Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.     

Comparison of rRNA and polar-lipid-derived fatty acid biomarkers for assessment of 13C-substrate incorporation by microorganisms in marine sediments

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13C-labeled compounds. Similar results were obtained with the 13C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13C analysis of the captured SSU rRNA.     

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.     

Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA)

Background

Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens.

Methods

Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.

Results

Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.

Conclusion

Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.     

Integrating microbial ecology into ecosystem models: challenges and priorities

Microbial communities can potentially mediate feedbacks between global change and ecosystem function, owing to their sensitivity to environmental change and their control over critical biogeochemical processes. Numerous ecosystem models have been developed to predict global change effects, but most do not consider microbial mechanisms in detail. In this idea paper, we examine the extent to which incorporation of microbial ecology into ecosystem models improves predictions of carbon (C) dynamics under warming, changes in precipitation regime, and anthropogenic nitrogen (N) enrichment. We focus on three cases in which this approach might be especially valuable: temporal dynamics in microbial responses to environmental change, variation in ecological function within microbial communities, and N effects on microbial activity. Four microbially-based models have addressed these scenarios. In each case, predictions of the microbial-based models differ—sometimes substantially—from comparable conventional models. However, validation and parameterization of model performance is challenging. We recommend that the development of microbial-based models must occur in conjunction with the development of theoretical frameworks that predict the temporal responses of microbial communities, the phylogenetic distribution of microbial functions, and the response of microbes to N enrichment.     

Unusual growth pattern in the Frasnian alveolitids (Tabulata) from the Holy Cross Mountains (Poland)

Abstract: Growth periodicity is a phenomenon occurring in fossil and modern corals. The most apparent feature is growth banding, and environmental changes are broadly accepted as controls on this phenomenon. If environment controls the growth, then all corallites within a colony should repeat the same growth pattern, as individuals are clones and must have shared the same environment. A study on several species of Alveolitidae (Anthozoa, Tabulata) from the Late Devonian (Early Frasnian) of the Holy Cross Mountains (Poland) shows that the growth pattern varies between neighbouring individuals within the same corallum. This contradicts observations of closely related Favositida as demonstrated on Pachyfavosites sp. from the Givetian of Avesnois, France, where neighbouring individuals repeat the same pattern. Therefore, environmental control on growth rhythm in Alveolitidae can be excluded; the causes of differences between individuals remain unknown.     

Phasic Firing in Vasopressin Cells: Understanding Its Functional Significance through Computational Models

Vasopressin neurons, responding to input generated by osmotic pressure, use an intrinsic mechanism to shift from slow irregular firing to a distinct phasic pattern, consisting of long bursts and silences lasting tens of seconds. With increased input, bursts lengthen, eventually shifting to continuous firing. The phasic activity remains asynchronous across the cells and is not reflected in the population output signal. Here we have used a computational vasopressin neuron model to investigate the functional significance of the phasic firing pattern. We generated a concise model of the synaptic input driven spike firing mechanism that gives a close quantitative match to vasopressin neuron spike activity recorded in vivo, tested against endogenous activity and experimental interventions. The integrate-and-fire based model provides a simple physiological explanation of the phasic firing mechanism involving an activity-dependent slow depolarising afterpotential (DAP) generated by a calcium-inactivated potassium leak current. This is modulated by the slower, opposing, action of activity-dependent dendritic dynorphin release, which inactivates the DAP, the opposing effects generating successive periods of bursting and silence. Model cells are not spontaneously active, but fire when perturbed by random perturbations mimicking synaptic input. We constructed one population of such phasic neurons, and another population of similar cells but which lacked the ability to fire phasically. We then studied how these two populations differed in the way that they encoded changes in afferent inputs. By comparison with the non-phasic population, the phasic population responds linearly to increases in tonic synaptic input. Non-phasic cells respond to transient elevations in synaptic input in a way that strongly depends on background activity levels, phasic cells in a way that is independent of background levels, and show a similar strong linearization of the response. These findings show large differences in information coding between the populations, and apparent functional advantages of asynchronous phasic firing.