Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.     

High-Resolution Taxonomic Profiling of the Subgingival Microbiome for Biomarker Discovery and Periodontitis Diagnosis

The oral microbiome plays a key role for caries, periodontitis, and systemic diseases. A method for rapid, high-resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here, we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S rRNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, 9 of whom had been diagnosed with chronic periodontitis. Five hundred twenty-three operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia could be identified at the species level with both primer sets. Principal coordinate analysis identified two outliers that were consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health- and periodontitis-related clusters of OTUs were identified using a connectivity analysis, and the results confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jackknife method). Using a combination of the 10 best biomarkers, 15 of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.     

The impact of a boosting immunogen on the differentiation of secondary memory CD8+ T cells

While recent studies have demonstrated that secondary CD8⁺ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens—recombinant vaccinia, recombinant adenovirus, and plasmid DNA—on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8⁺ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8⁺ T cells following a secondary immunization. In contrast, the functional profiles of both CD8⁺ and CD4⁺ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8⁺ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8⁺ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8⁺ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8⁺ T cells. These data therefore suggest that the phenotype of secondary CD8⁺ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.     

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.     

Association between random glucose and all-cause mortality: findings from the mortality follow-up of the German National Health Interview and Examination Survey 1998

Background

Random glucose is widely measured in epidemiological studies and in the clinical setting when standardized fasting protocols and oral glucose tolerance testing or HbA_1c measuring are not feasible. The relationship between random glucose and all-cause mortality has hardly been studied so far and was examined in the present study.

Methods

We ascertained mortality status among 5955 persons aged 18–79?years and free of known diabetes when participating in the German National Health Interview and Examination Survey 1998 (mean observation time 11.7?years, 458 deaths). Cox regression was applied to analyze the association of random serum glucose with all-cause mortality taken potential confounders into account. Relative mortality risks were estimated as hazard ratios (HRs) with 95% confidence intervals (CIs) modeling random glucose as categorical or continuous variable.

Results

Compared to random glucose levels of 4.3 -?<?5.3?mmol/L, HRs (95% CIs) were 1.94 (0.85–4.45) for levels <?4.3?mmol/L and 1.16 (0.89–1.50), 1.20 (0.91–1.58), 1.42 (0.88–2.29), 2.02 (1.26–3.25) and 4.71 (2.20–10.10) for levels 5.3 -?<?5.8, 5.8 -?<?6.8, 6.8 -?<?7.8, 7.8 -?<?11.1 and?≥?11.1?mmol/L, adjusted for age, sex, lifestyle, anthropometry and chronic diseases. An additional adjustment for fasting time or HbA_1c yielded similar estimates. Modeling continuous random glucose by restricted cubic spline functions revealed comparable findings.

Conclusions

In the present epidemiological study drawn from the general population, random glucose showed a significant association with all-cause mortality, independent of main potential confounders. Thus, random glucose measures are highly relevant to health risk assessment among people without known diabetes when fasting glucose or HbA_1c are difficult to obtain.

     

Antioxidant mechanisms of polyphenolic caffeic acid oligomers, constituents of Salvia officinalis

Caffeic acid, rosmarinic acid and oligomers of caffeic acid with multiple catechol groups are all constituents of Salvia officinalis. Their antioxidant potential was investigated with regard to their radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic studies revealed very high rate constants with hydroxyl radicals. Evidence from kinetic modeling calculations suggested an unusual complex behavior due to the presence of both O4- and O3-semiquinones and formation and decay of a hydroxyl radical adduct at the vinyl side chain. The radical structures observed by EPR spectroscopy after autoxidation in slightly alkaline solutions were only partially identified due to their instability and generally represented dissociated O4-semiquinones. Hybrid density-functional calculations of the potential radical structures showed distinct differences between the resonance stabilization of the O4- and O3-semiquinones of caffeic and dihydrocaffeic acids, reflected also in the considerably faster decay of the O3-semiquinone observed by pulse radiolysis. No evidence was found for dimerization reactions via Cbeta radicals typical for lignin biosynthesis.     

Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

Roles of Aquaporins in Root Responses to Irrigation

Due to current environmental issues concerning the use of water for irrigation, the improvement of crop water-use efficiency and a reduction in water consumption has become a priority. New irrigation methods that reduce water use, while still maintaining production have been developed. To optimise these techniques knowledge of above- and below-ground plant physiological responses is necessary. During growth, plant roots are exposed to cycles of wetting and drying in normal rain-fed and irrigation situations. This review concentrates on the below-ground aspects, in particular the water permeability of roots. Significant research has been conducted on the root anatomy and hydraulic conductivity of desert plants subjected to wetting and drying. Major intrinsic proteins (MIPs), most of which show aquaporin (water-channel) activity are likely to be involved in balancing the water relations of the plants during water deficit. However, many MIPs seem to allow permeation of other small neutral solutes and some may allow permeation of ions under certain conditions. The ability of the plant to rapidly respond to rewetting may be important in maintaining productivity. It has been suggested that aquaporins may be involved in this rapid response. The down-regulation of the aquaporins during dry conditions can also limit water loss to the soil, and intrinsic sensitivity of aquaporins to water potential is shown here to be very strong in some cases (NOD26). However, the response of aquaporins in various plant species to water deficits has been quite varied. Another component of aquaporin regulation in response to various stresses (hypoxia/anoxia, salinity and chilling) may be related to redistribution of flow to more favourable regions of the soil. Some irrigation techniques may be triggering these responses. Diurnal fluctuations of root hydraulic conductance that is related to aquaporin expression seem to match the expected transpirational demands of the shoot, and it remains to be seen if shoot-to-root signalling may be important in regulation of root aquaporins. If so, canopy management typical of horticultural crops may impact on root hydraulic conductance. An understanding of the regulation of aquaporins may assist in the development of improved resistance to water stress and greater efficiency of water use by taking into account where and when roots best absorb water.