The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup

Abstract The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.     

Data pattern analysis for the individualised pretherapeutic identification of high-risk diffuse large B-cell lymphoma (DLBCL) patients by cytomics.

BACKGROUND: Clinical outcome predictions in phase III studies are mostly derived for patient groups, but not for individual patients, although individualised predictions are an ultimate goal to permit a personalised fine tuning of therapy. This may permit earlier application of target therapies, minimise general damage to the organism, and result in improved complete remission rates in malignant diseases. METHODS: In this study, Lymphochip cDNA microarray gene expression results of DLBCL patients, from a published prospective meta-analysis study on the prediction of group prognosis, were analysed for individualised predictions using a nonstatistical data pattern classification approach. The training set was comprised of the same 160 DLBCL patients as in the prognosis study, with the validation set of 80 patients remaining unknown to the learning process. This permits the assessment of prospective classifier performance towards unknown patients. RESULTS: Pretherapeutic predictions for the training and validation set patients were correct in 98.1% and 78.3% of the cases for nonsurvival and in 67.3% and 45.3% for survival. The discriminatory data pattern consisted of 14 known and 10 unknown gene products. CONCLUSIONS: The better than 95% correct pretherapeutic prediction for about one-half of the ultimately nonsurviving high-risk patients of the training set is promising for clinical considerations about individualised therapy in such cases. Reliable individualised survival predictions are not possible with the information content of the present dataset. It seems necessary to investigate additional gene products, since survival may significantly depend on non-lymphocyte-associated genes that escape to the lymphocyte-oriented Lymphochip gene activation analysis.     

NADH oxidation and NAD+ reduction catalysed by tightly coupled inside-out vesicles from Paracoccus denitrificans.

Tightly coupled inside-out vesicles were prepared from Paracoccus denitrificans cells (SPP, sub-Paracoccus particles) and characterized kinetically. The rate of NADH oxidation, catalysed by SPP, increases 6-8 times on addition of gramicidin. The vesicles are capable of catalysing Delta micro H+-dependent reverse electron transfer from quinol to NAD+. The kinetic parameters of the NADH-oxidase and the reverse electron transfer carried out by membrane-bound P. denitrificans complex I were estimated and compared with those of the mitochondrial enzyme. The data demonstrate that catalytic properties of the dinucleotide-binding site of the bacterial and mitochondrial complex I are almost identical, pointing out similar organization of the site in mammals and P. denitrificans. Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme. The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nanomolar concentrations of rotenone. In contrast to the mitochondrial enzyme, rotenone, even at concentrations as high as 10 micro m, does not inhibit the reverse, Delta micro H+-dependent NAD+-reductase reaction on SPP.     

Carbohydrate moieties of rat MHC class I antigens

The rat major histocompatibility complex class I antigens RT1.A^u and RT1.E^u from the u haplotype and RT1.Aⁿ from the n haplotype were labeled with ¹⁴C-asparagine or with ³H-fucose, mannose, galactose, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed complete removal of radioactivity from the sugar-labeled antigen heavy chains by digestion with glycopeptidase F, an enzyme that removes N-linked glycans completely. High performance liquid chromatography analysis of the tryptic digests of the mixed sugar-labeled and asparagine-labeled antigens demonstrated that all the sugar-labeled peptides were coincident with asparagine-labeled peptides. The Aⁿ antigen showed three glycopeptides, each of which had different amounts of sugar radioactivity. The antigens A^u and E^u showed two glycopeptides with different amounts of radioactivity but at identical positions in the two antigens. Antigen E^u had an additional glycopeptide with a lower amount of radioactivity. The positions of the glycopeptides from the A^u and E^u antigens were different from those of the Aⁿ antigen. The peptide profiles of the ¹⁴C-asparagine-labeled A^u and E^u antigens demonstrated distinct differences between the molecules. The results of this study show that: (a) all the glycans on rat class I antigens are N-linked, as they are on H-2 and HLA class I antigens; (b) there are compositional differences among the glycans in each of the three antigens; (c) the glycosylation pattern of the rat class I antigens is similar to that of the mouse class I antigens, which contain two or three glycans, in contrast to that of the human class I antigens, which contain only one glycan; and (d) the antigens A^u and E^u from the same haplotype are more closely related to each other than they are to the Aⁿ antigen.