Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.     

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.     

Chromium determinations in blood cells: clinical relevance demonstrated in patients with diabetes mellitus type 2

As an essential nutrient involved in carbohydrate and lipid metabolism, chromium is of extraordinary importance for patients with diabetes. Plasma concentrations do not reflect the chromium supply; thus, we determined the element’s content in blood cells in order to evaluate the body status. We investigated 86 blood donors (C) and 35 diabetics type 2 (Dm2). After the isolation of the blood cells by using a density centrifugation, the chromium concentrations were determined by electrothermal atomic absorption spectrometry. Compared to C, Dm2 had higher values in plasma, erythrocytes, and platelets (248%, 61%, and 91%, respectively) and lower contents in polymorphonuclear and mononuclear leukocytes (each −35%, age- and sex-matched groups with n=35, each p<0.01). The poorer the metabolic control assessed by HbA1c, the higher were the chromium concentrations in plasma (r=+0.46, n=33, p=0.007, increase 11.1% per %HbA1c) and the lower were the values in mononuclear leukocytes (r=−0.45, n=33, p=0.008, decrease 17.8% per %HbA1c). The changed amounts in plasma and in mononuclear cells in increasing hyperglycemia could be the result of an intracellular/extracellular redistribution of the element. High plasma levels might explain the renal chromium losses of diabetics, whereas the lymphocytes could reflect a decreasing chromium body state.     

Towards Antiviral shRNAs Based on the AgoshRNA Design

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these “AgoshRNA” molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.     

Abscisic add levels of individual leaf cells

Abscisic acid (ABA) concentrations of guard cells, subsidiary cells and epidermis cells of Comrnelina communis L. and Tradescantia virginiana L. were determined in cell sap samples extracted by means of micro glass capillaries. Concentrations up to 6.7 m M were indicated by commercial immunoassay test kits. A gradient of ABA concentration was found between guard cells (2,49 ± 1.81 m M , n = 25), subsidiary cells (1.25 ± 1.46 m M , n = 21) and epidermis cells (0.86 ± 0.76 m M , n = 20; mean values ± SD).     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.