No abstract

At least three structural proteins in Paramecium bursaria Chlorella virus (PBCV-1) are glycosylated, including the major capsid protein Vp54. However, unlike other glycoprotein-containing viruses that use host-encoded enzymes in the endoplasmic reticulum-Golgi to glycosylate their proteins, PBCV-1 encodes at least many, if not all, of the glycosyltransferases used to glycosylate its structural proteins. As described here, PBCV-1 also encodes two open reading frames that resemble bacterial and mammalian enzymes involved in de novo GDP-L-fucose biosynthesis. This pathway, starting from GDP-D-mannose, consists of two sequential steps catalyzed by GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/ reductase, respectively. The two PBCV-1-encoded genes were expressed in Escherichia coli, and the recombinant proteins had the predicted enzyme activity. However, in addition to the dehydratase activity, PBCV-1 GMD also had a reductase activity, producing GDP-D-rhamnose. In vivo studies established that PBCV-1 GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are expressed after virus infection and that both GDP-L-fucose and GDP-D-rhamnose are produced in virus-infected cells. Thus, PBCV-1 is the first virus known to encode enzymes involved in nucleotide sugar metabolism. Because fucose and rhamnose are components of the glycans attached to Vp54, the pathway could circumvent a limited supply of GDP sugars by the algal host. Paramecium bursaria Chlorella virus (PBCV-1)1 is the prototypical member of a group of large polyhedral, plaque-forming viruses in the family Phycodnaviridae that replicate in certain unicellular, eukaryotic Chlorella-like green algae (1). The PBCV-1 genome, which has been sequenced, consists of a large (Ͼ330 kb) linear, non-permuted double-stranded DNA with covalently closed hairpin ends (2). Additional features of the virus have been reviewed recently (3). The PBCV-1 major capsid protein, Vp54, located on the viral surface, is one of three glycosylated viral proteins. The glycan portion of Vp54 contains seven neutral sugars, glucose, fucose, rhamnose, galactose, mannose, xylose, and arabinose (4) that contribute to the protease resistance and antigenicity of the virus. Recently, the Vp54 crystal structure was solved, and four N-linked glycosylation sites and two O-linked oligosaccharides were identified (5).

GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-D-mannose to GDP-L-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion protein has a K m of 0.22 ± 0.04 mM and a specific activity of 2.3 ±0.2 /ffliol/h/mg. Ca 2+ and Mg 2+activate GMD, while GDP-L-j3-fucose, the end-product of the pathway, inhibits it specifically. The GST-GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.

No abstract

Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialylLewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP-L-fucose biosynthesis from GDP-D-mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP-D-mannose-4,6-dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD-regulating protein.z 1998 Federation of European Biochemical Societies.

GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the`de novo' pathway of GDP-L-fucose biosynthesis. The reported cDNA sequences for human GMD predict three forms of different length, whose`in vivo' occurrence and molecular properties are completely undefined. Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form. Only the 42 kDa long GMD (L-GMD) and the 40.2 kDa (M-GMD) forms were recovered as soluble functional proteins, while the 38.7 kDa form, short GMD (S-GMD), lacking an N-terminal domain critical for dinucleotide binding, was inactive and formed a precipitate. Both L-GMD and M-GMD are homodimers and contain 1 mol of tightly bound NADP + . Their kinetic properties (K m , K cat ) are apparently identical and both forms are non-competitively feedbackinhibited by GDP-L-fucose to a similar extent. M-GMD is the predominant enzyme form expressed in several human cell lines. These data seem to suggest that modulation of the`de novo' pathway of GDP-L-fucose biosynthesis involves mechanisms other than differential`in vivo' expression of GMD forms.z 1999 Federation of European Biochemical Societies.

PurposeOur paper shows an empirical analysis of the European football companies to test the association between sport results, proxied by ranking position and financial performance in panel framework (starting from 59 firms over the 2013–2018 time span).Design/methodology/approachWe use panel data models for studying the relationship of our interest and we make no a priori assumption about the strict exogeneity of the covariates and estimate equation using both Random Effects GLS (RE-GLS) and Fixed Effects OLS (FE-OLS) estimations.FindingsOur results suggest there is stable and significant relationship between the two types of performance and that when detectable this is linked in a positive way to the profit maximization of the business model, suggesting that it is more useful for investor remuneration and to increase technical-tactical resources and therefore sports results. Not surprisingly, as for many clubs, concentration effect is relevant while the financial fair play regulation is not. In fact, the current regulation of UEFA authority does not seem to have an impact on sport and financial results.Originality/valueThis work complements literature in several ways. First, we offer new empirical evidence for the association between the sport and financial performance for a panel of the European football companies, listed and not. Second, we show that the persistence of the sport results is strongly correlated with financial performance.

Interest in the virtualization of human–robot interactions is increasing, yet the impact that collaborating with either virtual or physical robots has on the human operator’s mental state is still insufficiently studied. In the present work, we aimed to fill this gap by conducting a systematic assessment of a human–robot collaborative framework from a user-centric perspective. Mental workload was measured in participants working in synergistic co-operation with a physical and a virtual collaborative robot (cobot) under different levels of task demands. Performance and implicit and explicit workload were assessed as a function of pupil size variation and self-reporting questionnaires. In the face of a similar self-reported mental demand when maneuvering the virtual or physical cobot, operators showed shorter operation times and lower implicit workload when interacting with the virtual cobot compared to its physical counterpart. Furthermore, the benefits of collaborating with a virtual cobot most vividly manifested when the user had to position the robotic arm with higher precision. These results shed light on the feasibility and importance of relying on multidimensional assessments in real-life work settings, including implicit workload predictors such as pupillometric measures. From a broader perspective, our findings suggest that virtual simulations have the potential to bring significant advantages for both the user's mental well-being and industrial production, particularly for highly complex and demanding tasks.