Isolation and Purification of Enzymes from Ligninolytic Complex of the Basidial Fungus <Emphasis Type="Italic">Trametes pubescens</Emphasis> (Schumach.) Pilat and Study of Their Properties

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.     

Growth and fecundity of Daphnia after diapause and their impact on the development of a population

Laboratory and field investigations revealed that the life history traits of exephippial and parthenogenetic generations of Daphnia differ substantially. Daphniids hatching from resting eggs grow faster and their definitive body sizes are bigger than of hatchlings from subitaneous eggs. Size at maturity for exephippial animals is significantly larger. In spite of this, they mature a few days earlier than parthenogenetic females. In this study, the difference was 3–4 days for the laboratory experiments and 1–3 days for the field. Fecundity of the exephippial generation is markedly higher. Here, the clutch size for this generation was up to 3.5–4.0 times as large as for the parthenogenetic generation. Moreover, obtained results suggest that the relationship between clutch size and body length for both generations differ significantly.Estimates of the intrinsic rate of increase for field Daphnia populations demonstrated that life history traits of exephippial animals lead to a two or threefold higher rate of increase in the conditions of invertebrate predation pressure. Under moderate fish pressure, obtained r values for the daphniids hatching from resting eggs were larger than those from subitaneous. High growth rate of exephippial females is disadvantageous only under the conditions of severe pressure by fish. Obtained results suggest that hatchlings from diapausing eggs an acceleration of population increase by several times during the beginning of the development of a population with periodical re-establishment from resting eggs.     

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in sam- ples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by “sandwich”-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.     

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptide cyclo[-d-Val-Arg-Gly-Asp-Glu(-Ahx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugate BSA–peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated that covalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Blood parasites in owls with conservation implications for the Spotted Owl (Strix occidentalis)

The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls.     

Prioritizing Biosecurity Risks Using a Participatory Decision-Making Tool

A decision framework called Deliberative Multi-Criteria Evaluation (DMCE) was developed and deployed to prioritize biosecurity risks using a variety of subjective and objective information. To aid stakeholders in the prioritization of Emergency Plant Pest (EPP) species risk, we presented them with outputs from a Stella-based bio-economic pest risk model, and probable ecological and socioeconomic impacts. The stakeholder participants weighted the consequence criteria they deemed to have the highest expected impact. The methodology featured an uncertain set of parameters, multiple iterations of criteria weighting along with real-time sensitivity analysis. Of the five criteria, economic cost was weighted the highest at 26% while landscape amenity was weighted the lowest at the 10–12% range. The increased understanding and support gained by stakeholders through the DMCE process provided a greater likelihood of the sanctioning of the policies concerned and progress toward desired outcomes.