Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers

Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.

Inactive Matrix Gla-Protein Is Associated With Arterial Stiffness in an Adult Population-Based Study

Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor of mortality. Matrix Gla-protein (MGP) is a vascular calcification inhibitor that needs vitamin K to be activated. Inactive MGP, known as desphospho-uncarboxylated MGP (dp-ucMGP), can be measured in plasma and has been associated with various cardiovascular markers, cardiovascular outcomes, and mortality. In this study, we hypothesized that high levels of dp-ucMGP are associated with increased PWV. We recruited participants via a multicenter family-based cross-sectional study in Switzerland. Dp-ucMGP was quantified in plasma by sandwich ELISA. Aortic PWV was determined by applanation tonometry using carotid and femoral pulse waveforms. Multiple regression analysis was performed to estimate associations between PWV and dp-ucMGP adjusting for age, renal function, and other cardiovascular risk factors. We included 1001 participants in our analyses (475 men and 526 women). Mean values were 7.87±2.10 m/s for PWV and 0.43±0.20 nmol/L for dp-ucMGP. PWV was positively associated with dp-ucMGP both before and after adjustment for sex, age, body mass index, height, systolic and diastolic blood pressure (BP), heart rate, renal function, low- and high-density lipoprotein, glucose, smoking status, diabetes mellitus, BP and cholesterol lowering drugs, and history of cardiovascular disease (P≤0.01). In conclusion, high levels of dp-ucMGP are independently and positively associated with arterial stiffness after adjustment for common cardiovascular risk factors, renal function, and age. Experimental studies are needed to determine whether vitamin K supplementation slows arterial stiffening by increasing MGP carboxylation.

1D.03: Inactive Matrix Gla Protein is Assocated with Renal Resistive Index in a Population-Based Study

OBJECTIVE Renal resistive index (RRI) varies directly with renal vascular stiffness and pulse pressure. RRI correlates positively with arteriolosclerosis in damaged kidneys and predicts progressive renal dysfunction. Matrix Gla-protein (MGP) is a vascular calcification inhibitor that needs vitamin K to be activated. Inactive MGP, known as desphospho-uncarboxylated MGP (dp-ucMGP), can be measured in plasma and has been associated with various cardiovascular (CV) markers, CV outcomes and mortality. In this study we hypothesize that increased RRI is associated with high levels of dp-ucMGP. DESIGN AND METHOD We recruited participants via a multi-center family-based cross-sectional study in Switzerland exploring the role of genes and kidney hemodynamics in blood pressure regulation. Dp-ucMGP was quantified in plasma samples by sandwich ELISA. Renal doppler sonography was performed using a standardized protocol to measure RRIs on 3 segmental arteries in each kidney. The mean of the 6 measures was reported. Multiple regression analysis was performed to estimate associations between RRI and dp-ucMGP adjusting for sex, age, pulse pressure, mean pressure, renal function and other CV risk factors. RESULTS We included 1035 participants in our analyses. Mean values were 0.64 ± 0.06 for RRI and 0.44 ± 0.21 (nmol/L) for dp-ucMGP. RRI was positively associated with dp-ucMGP both before and after adjustment for sex, age, body mass index, pulse pressure, mean pressure, heart rate, renal function, low and high density lipoprotein, smoking status, diabetes, blood pressure and cholesterol lowering drugs, and history of CV disease (P < 0.001). CONCLUSIONS RRI is independently and positively associated with high levels of dp-ucMGP after adjustment for pulse pressure and common CV risk factors. Further studies are needed to determine if vitamin K supplementation can have a positive effect on renal vascular stiffness and kidney function.

The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hirae

Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu(+) to the CopY repressor, thereby releasing its bound zinc and abolishing repressor-DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis.

Habitat-specific demography and source-sink dynamics in a population of Siberian jays

P>1. There are a number of models describing population structure, many of which have the capacity to incorporate spatial habitat effects. One such model is the source-sink model, that describes a system where some habitats have a natality that is higher than mortality (source) and others have a mortality that exceeds natality (sink). A source can be maintained in the absence of migration, whereas a sink will go extinct. 2. However, the interaction between population dynamics and habitat quality is complex, and concerns have been raised about the validity of published empirical studies addressing source-sink dynamics. In particular, some of these studies fail to provide data on survival, a significant component in disentangling a sink from a low quality source. Moreover, failing to account for a density-dependent increase in mortality, or decrease in fecundity, can result in a territory being falsely assigned as a sink, when in fact, this density-dependent suppression only decreases the population size to a lower level, hence indicating a 'pseudo-sink'. 3. In this study, we investigate a long-term data set for key components of territory-specific demography (mortality and reproduction) and their relationship to habitat characteristics in the territorial, group-living Siberian jay (Perisoreus infaustus). We also assess territory-specific population growth rates (r), to test whether spatial population dynamics are consistent with the ideas of source-sink dynamics. 4. Although average mortality did not differ between sexes, habitat-specific mortality did. Female mortality was higher in older forests, a pattern not observed in males. Male mortality only increased with an increasing amount of open areas. Moreover, reproductive success was higher further away from human settlement, indicating a strong effect of human-associated nest predators. 5. Averaged over all years, 76% of the territories were sources. These territories generally consisted of less open areas, and were located further away from human settlement. 6. The source-sink model provides a tool for modelling demography in distinct habitat patches of different quality, which can aid in identifying key habitats within the landscape, and thus, reduce the risk of implementing unsound management decisions.

HMGA1 and HMGA2 protein expression correlates with advanced tumour grade and lymph node metastasis in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). The high mobility group A1 (HMGA1) and high mobility group A2 (HMGA2) proteins are architectural transcription factors that have been implicated in the pathogenesis and progression of malignant tumours, including pancreatic cancer. The aim of this study was to explore the role of HMGA1 and HMGA2 in pancreatic carcinogenesis.

Prostate specific antigen expression does not necessarily correlate with prostate cancer cell growth

PURPOSE: The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. MATERIALS AND METHODS: Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. RESULTS: Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. CONCLUSIONS: Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.

The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system

Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10(-3) of yeast cells survived low stringency selection and expressed beta-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.

Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.

Drug-specific in vitro release of IL-2, IL-5, IL-13 and IFN-gamma in patients with delayed-type drug hypersensitivity

BACKGROUND: The most prevalent drug hypersensitivity reactions are T-cell mediated. The only established in vitro test for detecting T-cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug-sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well-documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T-cell sensitization to drugs. METHODS: Peripheral blood mononuclear cell of 10 patients, five allergic to beta-lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17-plex bead-based immunoassay kits. RESULTS: Among the 17 cytokines/chemokines analysed, interleukin-2 (IL-2), IL-5, IL-13 and interferon-gamma (IFN-gamma) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide- and beta-lactam-reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. CONCLUSION: The measurement of IL-2, IL-5, IL-13 or IFN-gamma or a combination thereof might be a useful in vitro tool for detection of T-cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Species-specific stomatal response of trees to drought - a link to vegetation dynamics?

Question: Is stomatal regulation specific for climate and tree species, and does it reveal species-specific responses to drought? Is there a link to vegetation dynamics? Location: Dry inner alpine valley, Switzerland Methods: Stomatal aperture (θE) of Pinus sylvestris, Quercus pubescens, Juniperus communis and Picea abies were continuously estimated by the ratio of measured branch sap flow rates to potential transpiration rates (adapted Penman-Monteith single leaf approach) at 10-min intervals over four seasons. Results: θE proved to be specific for climate and species and revealed distinctly different drought responses: Pinus stomata close disproportionately more than neighbouring species under dry conditions, but has a higher θE than the other species when weather was relatively wet and cool. Quercus keeps stomata more open under drought stress but has a lower θE under humid conditions. Juniperus was most drought-tolerant, whereas Picea stomata close almost completely during summer. Conclusions: The distinct microclimatic preferences of the four tree species in terms of θE strongly suggest that climate (change) is altering tree physiological performances and thus species-specific competitiveness. Picea and Pinus currently live at the physiological limit of their ability to withstand increasing temperature and drought intensities at the sites investigated, whereas Quercus and Juniperus perform distinctly better. This corresponds, at least partially, with regional vegetation dynamics: Pinus has strongly declined, whereas Quercus has significantly increased in abundance in the past 30 years. We conclude that θE provides an indication of a species' ability to cope with current and predicted climate.