In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.     

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Atomic force microscopy measurement of leukocyte-endothelial interaction

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40-100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-alpha-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against beta1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-alphaVbeta3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50 approximately 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-beta1 and anti-alphaVbeta3 antibodies. In conclusion, these data demonstrate the role played by beta1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by alphaVbeta3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.     

Taxonomic affinities of the Eppelsheim femur

The taxonomic affinities of the Eppelsheim femur, known as Paidopithex, have been unclear for more than a century. Over the years, due to similarities with Pliopithecus, some authors have considered it a large pliopithecid, while others refer to it as Dryopithecus. The issue could not be resolved, because no definitive Dryopithecus femora were available. With the discovery of the Dryopithecus laietanus skeleton from Can Llobateres (CLl 18800), it has become possible to test the attribution of the Eppelsheim femur to Dryopithecus on the basis of direct morphological and metrical comparisons. By means of allometric techniques, we show that the Eppelsheim and D. laietanus femora fit different hindlimb morphologies with regard to relative length and relative head/neck size, with Paidopithex significantly differing from Dryopithecus, but more closely resembling Pliopithecus. Paidopithex also differs from Dryopithecus in other important aspects, such as its lower neck/shaft angle, lack of elevation of the femoral head above the greater trochanter, more posteriorly oriented lesser trochanter, and proximal shaft diameter thicker anteroposteriorly than mediolaterally. In these features, Paidopithex most closely resembles Pliopithecus in spite of differences in body mass (ca. 22 kg vs. ca. 10 kg, respectively). These features suggest that Paidopithex used a primitive locomotor pattern associated with arboreal quadrupedalism, instead of the more derived pattern displayed by Dryopithecus. Currently available evidence confirms that the attribution of Paidopithex to Dryopithecus can be rejected. Paidopithex could be a large and otherwise unknown pliopithecid, but the possibility cannot be ruled out that it represents a third kind of catarrhine.