Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation

Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT.Grb2.Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2.Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway.     

Micro-Geographical Heterogeneity in Schistosoma mansoni and S. haematobium Infection and Morbidity in a Co-Endemic Community in Northern Senegal

Background

Schistosoma mansoni and S. haematobium are co-endemic in many areas in Africa. Yet, little is known about the micro-geographical distribution of these two infections or associated disease within such foci. Such knowledge could give important insights into the drivers of infection and disease and as such better tailor schistosomiasis control and elimination efforts.

Methodology

In a co-endemic farming community in northern Senegal (346 children (0–19 y) and 253 adults (20–85 y); n = 599 in total), we studied the spatial distribution of S. mansoni and S. haematobium single and mixed infections (by microscopy), S. mansoni-specific hepatic fibrosis, S. haematobium-specific urinary tract morbidity (by ultrasound) and water contact behavior (by questionnaire). The Kulldorff''s scan statistic was used to detect spatial clusters of infection and morbidity, adjusted for the spatial distribution of gender and age.

Principal Findings

Schistosoma mansoni and S. haematobium infection densities clustered in different sections of the community (p = 0.002 and p = 0.023, respectively), possibly related to heterogeneities in the use of different water contact sites. While the distribution of urinary tract morbidity was homogeneous, a strong geospatial cluster was found for severe hepatic fibrosis (p = 0.001). Particularly those people living adjacent to the most frequently used water contact site were more at risk for more advanced morbidity (RR = 6.3; p = 0.043).

Conclusions/Significance

Schistosoma infection and associated disease showed important micro-geographical heterogeneities with divergent patterns for S. mansoni and S. haematobium in this Senegalese community. Further in depth investigations are needed to confirm and explain our observations. The present study indicates that local geospatial patterns should be taken into account in both research and control of schistosomiasis. The observed extreme focality of schistosomiasis even at community level, suggests that current strategies may not suffice to move from morbidity control to elimination of schistosomiasis, and calls for less uniform measures at a finer scale.     

Transfer of the Pheromone-Inducible Plasmid pCF10 among Enterococcus faecalis Microorganisms Colonizing the Intestine of Mini-Pigs

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10⁶ and 10⁷ CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10⁶ CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10³ and 10⁴ CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Effect of cheese and butter intake on metabolites in urine using an untargeted metabolomics approach

Cheese intake has been shown to decrease total cholesterol and LDL cholesterol concentrations when compared to butter of equal fat content. An untargeted metabolite profiling may reveal exposure markers of cheese but may also contribute with markers which can help explain how the intake of cheese affects cholesterol concentrations. Twenty-three subjects collected 2 × 24 h urine samples after 6 weeks of cheese and 6 weeks of butter intake with equal amounts of fat in a cross-over intervention study. The samples were analyzed by UPLC-QTOF/MS. A two-step univariate data analysis approach using linear mixed model was applied separately for positive and negative ionization mode: In the first step a total of 44 features related to treatment were identified and in the second step 36 of these features were related to total cholesterol concentrations. Cheese intake resulted in increased urinary indoxyl sulfate, xanthurenic acid, tyramine sulfate, 4-hydroxyphenylacetic acid, isovalerylglutamic acid and several acylglycines including isovalerylglycine, tiglylglycine and isobutyrylglycine when compared to butter intake of equal fat content. The biological mechanisms of action linking the metabolites to cholesterol concentrations need to be further explored.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection

rac-Isradipine is a dihydropyridine type calcium antagonist. Its calcium entry blocking effect is due primarily to the (+)-(S)-enantiomer. This study describes a sensitive enantioselective method for the determination of isradipine in human serum. Following alkaline extraction into hexane, the enantiomers of isradipine are separated quantitatively by high-performance liquid chromatography on a Chiralcel OJ column at 39°C. The collected fractions were evaporated and assayed using capillary gas chromatography on a HP 50+ column with nitrogen selective detection. Using 2.0 ml of serum, 0.7 nmol/1 (0.26 ng/ml) of each enantiomer could be determined with acceptable precision. The method has successfully been used to measure (+)-(S)- and (−)-(R)-isradipine concentrations in samples from volunteers after intravenous and oral administration of isradipine. Chirality 10:808–812, 1998. © 1998 Wiley-Liss, Inc.