Laccase assay by means of high-performance liquid chromatography

Spectrophotometric determination of laccase activity may be affected by the formation of quinoid chromophores arising from nonenzymatic oxidations interfering with enzymatic reactions. Km values for guaiacol obtained by spectrophotometric and HPLC methods confirm the above hypothesis. HPLC results are particularly useful for the assay of laccase activity on natural phenolic extracts.     

Substrate specificity of laccase from Lentinus edodes

In previous studies, the white-rot basidiomycete Lentinus edodes, strain SC-495, was proved to be a “selective” lignin degrader and its extracellular crude preparations arising from solid-state cultures were successfully employed in biopulping experiments on annual plants. This fungus produced extracellular laccase as the predominant phenoloxidase when growing in solid-state fermentation on corn stalks. Laccase from this strain was purified and partially characterized, as an initial approach towards the study of its ligninolytic complex. Laccase was purified 69.6-fold by anion-exchange chromatography and two affinity-chromatography steps with an overall yield of 7.45%. The native enzyme exhibited a molecular mass of 74 kDa, an isoelectric point of 3.42 and a carbohydrate content of 7.5%. The absorption spectrum of laccase showed a maximum at 605 nm, typical of blue-copper oxidases. The optimum pH and temperature for the activity of laccase were 4.0–4.2 and 50°C, respectively. Kinetic experiments, performed with a wide range of phenolic compounds, showed that the reaction rate and the substrate affinity greatly varied depending on the nature of substituents and their reciprocal positions on the aromatic ring. In particular, the enzyme showed high affinity to phenolic compounds bearing methoxyl or methyl groups, but no affinity to those bearing the nitro group directly attached to the benzene ring, nor to non-phenolic lignin-related compounds, such as trans-cinnamic acid or 3,4-dimethoxycinnamic acid. The huge differences in terms of reactivity of the enzyme towards phenolic compounds suggests that a preliminary systematic screening should be advisable when using laccase in effluent treatment applications.     

Glycoproteins gB, gD, and gHgL of Herpes Simplex Virus Type 1 Are Necessary and Sufficient To Mediate Membrane Fusion in a Cos Cell Transfection System

Herpes simplex virus type 1 glycoproteins gB, gD, and gHgL were expressed by transient transfection of Cos cells. Polykaryocyte formation above the background level seen in untransfected controls was observed only if all three components were expressed. Thus, gB, gD, and gHgL are necessary and sufficient to induce membrane fusion.     

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.     

Chemical characterization of new oxylipins from Cestrum parqui, and their effects on seed germination and early seedling growth

Isolation, chemical characterization, and phytotoxicity of five new oxylipins, together with seven already known related compounds, from Cestrum parqui L' Hérl. is reported. All the structures were elucidated on the basis of their spectral data, especially 1D-(1H- and 13C-NMR, DEPT) and 2D-NMR (COSY, TOCSY, HSQC, HMBC, and NOESY). The configurations of the stereogenic C-atoms were determined by the Mosher's method. The compounds have been assayed for their phytotoxicity on Lactuca sativa at concentrations ranging between 10(-4) and 10(-8) M. The results of the phytotoxicity tests on the germination and growth of the test species, obtained by a cluster analysis, showed interesting relationship between the chemical structures of the compounds and their biological effects.     

Broussonetia papyrifera controls nutrient return to soil to facilitate its invasion in a tropical forest of Ghana

Aims Non-native invasive plants can alter soil chemistry through litter production and decomposition to facilitate their invasion. However, the important roles of these underlying processes in plant invasion remain poorly understood, particularly in tropical forest ecosystems. Here, we compared litter production, quality and decomposition of two invasive species (Broussonetia papyriferaandCedrela odorata) and two co-occurring native species (CeltismildbraediiandFuntumia elastica), and soil properties under them to elucidate their roles in the invasion of a tropical forest in Ghana.     

Cost of walking and locomotor impairment.

The aim of the present study was to evaluate the importance and the necessity of metabolic measurements to quantify locomotor impairment in a clinical context. Oxygen consumption, heart rate, pulmonary ventilation and walking speed were measured during locomotion in 14 normal subjects, used as a control group, and 82 patients with different pathologies [hemiparetic, paraparetic, tetraparetic, orthopaedic and paraplegic patients, who walked using a reciprocating gait orthosis (RGO)]. The subjects were characterized on the basis of a cumulative impairment score (CIS), based on clinical scales commonly used to evaluate impairment and disability in locomotion. Appropriate indices of energy, cardiac and ventilatory costs expressed per metre walked, globally called physiological costs, were obtained. It resulted that the most comfortable speed (MCS) of normal subjects was significantly higher than that of each group of patients. Normal subjects' physiological costs were found to be significantly lower than those of patients who needed either a device or the help of a person to walk. All measured parameters correlated significantly with each other. The MCS was found to be the most correlated parameter with the CIS (r = 0.8), and therefore it must be considered the best single measurement, if only one is to be used. Measurements more precise than MCS, such as the physiological costs, may be necessary in clinical trials.     

Carbon mineralization in the southern Sonoran Desert

We measured carbon mineralization in four different desert habitats (Arroyos, Hillsides, Canopies-Plains and Open-Plains) and the separate effect of litter addition from annual and perennial plants on soil microbial respiration using two laboratory soil incubation experiments. The differences in total aboveground phytomass among habitats correlates with soil nutrient content, soil particulate organic matter (POM) and consequently, C mineralization. The Arroyos habitat with the highest perennial plant phytomass and litter production, had the highest soil nutrient content, soil POM and C mineralization. Litter from annual plants had twice the P concentration than litter from the perennials, but only half the N concentration. Soil microbial respiration was higher with annual plant litter than with perennial plant litter in the Hillsides and Canopies-Plains, suggesting that microbial activity in both habitats was improved by litter with a higher C quality. In contrast, in the poorest habitat, the Open-Plains, the better response to the addition of perennial plant litter suggests that microbial activity may have been constrained by N input.     

IFN-alpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-alpha/beta receptor (IFN-alpha/betaR) or IFN-beta died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-alpha/betaR-/- mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-alpha, IFN-gamma, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-alpha/betaR-/- mice, although this change was less extensive than that observed in similarly infected IFN-gammaR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-beta levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.