Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.     

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.     

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.     

The mycetisms and their relevancy in medicine.

Poisonous mushrooms play an important role in public health, since the poisonings caused by their toxins have very high morbidity-mortality rates. No real data are available on this problem because diagnosis is often difficult. This work points out the important role of poisonous mushrooms. An analysis is included of the clinical features of the most important mycetisms, the mushrooms that produce them and the toxins that cause this pathology. A guide to aid clinical doctors in the diagnosis and treatment of mycetisms and recomendations to prevent these poisonings are provided.     

Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.     

Ultrastructural Study of the Filum Terminate and Caudal Ampulla of the Spinal Cord of Amphioxus (Branchiostoma lanceolatum Pallas)

The filum terminale and caudal ampulla of amphioxus were studied by electron microscopy. The filum terminale consists of ependymal cells whose cilia are directed caudally. Remarkably, nerve fibres course through the filum terminale and caudal ampulla and end on the basal lamina forming neuro-connective structures. Moreover, these nerve boutons are divisible into several classes according to their vesicle content. Boutons containing large dense-cored vesicles are very similar in appearance to the neurosecretory terminals found in the caudal spinal cord of some vertebrates. These observations on nerve fibres suggest that a primitive neurosecretory system similar to the fish urophysis is present in the amphioxus.     

Preliminary studies of the 2D crystallization of Omp1 of Serratia marcescens: observation by atomic force microscopy in native membranes environment and reconstituted in proteolipid sheets

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.     

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate

DNA polymerase μ (Pol μ) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol μ protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol μ in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol μ to mutation of G and C residues during SHM. In vitro analysis of Pol μ misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol μ to induce transient template/primer misalignments.