A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.     

Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates.

Antioxidant protein 2 (AOP2) is a member of a family of thiol-specific antioxidants, recently renamed peroxiredoxins, that evolved as part of an elaborate system to counteract and control detrimental effects of oxygen radicals. AOP2 is found in endothelial cells, erythrocytes, monocytes, T and B cells, but not in granulocytes. AOP2 was found solely in the cytoplasm and was not associated with the nuclear or membrane fractions; neither was it detectable in plasma. Further experiments focused on the function of AOP2 in erythrocytes where it is closely associated with the hemoglobin complex, particularly with the heme. An investigation of the mechanism of this interaction demonstrated that the conserved cysteine-47 in AOP2 seems to play a role in AOP2-heme interactions. Recombinant AOP2 prevented induced as well as noninduced methemoglobin formation in erythrocyte hemolysates, indicating its antioxidant properties. We conclude that AOP2 is part of a sophisticated system developed to protect and support erythrocytes in their many physiological functions.     

The photobiont determines the pattern of photosynthetic activity within a single lichen thallus containing cyanobacterial and green algal sectors (photosymbiodeme)

Photosystem activity status of the green algal (Pseudocyphellaria lividofusca) and cyanobacterial (P. knightii) components of a photosymbiodeme were continuously monitored in the field over a period of 35 days. The photosymbiodeme grew on a Nothofagus menziesii tree at Lake Waikaremoana, Urewera National Park, North Island, New Zealand. Two Mini-PAM fluorometers were placed so that the chlorophyll a fluorescence, temperature and PPFD (photosynthetically active photon flux density) could be recorded every 30 min for green algal and cyanobacterial parts of the thallus. Microclimate conditions were also recorded with a datalogger. The study confirmed the already known ability of green algal lichens to reactivate from high humidity alone whilst cyanobacterial species need liquid water, here obtained from rainfall. The photosystems of P. lividofusca were activated on every day and positive ETR (relative electron transport rate) occurred on all but 3 days. Activation level depended on the overnight relative humidity. P. knightii was activated and had positive ETR on only 13 days when rainfall had occurred. Both species were mostly inactive above 12°C but differed at low temperatures. P. knightii showed no activation at very low temperatures, -2 to 0°C, since these only occurred on clear, rain-free nights. PPFD was always very low, mostly below 80 µmol m^-2 s^-1, and both species were inactive at higher PPFD. The three-dimensional structure of the thallus seemed to contribute to the hydration. The cyanobacterial sectors were more appressed to the trunk and needed substantial rainfall to rewet whereas the green algal lobes were more distant from the trunk and this probably caused more rapid desiccation as well as lower temperatures. It is suggested that the longer active periods for photosynthesis by P. lividofusca are balanced by several factors: first, depressed net photosynthesis at high thallus water contents after rainfall, a feature not shown by P. knightii; second, possible lower maximal net photosynthetic rates; and third, the possibility of greater respiratory rates when thalli have been hydrated by high relative humidity. There is little evidence for high PPFD differently affecting the photosymbiodeme components since sustained, high PPFD did not occur. It has been reported that the photosystems of cyanobacterial species from photosymbiodemes can reactivate at high relative humidity but the results obtained here suggest that it is not ecologically significant.     

Nitric oxide synthase inhibitor and IL-18 enhance the anti-tumor immune response of rats carrying an intrahepatic colon carcinoma

The role of nitric oxide (NO) produced by adherent spleen cells in the systemic immunosuppression developing in tumor-bearing hosts was investigated. After therapeutic immunization of rats carrying an intrahepatic colon carcinoma, H1D2, the spleen cell antitumor immune responsiveness was analyzed. Compared to parallel immunized tumor-free rats, tumor-bearing rats (TB rats) had a greatly reduced proliferative T-cell response to wild-type tumor stimulator cells. The TB rats had a depressed proliferative response to anti-CD3 and to the superantigen SEA. TB rats with small tumors had a stronger response to IL-18-producing H1D2 stimulator cells than to wild type H1D2 cells. This was not the case with TB rats carrying larger tumors. Also the IFN-gamma production and cytotoxicity against the wild-type tumor cells and the NK sensitive YAC cells were depressed in spleen cells of TB rats after 5-day restimulation with wild-type tumor cells. A part of this immunosuppression was mediated by adherent spleen cells, mostly consisting of macrophages. An important mode of action appears to involve their production of an enhanced level of nitric oxide, since the competitive nitric oxide synthase (NOS) inhibitor L-NAME could partially counteract the suppression in vitro. We conclude that NOS inhibitors in combination with immunostimulatory cytokines, such as IL-18, could be useful tools to enhance anti-tumor immune responses in TB rats and therefore to increase the efficiency of immunotherapies.     

Net efflux rate of norepinephrine from platelets in DOCA- and salt-treated rats

39 Wistar Kyoto rats were treated for 4 weeks with weekly injections of DOCA and extra sodium chloride in drinking water. The efflux rate of norepinephrine from platelets (k) was determined in 13 samples, each consisting of pooled blood from 3 animals. The efflux rate of norepinephrine was significantly higher (k=24.7±4.9) in these animals than in the 29 controls in which 10 k-determinations were made (k=17.0±4.5) (p<0.001). The heart weight was significantly higher in the DOCA-salt treated group and there was a significant correlation between heart weight and k (r9 p<0.05). As earlier studies have demonstrated a decrease in nonepinephrine in the granular fraction from heart tissue in DOCA-salt treated and hypertensive rats these findings suggest close similarities between norepinephrine storage in platelets and in neuronal cells.     

Cultures grown and embedded in situ on Spurr discs, a low viscosity epoxy resin for electron microscopy

The ultrastructural study of cellular inter-relationships in vitro requires methods of embedding in situ. The present technic combines the advantages of growing the cells directly on the substrate with subsequent embedding in situ. In addition it uses Spurr medium which provides qualities of low viscosity, high penetrability and easy handling.     

Synthesis of Modified Building Blocks Containing Amino or Thiol Moieties: Application of Modified Oligodeoxyribonucleotides

Abstract

Modified building blocks have been synthesised and used to prepare 5′- amino and 5′-mercapto-oligodeoxyribonucleotides. Subsequent labelling with fluorophores or metal cluster derivatives generates a range of very useful probes.     

Accumulation of the chalcone isosalipurposide in primary leaves of barley flavonoid mutants indicates a defective chalcone isomerase

Mutants defective in flavonoid biosynthesis have become increasingly useful in elucidating the potential functions of these compounds in plants. To define the role of flavonoids as UV-B protectants in barley, we have screened part of the collection of proanthocyanidin-free barley mutants at the Carlsberg Research Laboratory, Copenhagen, Denmark. The four mutants ant 30–245, ant 30–272, ant 30–287 and ant 30–310 showed drastically reduced flavonoid levels in the primary leaf as compared to their corresponding parent varieties, and in addition accumulated a new mutant-specific phenolic compound which was identified as the chalcone glucoside isosalipurposide. Results from diallelic crosses indicate that all four mutants belong to the same new complementation group, which is designated as the Ant 30 locus. This gene has not earlier been described in barley. The data presented suggest a defective chalcone isomerase gene for the observed flavonoid pattern in leaves of ant 30 mutants.     

Biodistribution of 68Ga-labeled LNA-DNA mixmer antisense oligonucleotides for rat chromogranin-A

In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with 68Ga and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2--differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched--differing only in two nucleotides from the two Antisenses--was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after 68Ga-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.