Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens

Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node, for detection of HIV-specific B-cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection strategies for improved biocatalysts

Enzymes have become an attractive alternative to conventional catalysts in numerous industrial processes. However, their properties do not always meet the criteria of the application of interest. Directed evolution is a powerful tool for adopting the characteristics of an enzyme. However, selection of the evolved variants is a critical step, and therefore new strategies to enable selection of the desired enzymatic activity have been developed. This review focuses on these novel strategies for selecting enzymes from large libraries, in particular those that are used in the synthesis of pharmaceutical intermediates and pharmaceuticals.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Does the nutrient stoichiometry of primary producers affect the secondary consumer Pleurobrachia pileus?

We investigated whether phosphorus limitations of primary producers propagate upwards through the food web, not only to the primary consumer level but also onto the secondary consumers’ level. A tri-trophic food chain was used to assess the effects of phosphorus-limited phytoplankton (the cryptophyte Rhodomonas salina) on herbivorous zooplankters (the copepod Acartia tonsa) and finally on zooplanktivores (the ctenophore Pleurobrachia pileus). The algae were cultured in phosphorus-replete and phosphorus-limited media before being fed to two groups of copepods. The copepods in turn were fed to the top predator, P. pileus, in a mixture resulting in a phosphorus-gradient, ranging from copepods having received only phosphorus-replete algae to copepods reared solely on phosphorus-limited algae. The C:P ratio of the algae varied significantly between the two treatments, resulting in higher C:P ratios for those copepods feeding on phosphorus-limited algae, albeit with a significance of 0.07. The differences in the feeding environment of the copepods could be followed to Pleurobrachia pileus. Contrary to our expectations, we found that phosphorus-limited copepods represented a higher quality food source for P. pileus, as shown by the better condition (expressed as nucleic acid content) of the ctenophore. This could possibly be explained by the rather high C:P ratios of ctenophores, their resulting low phosphorus demand and relative insensitivity to P deficiency. This might potentially be an additional explanation for the observed increasing abundances of gelatinous zooplankton in our increasingly phosphorus-limited coastal seas.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Psychological consequences of myocardial infarction: a self-regulation perspective on health-related quality of life and cardiac rehabilitation

The aim of this review was to gain insight into prevalence of and interventions targeted specifically at psychological distress and health-related quality of life (HRQL) after a myocardial infarction (MI). For this purpose, self-regulation theory was introduced as frame of reference. Psychological distress and a reduction in HRQL after an MI are prevalent and can, for some patients, be persistent. This can negatively influence secondary prevention efforts, adherence, return to work and progression of the underlying coronary heart disease. At the same time, the effectiveness of cardiac rehabilitation programmes in improving HRQL is inconclusive. By starting off from a theoretical framework, effective strategies can be either identified or developed. Self-regulation theory is concerned with the process of goal setting and goal attainment and offers a model for explaining well-being and quality of life. The usefulness of this theory is supported by empirical evidence. Psychological factors derived from this theoretical framework (e.g. higher order goal disturbance) are associated with psychological distress and HRQL in the short and medium term after an MI and should thus be the target of cardiac rehabilitation programmes.     

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

Predator-mediated plasticity in morphology, life history, and behavior of Daphnia: the uncoupling of responses

We studied the way 12 traits responded to fish kairomones in a set of 16 Daphnia magna clones derived from four different habitats-two where daphnids co-occur with fish and two without fish. These clones differed widely in their response to predator kairomones, with none of the clones showing a significant response in all traits and all clones showing a response for at least one trait. Most of the clones showed a significant response in one to four traits, with no evidence for an association between different traits. Clones from fish habitats were slightly more responsive to the presence of fish kairomones than clones from fishless locations. We conclude that most clones show an induced response to the presence of their predators (fish) but that there is a large genetic variability with respect to the traits for which clones show a response. Our results indicate that the major distinction is not between inducible and noninducible genotypes but rather that the genotypes differ in the combination of traits for which they show inducible responses.