry, extent of damage and stress-mediated turnover as a step towards patient-specific modelling.     

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.     

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

Olfactory response of Phytoseiulus persimilis (Acari: Phytoseiidae) to untreated and Beauveria bassiana-treated Tetranychus urticae (Acari: Tetranychidae)

Determination of attraction and avoidance behavior of predators is important in concomitant use of multiple natural enemies to control a pest. The olfactory response of the predatory mite Phytoseiulus persimilis was studied to odors related to Tetranychus urticae adults infected by Beauveria bassiana DEBI008 in 0, 24, 48 and 72 h intervals, both in absence and in presence of plants. In plant-present experiments, P. persimilis attraction was neither towards adults of T. urticae infected by 0.02 % Tween 80 (as control), nor to the ones infected by B. bassiana for 0 or 24 h, whereas significant attraction towards the control was observed when tested against T. urticae infected by B. bassiana for 48 or 72 h. In absence of plants, P. persimilis displayed significant avoidance of T. urticae infected by B. bassiana for 48 or 72 h, when their alternative option was 0.02 % Tween 80-infected T. urticae adults. These results indicate that P. persimilis can recognize the presence of B. bassiana and that the predator avoids the fungus. This suggests that the two natural enemy species can be used together in biological control programmes.     

Modeling of cell adhesion and deformation mediated by receptor–ligand interactions

The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor–ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor–ligand interaction via Fick’s Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell.     

Design and characterization of electrochemical dopamine–aptamer as convenient and integrated sensing platform

Here, an ultrasensitive label-free electrochemical aptasensor was developed for dopamine (DA) detection. Construction of the aptasensor was carried out by electrodeposition of gold–platinum nanoparticles (Au–PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs–COOH). A designed complementary amine-capped capture probe (ssDNA1) was immobilized at the surface of PtNPs/CNTs–COOH/GC electrode through the covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides. DA-specific aptamer was attached onto the electrode surface through hybridization with the ssDNA1. Methylene blue (MB) was used as an electrochemical indicator that was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to a decreased amount of adsorbed MB. This phenomenon can be applied for DA detection. The peak current of probe (MB) linearly decreased over a DA concentration range of 1–30 nM with a detection limit of 0.22 nM.