Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

A recurring theme in protein engineering: the design, stability and folding of repeat proteins

Repeat proteins are ubiquitous and are involved in a myriad of essential processes. They are typically non-globular structures that act as diverse scaffolds for the mediation of protein-protein interactions. These excitingly different structures, which arise from tandem arrays of a repeated structural motif, have generated significant interest with respect to protein engineering and design. Recent advances have been made in the design and characterisation of repeat proteins. The highlights include re-engineering of binding specificity, quantitative models of repeat protein stability and kinetic studies of repeat protein folding.     

Determinants of diastolic myocardial tissue Doppler velocities: influences of relaxation and preload

Myocardial tissueDoppler echocardiography (TDE) has been proposed as a tool for theassessment of diastolic function. Controversy exists regarding whetherTDE measurements are influenced by preload. In this study, leftventricular volume and high-fidelity pressures were obtained ineight closed-chest dogs during intermittent caval occlusion. The timeconstant of isovolumic ventricular relaxation () was alteredwith varying doses of dobutamine and esmolol. Peak early diastolicmyocardial (E_m) and transmitral (E)velocities were measured before and after preload reduction. Therelative effects of changes in preload and relaxation were determinedfor E_m and compared with their effects onE. The following results were observed: caval occlusionsignificantly decreased E (E = 16.4 ± 3.3 cm/s, 36.6 ± 13.7%, P < 0.01) andE_m (E_m = 1.3 ± 0.4 cm/s, 32.5 ± 26.1%, P < 0.01) underbaseline conditions. However, preload reduction was similar forE under all lusitropic conditions (P = notsignificant), but these effects on E_m decreasedwith worsening relaxation. At  < 50 ms, changes inE_m with preload reduction were significantlygreater (E_m = 2.8 ± 0.6 cm/s) than at  = 50-65 ms (E_m = 1.2 ± 0.2 cm/s) and at  >65 ms(E_m = 0.5 ± 0.1 cm/s,P < 0.05). We concluded that TDEE_m is preload dependent. However, this effectdecreases with worsening relaxation.

     

Cytotaxonomic studies in the Adiantum caudatum complex of Africa and Asia

Study of meiosis in synthesized hybrids has permitted a genome analysis to be carried out with respect to the holotype of A. indicum Ghatak, 4x, originally found wild near Calcutta. The two differentiated diploids from which A. indicum , 4x, seems to have arisen are represented in the analysis by A. zollingeri from Ceylon and by two strains of an Indian diploid provisionally designated A. incisum sens. lat. 2x. Supplementary information excluding an autopolyploid origin for A. indicum is provided by hybrids between A. indicum 4x and an unrelated tetraploid, A. malesianum Ghatak. Ecological information is supplied for the material of A. zollingeri , indicating that it occupies a specialized habitat which might be found in India if search is made in the right kind of territory. A. incisum 2x is shown to be much more widespread and to be present in hill country, or occasionally in lowlands, from the Himalayas to South India inclusive. It contains a number of genetically determined biotypes differing slightly in morphology but behaving as members of one species when combined into hybrids. Further morphological information and a revised key for separating the specimens used in the investigation is provided in an Appendix.     

Epizootic coronaviral typhlocolitis in suckling mice

Multiple epizootics of typhlocolitis associated with high morbidity and mortality occurred among suckling mice in an arbovirology research laboratory. Affected mice had necrosis and hyperplasia of cecal, colonic, and less often, small intestinal mucosa. Epithelial syncytia were present throughout the affected areas. Other organs generally were not involved. The etiologic agent was a coronavirus antigenically related to mouse hepatitis virus strains 1 and S. Sera from dams of affected litters and recovered animals did not contain detectable complement fixing antibody to coronavirus antigen. The lesions and the causative agent differed from previously reported coronaviral syndromes in mice. The source of the infection was not definitely found.     

Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease

Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids’ MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35 % identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.

Structural characteristics and aboveground biomass of old‐growth spruce–fir stands in the eastern Carpathian mountains,Ukraine

Abstract

Temperate old‐growth forests are known to have ecological characteristics distinct from younger forests, but these have been poorly described for the remaining old‐growth Picea abies–Abies alba forests in the eastern Carpathian mountains. In addition, recent studies suggest that old‐growth forests may be more significant carbon sinks than previously recognized. This has stimulated interest in quantifying aboveground carbon stocks in primary forest systems. We investigated the structural attributes and aboveground biomass in two remnant old‐growth spruce–fir stands and compared these against a primary (never logged) mature reference stand. Our sites were located in the Gorgany Nature Reserve in western Ukraine. Overstory data were collected using variable radius plots; coarse woody debris was sampled along line intercept transects. Differences among sites were assessed using non‐parametric statistical analyses. Goodness‐of‐fit tests were used to evaluate the form of diameter distributions. The results strongly supported the hypothesis that old‐growth temperate spruce–fir forests have greater structural complexity compared to mature forests, including higher densities of large trees, more complex horizontal structure, and elevated aboveground biomass. The late‐successional sites we sampled exhibited rotated sigmoid diameter distributions; these may reflect natural disturbance dynamics. Old‐growth Carpathian spruce–fir forests store on average approximately 155–165 Mg ha^?1 of carbon in aboveground tree parts alone. This is approximately 50% higher than mature stands. Given the scarcity of primary spruce–fir forests in the Carpathian region, remaining stands have high conservation value, both as habitat for late‐successional species and as carbon storage reservoirs.