Endoscopy in humans is a powerful method for physicians to examine the gut for inflammatory or neoplastic changes. In medical and immunological research, animal models of intestinal diseases are established key tools to investigate the mucosal immune system, colitis and cancer development in the gut. Moreover, such models represent valid systems for testing of novel drugs. In the past, mice had to be killed in order to analyze colitis activity and tumor development. The following protocol describes a method to perform high resolution endoscopic monitoring of live mice. Mice developing colitis or colonic tumors are anesthetized and examined with a miniendoscope. The endoscope is introduced via the anus and the colon is carefully insufflated with an air pump. Endoscopic pictures obtained are of high quality and allow the monitoring and grading of tumors and inflammation. In addition, colonic biopsies can be taken. This protocol can be completed within 1 h. 相似文献
The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development. 相似文献
Alzheimer β‐amyloid (Aβ) peptides can self‐organize into oligomeric ion channels with high neurotoxicity potential. Cholesterol is believed to play a key role in this process, but the molecular mechanisms linking cholesterol and amyloid channel formation have so far remained elusive. Here, we show that the short Aβ22‐35 peptide, which encompasses the cholesterol‐binding domain of Aβ, induces a specific increase of Ca2+ levels in neural cells. This effect is neither observed in calcium‐free medium nor in cholesterol‐depleted cells, and is inhibited by zinc, a blocker of amyloid channel activity. Double mutations V24G/K28G and N27R/K28R in Aβ22‐35 modify cholesterol binding and abrogate channel formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α‐helical topology of Aβ22‐35. This facilitates the establishment of an inter‐peptide hydrogen bond network involving Asn‐27 and Lys‐28, a key step in the octamerization of Aβ22‐35 which proceeds gradually until the formation of a perfect annular channel in a phosphatidylcholine membrane. Overall, these data give mechanistic insights into the role of cholesterol in amyloid channel formation, opening up new therapeutic options for Alzheimer's disease.
Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.Bacterial lipoproteins (Lpps)1 are a subset of membrane proteins that are covalently modified with a lipidic moiety at their N-terminal cysteine residue. It is commonly reported that Lpps of Gram-positive bacteria are processed by two key enzymes; the prolipoprotein diacylglyceryl transferase (Lgt) and the lipoprotein signal peptidase (Lsp). The Lgt enzyme recognizes a so-called lipobox motif in the C-terminal region of the signal peptide of a premature lipoprotein and transfers a diacylglyceryl moiety to the cysteine residue of the lipobox (1), (2). Subsequently, the Lsp enzyme cleaves the signal peptide resulting in a mature Lpp (3), (4). Nevertheless, recent reports have suggested that N-acylation occurs in bacteria that lack the Gram-negative homologous apolipoprotein N-acyltransferase (Lnt) gene responsible for this modification (5, 6), and that Lpp N-terminal could also be modified with an acetyl group in some Gram-positive (7).Lpps have been described as virulence factors because they play critical roles in membrane stabilization, nutrient uptake, antibiotic resistance, bacterial adhesion to host cells, protein maturation and secretion and many of them still have unknown function (8). Several studies have suggested that bacterial Lpps are pathogen-associated molecular patterns (PAMPs) sensed by the mammalian host through Toll-like receptor 2 (TLR2) heterodimerized with TLR1 or TLR6 to induce innate immunity activation and to control adaptive immunity (9–12). TLR2 plays a critical role in the host response to the Gram-positive bacteria Staphylococcus aureus (13) and Streptococcus agalactiae (14). Although TLR2 has been considered a receptor for various structurally unrelated PAMPs, recent studies have suggested that, via their lipid moiety, bacterial Lpps function as the major, if not the sole, ligand molecules responsible for TLR2 activation (15). Although Gram-negative Lpps have been widely studied, little information is available for Gram-positive Lpps (16) and the ways they are released into the bacterial extracellular compartment and reach the host immune system remain unclear.We focused our attention on Lpps release by Streptococcus pyogenes. This Gram-positive bacterium is an important human pathogen that causes a wide range of diseases from superficial and self-limiting infection, e.g. pharyngitis and impetigo, to more systemic or invasive diseases like necrotizing fasciitis and septicemia (17). Understanding the role of bacterial Lpps in mediating innate and acquired immunity can be instrumental for the therapy and prophylaxis of human S. pyogenes infections. In this study, we showed that in S. pyogenes Lpps are released into the growth medium within vesicle-like structures in minute amounts. Conditions weakening the bacterial cell wall, such as the addition of sublethal concentrations of penicillin to the bacterial growth medium enhanced this phenomenon and allowed the recovery of sufficient material to enable an in-depth characterization. Proteomic analysis of the vesicles revealed that they were almost exclusively constituted of Lpps. A total of 28 Lpps were identified, representing more than 72% of the Lpps predicted from the genome of the strain under investigation. In addition, multiple transmembrane domain proteins were not found in abundance associated to the vesicles, indicating that vesicles were not representative of the bacterial membrane. We defined these vesicles as Lipoprotein-rich Membrane Vesicles (LMVs).Common characteristics are shared between the LMVs and the ExPortal described for the first time by Rosch and Caparon (18). This asymmetric and distinct membrane microdomain has been reported to be enriched in anionic phospholipids and acts in promoting the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and the accessory factors required for their maturation (19–21). An association between ExPortal and peptidoglycan synthesis has also been reported (22). Similarly, LMVs are enriched in anionic phosphatidylglycerol, enzymes involved in protein maturation/secretion and cell wall biogenesis, suggesting that LMVs might derive from the ExPortal. Finally, we showed that LMVs do not induce TLR2 activation, indicating that the Lpps did not act as PAMPs when integrated into the LMVs. 相似文献
Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy. 相似文献
Lepidaploa belongs to tribe Vernonieae, one of the most complex tribes of Asteraceae, and the relationships within Lepidaploa and among related genera are poorly understood. Microcharacters may be of taxonomic value and may be used in the identification of taxa at different ranks. To evaluate the reliability of microcharacters as taxonomic markers in this group, we analysed the micromorphology of phyllaries, florets and cypselae in detail in 23 species of Lepidaploa .The species were studied using stereo, light, and scanning electron microscopy. Eight trichome types (eglandular and glandular) were observed on phyllaries, florets and cypselae, in addition to crystals, idioblasts and other microstructures. The results demonstrates that the ocurrence of different combinations of trichome types and crystals, presence of a stylar basal node, idioblasts and glandular apical anther appendages are highly useful to differentiate between related species of Lepidaploa and a diagnostic key using these characters is presented. However, these characters are not of much use to distinguish between closely related genera of Vernonieae since most characters appear homeoplasic and are found in representatives of different genera. 相似文献
The protein composition of the outer membrane of Gram-negative bacteria consists of about 20 immunochemically distinct proteins, termed outer membrane proteins (OMPs). Apart from their structural role, OMPs have been shown to have other functions, particularly with regard to transport, and have been classified as permeases and porins. Porins, during their interaction with the host, are immunogenic and also directly stimulate several cellular functions. Porins work both as molecules present on the bacterial surface and as molecules released by bacteria. Lipopolysaccharide and OMPs, the major structural macromolecular constituents of the outer membrane, carry out a fundamental role in the pathogenesis of Gram-negative infections. This brief review describes the multiple facets of the biological activities of porins both in vitro and in vivo, particularly focusing on their ability to induce the expression of cytokines and other factors that modulate cellular activities with either pathological or adaptive consequences. This brief discussion will focus on the signal transmission mechanisms induced by bacterial porins. 相似文献
Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP+) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP+, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.HMG-CoA reductase (HMGR) is the enzyme that catalyze the four-electron reductive deacylation of HMG-CoA to CoA and mevalonate (Fig. 1) (1). This reaction is the controlling step in the biosynthesis of sterols and isoprenoids (2, 3); hence, a large number of studies on the modulation of HMGR activity are continuously performed in the effort of developing new drugs in the treatment of hypercholesterolemic disorders (1).Open in a separate windowFig. 1.Schematic representation of HMGR enzymatic reaction.HMGR activity is conventionally assayed using elaborate radiochemical assay (4–9), chromatographic techniques coupled with mass spectrometry (10–15), or spectrophotometrically by monitoring the decrease in the absorbance of cofactor NADPH at 340 nm (16).Herein, as an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions. 相似文献
The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600+/-98 U l(-1) on day 13) and in the air-lift reactor (410+/-22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities. 相似文献
