Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.     

Analysis of vertical ozone and nitrogen oxides profiles in aPrunus cerasifera canopy

An automatic system was installed for continuous analyses of ozone, sulphur dioxide, nitrogen monoxide and nitrogen dioxide in an experimental orchard with a canopy ofPrunus cerasifera plants in summer 1993. Air samples from three elevations (0.8 m, 1.6 m and 3 m above ground) were sequentially analyzed. Ozone concentrations above the canopy were usually higher than within the canopy; their relationships with stomatal resistance have been investigated. Sulphur dioxide levels were negligible. Nitrogen oxides showed a complex profile, with no particular trend, likely due to a reciprocal exchange between the atmosphere and the ground surface.     

Enzyme-enzyme interactions and control analysis. 2. The case of non-independence: heterologous associations

The association of different enzymes into a complex may induce changes in the kinetic parameters of its component enzymes. This implies that they cannot be treated as independent catalysts. It will affect the formulations and theorems of control analysis and necessitates the introduction of additional elasticities reflecting the effect of one enzyme on the rate of another. We show how this is achieved as an extension of the classical treatment. We present modified summation and connectivity theorems incorporating both homologous and heterologous interactions. The case of channelling of metabolites in such complexes is considered and an experimental method for its detection is suggested.     

Chemopreventive and Antioxidant Activity of the Chamazulene‐Rich Essential Oil Obtained from Artemisia arborescens L. Growing on the Isle of La Maddalena,Sardinia, Italy

The essential oils of Artemisia arborescens growing in Sardinia (Italy), collected during three plant growth stages, i.e., from the vegetative stage to post‐blooming time, were characterized. Moreover, the in vitro antiproliferative and antioxidant activities of the oil isolated from aerial parts collected in February were evaluated. The essential oils belonged to the β‐thujone/chamazulene chemotype, notably with the highest amount of chamazulene (ca. 52%) ever detected up to now in the genus Artemisia and, in general, in essential oils. Quantitative variations in the oil composition were observed as the plant passes from the vegetative to the blooming stage. The oil was tested for its potential tumor cell growth‐inhibitory effect on T98G, MDA‐MB 435S, A375, and HCT116 human cell lines, using the MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) assay. The highest activity was observed on A375 and HCT116 cell lines, with IC₅₀ values of 14 μg/ml. Moreover, the in vitro antioxidant and free radical‐scavenging assays revealed the oil to be an effective scavenger of the ABTS radical cation, with an activity comparable to that of Trolox^®. These results support the use of A. arborescens oil for the treatment of inflamed skin conditions. Finally, the composition of the polar fraction of the A. arborescens aerial parts was also examined, and the main component detected was 5‐O‐caffeoylquinic acid, which was identified for the first time in this plant.     

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.     

Conservation analysis in biochemical networks: computational issues for software writers

Large scale genomic studies are generating significant amounts of data on the structure of cellular networks. This is in contrast to kinetic data, which is frequently absent, unreliable or fragmentary. There is, therefore, a desire by many in the community to investigate the potential rewards of analyzing the more readily available topological data. This brief review is concerned with a particular property of biological networks, namely structural conservations (e.g. moiety conserved cycles). There has been much discussion in the literature on these cycles but a review on the computational issues related to conserved cycles has been missing. This review is concerned with the detection and characterization of conservation relations in arbitrary networks and related issues, which impinge on simulation simulation software writers. This review will not address flux balance constraints or small-world type analyses in any significant detail.     

In vitro effect of indomethacin and interferon-alpha on Th1 and Th2 cytokine synthesis in patients with chronic hepatitis C

Current evidences suggest that non-steroidal anti-inflammatory drugs could enhance the antiviral activity of interferon-alpha in chronic HCV infection. In this study, we investigated the effect of indomethacin, a non-steroidal anti-inflammatory drug, and interferon-alpha on cytokine production by peripheral blood mononuclear cells from 12 untreated patients with chronic hepatitis C. We evaluated the effect of incubation with indomethacin, interferon-alpha or both on synthesis of Th1- (interleukin-2, interferon-gamma) and Th2-associated cytokines (interleukin-4, interleukin-10), and of the antiviral protein 2',5'-oligoadenylate synthetase. Interferon-alpha induced a significant increase in production of interleukin-2. Smaller increases were also seen in the presence of indomethacin, while incubation with both indomethacin and interferon-alpha leads to a synergistic effect. Incubation with indomethacin decreased both interleukin-4 and interleukin-10, whereas interferon-alpha increased these cytokines. The addition of indomethacin to interferon-alpha significantly reversed this interferon-induced increase. Finally, both indomethacin and the association interferon-alpha plus indomethacin determined a significant increase in 2',5'-oligoadenylate synthetase production compared to both baseline and interferon-alpha alone. In conclusion, indomethacin was able to enhance the antiviral activity of interferon-alpha and to modulate the interferon-induced Th1 and Th2 cytokine response by increasing the Th1-response, fundamental for sustained clearance of HCV, and by decreasing the Th-2 type response, associated with HCV persistence.