Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY

In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.     

Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.     

Calbindin and calretinin immunoreactivities in the retina of a chondrostean,Acipenser baeri

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.     

Immunocytochemical investigation of the hypothalamo-neurohypophysial system in birds

Summary The results of an immunohistochemical investigation of the hypothalamo-neurohypophysial system in several species of birds have shown that: (1) mesotocin and vasotocin are synthesized in separate neurons; (2) in all species investigated the distribution of mesotocinergic and vasotocinergic perikarya follows a common pattern; (3) the external zone of the avian anterior median eminence contains exclusively vasotocinergic nerve fibers, originating in supraoptic and ventral paraventricular regions; (4) the distribution of immunoreactive elements in the neural lobe shows a definite species-dependent pattern.     

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.     

Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes

Recently, vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. In this study, we examined whether vaspin mRNA expression is a marker of visceral obesity and correlates with anthropometric and metabolic parameters in paired samples of visceral and subcutaneous adipose tissue from 196 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Vaspin mRNA expression was only detectable in 23% of the visceral and in 15% of the subcutaneous (SC) adipose tissue samples. Vaspin mRNA expression was not detectable in lean subjects (BMI<25) and was more frequently detected in patients with type 2 diabetes. No significant correlations were found between visceral vaspin gene expression and visceral fat area or SC vaspin expression. However, visceral vaspin expression significantly correlates with BMI, % body fat, and 2 h OGTT plasma glucose. Subcutaneous vaspin mRNA expression is significantly correlated with WHR, fasting plasma insulin concentration, and glucose infusion rate during steady state of an euglycemic-hyperinsulinemic clamp. Multivariate linear regression analysis revealed % body fat as strongest predictor of visceral vaspin and insulin sensitivity as strongest determinant of SC vaspin mRNA expression. In conclusion, our data indicate that induction of human vaspin mRNA expression in adipose tissue is regulated in a fat depot-specific manner and could be associated with parameters of obesity, insulin resistance, and glucose metabolism.     

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non-toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an alpha-motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone-rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m-Carboxycinnamic acid bis-Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS-275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood-brain barrier, oral administration may allow deceleration of progressive alpha-motoneurone degeneration by epigenetic SMN2 gene activation.     

Phosphoenolpyruvate Carboxylase Purified from Leaves of C3, C4, and C3-C4 intermediate species of Alternanthera: Properties at Limiting and Saturating Bicarbonate

Phosphoenolpyruvate carboxylase (PEPC) was purified from leaves of four species of Alternanthera differing in their photosynthetic carbon metabolism: Alternanthera sessilis (C₃), A. pungens (C₄), A. ficoides and A. tenella (C₃-C₄ intermediates or C₃-C₄). The activity and properties of PEPC were examined at limiting (0.05 mM) or saturating (10 mM) bicarbonate concentrations. The V_max as well as K_m values (for Mg²⁺ or PEP) of PEPC from A. ficoides and A. tenella (C₃-C₄ intermediates) were in between those of C₃ (A. sessilis) and C₄ species (A. pungens). Similarly, the sensitivity of PEPC to malate (an inhibitor) or G-6-P (an activator) of A. ficoides and A. tenella (C₃-C₄) was also of intermediate status between those of C₃ and C₄ species of A. sessilis and A. pungens, respectively. In all the four species, the maximal activity (V_max), affinity for PEP (K_m), and the sensitivity to malate (K_I) or G-6-P (K_A) of PEPC were higher at 10 mM bicarbonate than at 0.05 mM bicarbonate. Again, the sensitivity to bicarbonate of PEPC from C₃-C₄ intermediates was in between those of C₃- and C₄-species. Thus the characteristics of PEPC of C₃-C₄ intermediate species of Alternanthera are intermediate between C₃- and C₄-type, in both their kinetic and regulatory properties. Bicarbonate could be an important modulator of PEPC, particularly in C₄ plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis

CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47^-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice. Despite normal fungal colonization at earlier times, cd47^-/- mice at four days post-infection had increased colonization of brain and kidneys accompanied by stronger inflammatory reactions. Neutrophil and macrophage numbers were significantly elevated in kidneys and neutrophils in the brains of infected cd47^-/- mice. However, no defect in phagocytic activity towards C. albicans was observed in cd47^-/- bone-marrow-derived macrophages, and neutrophil and macrophage killing of C. albicans was not impaired. CD47-deficiency did not alter the early humoral immune response to C. albicans. Th1, Th2, and Th17 population of CD4⁺ T cells were expanded in the spleen, and gene expression profiles of spleen and kidney showed stronger pro-inflammatory signaling in infected cd47^-/- mice. The chemoattractant chemokines MIP-2α and MIP-2β were highly expressed in infected spleens of cd47^-/- mice. G-CSF, GM-CSF, and the inflammasome component NLRP3 were more highly expressed in infected cd47^-/- kidneys than in infected wild-type controls. Circulating pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) were significantly elevated, but IL-17 was decreased. These data indicate that CD47 plays protective roles against disseminated candidiasis and alters pro-inflammatory and immunosuppressive pathways known to regulate innate and T cell immunity.