The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Two Proton Pumps Operate in Parallel Across the Tonoplast of Vacuoles Isolated from Suspension Cells of Chenopodium rubrum L.*

The kinetics of vacuolar acidification upon addition of ATP and/or pyrophosphate (PPi) has been assayed on single immobilized vacuoles by computer-aided microfluorimetry of 9-aminoacridine, and by acridine orange absorption photometry on vacuole suspensions isolated from green suspension cells of Chenopodium rubrum L. Two proton pumps at the tonoplast, an ATPase and a pyrophosphatase (PPase), operate in parallel to acidify the vacuole with different contributions adding up to a transtonoplast Δ pH of 2.6 pH units at external pH 7.2. The saturable components of proton pumping reach half maximal velocity with 0.32 ± 0.06 mM ATP and 23 ± 2.5 μM PPi, respectively. At saturating substrate concentrations, ATPase and PPase hydrolyse ATP and PPi, respectively, at a ratio of 2.3. The same ratio holds for the corresponding proton fluxes maintaining a given steady-state vacuolar pH. We conclude that both pumps operate at the same stoichiometry.     

Validation of a Commercially Available Indirect Elisa Using a Nucleocapside Recombinant Protein for Detection of Schmallenberg Virus Antibodies

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-flurorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.     

HLA as a marker of the hemochromatosis gene in Sweden

Summary The frequency of HLA-A3 and HLA-B14 antigens was found to be significantly (P=<0.0001) higher in a series of 50 unrelated and unselected Swedish patients with idiopathic hemochromatosis (IH) than in controls, being 66% and 32% for A3 and 22% and 2% for B14. The haplotype A3B14 was associated with the highest risk in this material (relative risk 23.4). One family with this haplotype was traced back to the end of the seventeenth century. The pattern of HLA antigens associated with IH in Sweden shows remarkable similarity to those reported from England and Brittany.     

BUdR-Giemsa labeling and satellite association in human leukocytes

Summary Satellite associations were analysed in differentially stained human leukocyte chromosomes, obtained from four patients with Down's syndrome and four normal probands. A particular type of close association between two acrocentrics, showing a non-random arrangement of sister chromatids in a concordant dark-to-dark and light-to-light alignment, was found to be more common in patients with Down's syndrome compared with the normal controls. Apart from this particular type of association, sister chromatids are randomly arranged in satellite associations between two acrocentrics in both groups of probands. Considerable differences in the mean frequencies of satellite associations between first and second metaphases of the same individual were found in some probands of both groups of individuals. Since a high degree of inter-individual variability in the proliferative response of human leukocytes in culture is well established, the use of BUdR-Giemsa labeling for comparative analysis of satellite association frequencies is suggested.     

Charakterisierung des Regenerationsprozesses im Caulonema eines Mooses durch autoradiographische Bestimmung der DNS-Synthese

Summary Ten-cell-long filaments of the caulonema of Funaria hygrometrica were isolated and labeled with ³H-thymidine. During the process of regeneration this precursor is incorporated into the nucleus and the chloroplasts. The nuclei of aged cells are preferentially labeled, even the nuclei of such cells which probably will no longer divide.From these facts it is concluded that DNA synthesis can occur during the process of regeneration irrespectively of a following cell division.     

Communality in microbial stress response and differential metabolic interactions revealed by time-series analysis of sponge symbionts

The diversity and function of sponge-associated symbionts is now increasingly understood; however, we lack an understanding of how they dynamically behave to ensure holobiont stability in the face of environmental variation. Here, we performed a metatransciptomic analysis on three microbial symbionts of the sponge Cymbastela concentrica in situ over 14 months and through differential gene expression and correlation analysis to environmental variables uncovered differences that speak to their metabolic activities and level of symbiotic and environmental interactions. The nitrite-oxidizing Ca. Porinitrospira cymbastela maintained a seemingly stable metabolism, with the few differentially expressed genes related only to stress responses. The heterotrophic Ca. Porivivens multivorans displayed differential use of holobiont-derived compounds and respiration modes, while the ammonium-oxidizing archaeon Ca. Nitrosopumilus cymbastelus differentially expressed genes related to phosphate metabolism and symbiosis effectors. One striking similarity between the symbionts was their similar variation in expression of stress-related genes. Our time-series study showed that the microbial community of C. concentrica undertakes dynamic gene expression adjustments in response to the surroundings, tuned to deal with general stress and metabolic interactions between holobiont members. The success of these dynamic adjustments likely underpins the stability of the sponge holobiont and may provide resilience against environmental change.