Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.     

Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats

Objectives

Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis.

Methods

A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey’s test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05.

Results

hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48h. The treated group had less fibrosis and proteinuria at 2 months after injury.

Conclusion

hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.     

Human Gastroenteropancreatic Expression of Melatonin and Its Receptors MT1 and MT2

Background and Aim

The largest source of melatonin, according to animal studies, is the gastrointestinal (GI) tract but this is not yet thoroughly characterized in humans. This study aims to map the expression of melatonin and its two receptors in human GI tract and pancreas using microarray analysis and immunohistochemistry.

Method

Gene expression data from normal intestine and pancreas and inflamed colon tissue due to ulcerative colitis were analyzed for expression of enzymes relevant for serotonin and melatonin production and their receptors. Sections from paraffin-embedded normal tissue from 42 individuals, representing the different parts of the GI tract (n=39) and pancreas (n=3) were studied with immunohistochemistry using antibodies with specificity for melatonin, MT₁ and MT₂ receptors and serotonin.

Results

Enzymes needed for production of melatonin are expressed in both GI tract and pancreas tissue. Strong melatonin immunoreactivity (IR) was seen in enterochromaffin (EC) cells partially co-localized with serotonin IR. Melatonin IR was also seen in pancreas islets. MT₁ and MT₂ IR were both found in the intestinal epithelium, in the submucosal and myenteric plexus, and in vessels in the GI tract as well as in pancreatic islets. MT₁ and MT₂ IR was strongest in the epithelium of the large intestine. In the other cell types, both MT₂ gene expression and IR were generally elevated compared to MT₁. Strong MT₂, IR was noted in EC cells but not MT₁ IR. Changes in gene expression that may result in reduced levels of melatonin were seen in relation to inflammation.

Conclusion

Widespread gastroenteropancreatic expression of melatonin and its receptors in the GI tract and pancreas is in agreement with the multiple roles ascribed to melatonin, which include regulation of gastrointestinal motility, epithelial permeability as well as enteropancreatic cross-talk with plausible impact on metabolic control.     

The NLRP3 inflammasome is activated by nanoparticles through ATP,ADP and adenosine

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO₂) and titanium dioxide (nano-TiO₂) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO₂ or nano-TiO₂ caused a significant increase in P2Y1, P2Y2, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

Impaired Innate Immunity in Tlr4 ?/? Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8⁺ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8⁺ T cells specific for H-2K^b-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2^−/−, Tlr4^−/−, Tlr9^{−/ −} or Myd88^−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8⁺ T cells at levels comparable to WT mice, although the frequency of IFN-γ⁺CD4⁺ cells was diminished in infected Myd88^−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4^−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.     

Verbal cues modulate hedonic perception of odors in 5-year-old children as well as in adults

The judgment of pleasantness/unpleasantness is the prominent reaction to the olfactory world. In human adults, the hedonic valence of odor perception is affected by various factors, among which is an individual's lexical knowledge about smells. The present study examined whether such top-down effects of lexical knowledge on hedonic judgment of olfactory input are similar in children (5-6 years) and adults (20-25 years). In both groups, the lexical knowledge was found to influence the perception of the least emotional (or most neutral) odors: the pleasantness of the smells of banana and mint was enhanced when participants were given the corresponding odor label before olfactory sensation. These results lend support to the notion that, during childhood, smells are not only encoded perceptually but that verbal encoding also steers contextual effects that may be prominent factors in the early memorization and categorization of odors.     

Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

Background  

Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres.