Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution.

A method was developed for the quantification of protein-ligand interactions in which the free protein present in homogeneous reaction mixtures was separated and quantified using a KinExA immunoassay instrument. Separation was achieved by rapid percolation of the reaction mixture over a column of microbeads whose surfaces were coated with an immobilized form of the ligand. The protein thus captured was quantified using a fluorescently labeled anti-protein antibody. The features of this new method were illustrated using a model system in which each of the principal reagents was covalently labeled with a different fluorescent molecule: mouse monoclonal anti-biotin primary antibody (fluorescein), biotin (B-phycoerythrin), and goat anti-mouse polyclonal secondary antibody (indodicarbocyanin). Values for the equilibrium and kinetic rate constants for the binding between the anti-biotin antibody and biotin conjugated with B-phycoerythrin were determined and shown to be independent of whether the fluorescent label was located on the primary or secondary antibody. Equilibrium binding experiments conducted with (F(AB))(2) and corresponding F(AB) fragments showed that the valency of the binding protein had no influence on the value of the dissociation constant. The values of the equilibrium and rate constants obtained by this new method are those for the binding reaction in homogeneous solution; the immobilized ligand is only a tool exploited for the separation and quantification of the free protein.     

Haemosporidian infections in skylarks (<Emphasis Type="Italic">Alauda arvensis</Emphasis>): a comparative PCR-based and microscopy study on the parasite diversity and prevalence in southern Italy and the Netherlands

Changes in agricultural management have been identified as the most probable cause for the decline of Skylark (Alauda arvensis) populations in Europe. However, parasitic infections have not been considered as a possible factor influencing this process. Four hundred and thirty-four Skylarks from the Southern Italy and the Netherlands were screened for haemosporidian parasites (Haemosporida) using the microscopy and polymerase chain reaction (PCR)-based methods. The overall prevalence of infection was 19.5%; it was 41.8% in Italian birds and 8.3% in Dutch birds. The prevalence of Plasmodium spp. was 34.1% and 6.5% in Skylarks from Italy and Netherlands, respectively. Approximately 15% of all recorded haemosporidian infections were simultaneous infections both in Italian and Dutch populations. Six different mitochondrial cytochrome b (cyt b) lineages of Plasmodium spp. and three lineages of Haemoproteus tartakovskyi were found. The lineage SGS1 of Plasmodium relictum was the most prevalent at both study sites; it was recorded in 24.7% of birds in Italy and 5.5% in the Netherlands. The lineages SYAT05 of Plasmodium vaughani and GRW11 of P. relictum were also identified with a prevalence of <2% at both study sites. Two Plasmodium spp. lineages (SW2 and DELURB4) and three H. tartakovskyi lineages have been found only in Skylarks from Italy. Mitochondrial cyt b lineages SYAT05 are suggested for molecular identification of P. vaughani, a cosmopolitan malaria parasite of birds. This study reports the greatest overall prevalence of malaria infection in Skylarks during the last 100 years and shows that both Plasmodium and Haemoproteus spp. haemosporidian infections are expanding in Skylarks so it might contribute to a decrease of these bird populations in Europe.     

On the origin of charrs of the genus Salvelinus of the Kronotskoe Lake and their relationships with other charr populations of the Kamchatka peninsula

To reveal genetic differentiation and origin of sympatric Lake Kronotskoe charr morphs, we sequenced a fragment of control region (558-bp) and Cytochrome B gene (1015-bp) (combined length 1573-bp) in 232 specimens from the lake and, as an outgroup, 57 specimens from the Kronotskaya River below water-falls (anadromous malma) and 77 specimens from other large water bodies from the Kamchatka. A total of 52 nucleotide positions were polymorphic, and these defined 67 haplotypes. Our data does not allow to estimate genetic differentiation among different lake-dwelling morphs, but strongly supports the monophyletic origin of all morphs within the lake. To estimate genetic differentiation and population structure, an extensive study of microsatellite variation has to be conducted.     

Regulation of aldose reductase activity. Mechanism of action of an activated form of the enzyme]

Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was studied. The activated form of the enzyme was isolated, and the rate of glucose reduction measured within a broad range of substrate concentrations. Spectrophotometric titration and equilibrium gel-filtration were used to study the interaction of the enzyme active center with substrates. It was found that the reaction kinetics obeys the mechanism of a quasi-equilibrium binding of substrates with isomerization of the enzyme complexes with nicotinamide dinucleotide phosphates. This activation is accompanied by a transition from non-ordered to highly ordered binding of the substrates. The effect of ligands in the catalytic and inhibitory centers of the activated enzyme on the catalytic reaction was examined. It was found that the activated form of aldose reductase is characterized by a lower affinity of the inhibitory center for the flavonoid, morin. Morin binding not only inhibits the reaction but also prevents the activation of the enzyme.     

100 kGy gamma-affected microbial communities within the ancient Arctic permafrost under simulated Martian conditions

Extremophiles - This research aimed to investigate the viability and biodiversity of microbial communities within ancient Arctic permafrost after exposure to a gamma-radiation dose of...     

Hormonal status in different phenotypic forms of Black Sea trout <Emphasis Type="Italic">Salmo trutta labrax</Emphasis>

In three phenotypic forms (parrs, trout form, and kumzha form) of fish-farm Black Sea trout (age 1+) and wild Black Sea trout (age 1+ and 2+), Salmo trutta labrax, inhabiting the basin of the Mzymta River, the content of monoamines (dopamine, noradrenalin, and serotonin) and of their metabolites (3,4-dihydroxyindolacetic acid, 3-methoxy-4-hydroxyphenyl glycol, and 5-hydroxyindolacetic acid) was investigated in brain regions (forebrain, optic tectum, brainstem, hypothalamus, and hypophysis) and the level of cortisol, thyroxin, triiodothyronine, growth hormone, and adrenocorticotropic hormone was investigated in blood. Data on length and weight of fish body and maturation stage of gonads are indicated. The hormonal level differs in the fish of different phenotypic forms. It is the highest in specimens of the kumzha form which completed parr-smolt transformation. Among wild fish, numerous mature females at age 1+ were found which indicated the presence of a dwarf form of the Black Sea trout in the Caucasus in the Mzymta River.     

A method for the assessment of sperm quality in fish

A method of computer assisted sperm analysis in fish is described. This method is used for assessing the main parameters of sperm motility by means of a microscope and video camera connected to a computer with easily available software. The results obtained during the analysis noticeably depend on the maximum velocity of spermatozoa (the maximum distance covered by a spermatozoon between two subsequent frames of the videotape, which is set in the control window of the plugin “MTrack2” included into the image analyzing software “ImageJ”). The method is illustrated with reference to the analysis of sperm activity in Zebrasoma scopas (Acanthuridae), a representative of the ichthyofauna of coral reefs.     

Diet and dietary selectivity of Cane Toads (Rhinella marina) in restoration sites: a case study in Far North Queensland,Australia

Cane Toads (Rhinella marina, formerly Bufo marinus) in restoration sites on the Atherton Tableland in NE Australia consumed invertebrates belonging to 11 different taxa with ants being the most abundant prey item. Principal component analyses showed that the composition of invertebrates in Cane Toad diet is largely a reflection of invertebrates found in pitfall and leaf litter samples suggesting that the species is an indiscriminant feeder. However, pitfall samples contained more Collembola and Isopoda than were found in Cane Toad stomachs. The Cane Toad may benefit from restoration management practices by utilizing food resources enhanced by mulching and providing microhabitats (e.g. rock piles, logs) as shelter. While further studies would be needed to test this practitioners working in areas where the Cane Toad is problematic may consider trade‐offs between attracting invertebrates and Cane Toads by monitoring provided microhabitat features.