Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.     

Protective role of calreticulin in HFE hemochromatosis

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.     

p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping

Reverse signaling by ephrin-As upon binding EphAs controls axon guidance and mapping. Ephrin-As are GPI-anchored to the membrane, requiring that they complex with transmembrane proteins that transduce their signals. We show that the p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75(NTR) and ephrin-A colocalize within caveolae along retinal axons and form a complex required for Fyn phosphorylation upon binding EphAs, activating a signaling pathway leading to cytoskeletal changes. In vitro, retinal axon repulsion to EphAs by ephrin-A reverse signaling requires p75(NTR), but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75(NTR) knockout mice have aberrant anterior shifts in retinal axon terminations in superior colliculus, consistent with diminished repellent activity mediated by graded ephrin-A reverse signaling induced by graded collicular EphAs. We conclude that p75(NTR) is a signaling partner for ephrin-As and the ephrin-A- p75(NTR) complex reverse signals to mediate axon repulsion required for guidance and mapping.     

Gαo/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

The G_s and G_i pathways interact to control the levels of intracellular cAMP. Although coincident signaling through G_s and G_i-coupled receptors can attenuate G_s-stimulated cAMP levels, it is not known if prior activation of the G_i pathway can affect signaling by G_s-coupled receptors. We have found that activated Gα_o/i interact with RGS20, a GTPase activating protein for members of the Gα_ο/i family. Interaction between Gα_o/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gα_o and Gα_i2 but not by Gα_q, Gα_i1 or Gα_i3. The Gα_o/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gα_o stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to G_o/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that G_o/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that G_o/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the G_s and G_i pathways controls cellular cAMP levels, thus allowing for signal integration.     

Contribution of planktonic and detritic fractions to the natural diet of mesozooplankton in Bahía Blanca Estuary

The pike, Esox lucius Linnaeus, is a predatory fish that supports important fisheries and could substantially impact prey populations around the temperate northern hemisphere. Consumption of prey by pike is most readily estimated using the energy budget to calculate food intake indirectly using estimates of growth rate and metabolism. Resting metabolic rate, R _s, is a particularly important component of such calculations. Here, the available estimates of R _s are reviewed and compared. Scaling coefficients for variation with body mass are consistent between the two studies in which they have been derived (0.81, 0.82). However, the effect of temperature on R _s markedly varies among studies (Q ₁₀ from 1.73 to 4.80). There is substantial variation in R _s (twofold to fourfold) among studies when temperature and fish size are accounted for. This variation is shown to have a large effect on energy budget calculations of energy intake and to be sufficient to account for imbalances in published budgets. These effects depend on age of pike and season; in one energy budget model, a 50% reduction in R _s resulted in decrease of 19–42% in estimated energy intake of pike. Potential causes of among-study variation in R _s are discussed and it is recommended that standard techniques by applied in the future to differentiate between genuine biological variation among populations and experimental factors. Guest editors: J. M. Farrell, C. Skov, M. Mingelbier, T. Margenau & J. E. Cooper International Pike Symposium: Merging Knowledge of Ecology, Biology, and Management for a Circumpolar Species     

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.     

Arabidopsis sucrose transporter AtSUC1 is important for pollen germination and sucrose-induced anthocyanin accumulation

The Arabidopsis (Arabidopsis thaliana) sucrose transporter AtSUC1 (At1g71880) is highly expressed in pollen; however, its function has remained unknown. Here, we show that suc1 mutant pollen is defective in vivo, as evidenced by segregation distortion, and also has low rates of germination in vitro. AtSUC1-green fluorescent protein was localized to the plasma membrane in pollen tubes. AtSUC1 is also expressed in roots and external application of sucrose increased AtSUC1 expression in roots. AtSUC1 is important for sucrose-dependent signaling leading to anthocyanin accumulation in seedlings. suc1 mutants accumulated less anthocyanins in response to exogenous sucrose or maltose and microarray analysis revealed reduced expression of many genes important for anthocyanin biosynthesis. The results indicate that AtSUC1 is important for sugar signaling in vegetative tissue and for normal male gametophyte function.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.