Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: Focus on PI3K/Akt signaling pathway

In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8–12 weeks in the presence of 1, 2.5 and 5 μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a possibility of induction of different mechanisms in development of resistance to ATO depending on the drug concentration and thus involvement of different signaling mediators.     

Behavioural and physiological mechanisms behind extreme longevity in Daphnia

The combined effect of external environment and energy allocation strategy of the organism on longevity can be exceptional. In a cold oligotrophic fishless habitat, individual Daphnia can live for over a year, several times the usual Daphnia lifespan. This extreme lifespan is in part a consequence of the overwintering strategy which includes storing resources and delaying reproduction until another spring. Yet, contrasting strategies may be applied by Daphnia, resulting in over twofold differences in lifespan within a single habitat. We identify physiological mechanisms mediating such differences in longevity in closely related Daphnia of two lineages coexisting within a high altitude lake, testing the predictions that long-lived animals stay in colder waters and have lower metabolic rates, irrespective of temperature. Vertical distribution of the animals was assessed during three summer stratification seasons, and metabolic activity was measured as oxygen consumption and RNA:DNA ratio. The results not only support our predictions but also reveal that habitat choice is dependent on reproductive status rather than genotype. The young individuals of the overwintering lineage may delay reproduction in part by staying in colder waters than the reproducing adults, which together with low intrinsic metabolic rates may underlie the longevity of Daphnia of this lineage.     

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (²H/¹⁵N/¹³C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K_D values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Skeletal Lesions in Human Tuberculosis May Sometimes Heal: An Aid to Palaeopathological Diagnoses

In three to five percent of active cases of tuberculosis, skeletal lesions develop. Typically, these occur on the vertebrae and are destructive in nature. In this paper, we examined cases of skeletal tuberculosis from a skeletal collection (Galler Collection) with focus on the manifestation of bony changes due to tuberculosis in various body regions in association with antibiotic introduction. This skeletal collection was created in 1925–1977 by a pathologist at the University Hospital in Zürich, Ernst Galler. It includes the remains of 2426 individuals with documented clinical histories as well as autopsies. It contained 29 cases of skeletal tuberculosis lesions. We observed natural healing of vertebral lesions through several processes including fusion of vertebrae, bone deposition and fusion of posterior elements. In these cases, we observed a higher frequency and proportion of bone deposition and fusion of posterior vertebral elements where pharmacological agents were used. There were also four cases of artificial healing through surgically induced posterior spinal fusion. With the introduction of pharmaceutical treatments, the number of individuals with multiple tuberculous foci decreased from 80% to 25% when compared to individuals who did not receive any drug therapy. Investigation of comorbidities showed that pneumonia, pleuritis and being underweight were consistently present, even with pharmaceutical treatment. Our results have applications in palaeopathological diagnoses where healing and consequent bone deposition may complicate differential diagnoses.     

Exploring the cocrystallization potential of urea and benzamide

The cocrystallization landscape of benzamide and urea interacting with aliphatic and aromatic carboxylic acids was studied both experimentally and theoretically. Ten new cocrystals of benzamide were synthesized using an oriented samples approach via a fast dropped evaporation technique. Information about types of known bi-component cocrystals augmented with knowledge of simple binary eutectic mixtures was used for the analysis of virtual screening efficiency among 514 potential pairs involving aromatic carboxylic acids interacting with urea or benzamide. Quantification of intermolecular interaction was achieved by estimating the excess thermodynamic functions of binary liquid mixtures under supercooled conditions within a COSMO-RS framework. The smoothed histograms suggest that slightly more potential pairs of benzamide are characterized in the attractive region compared to urea. Finally, it is emphasized that prediction of cocrystals of urea is fairly direct, while it remains ambiguous for benzamide paired with carboxylic acids. The two known simple eutectics of urea are found within the first two quartiles defined by excess thermodynamic functions, and all known cocrystals are outside of this range belonging to the third or fourth quartile. On the contrary, such a simple separation of positive and negative cases of benzamide miscibility in the solid state is not observed. The difference in properties between urea and benzamide R2,2(8) heterosynthons is also documented by alterations of substituent effects. Intermolecular interactions of urea with para substituted benzoic acid analogues are stronger compared to those of benzamide. Also, the amount of charge transfer from amide to aromatic carboxylic acid and vice versa is more pronounced for urea. However, in both cases, the greater the electron withdrawing character of the substituent, the higher the binding energy, and the stronger the supermolecule polarization via the charge transfer mechanism.