Amyloid ? peptides (A?) aggregation is considered as a crucial pathological biomarker of Alzheimer's disease (AD). It was found that A? and heme can form A?-heme complex, which results in increase of heme pseudo-peroxidase activity. Recently, we found that the increase of pseudo-peroxidase activity would induce an elevated tyrosine nitration on A? in the presence of nitrite and hydrogen peroxide. However, the nature of tyrosine nitration of A? and its physiologic significance are still unknown. In this study, we revealed that A?1-40 can be nitrated in vitro by binding to heme with the presence of nitrite and hydrogen peroxide. Moreover, we found that tyrosine nitration had little effect on A?1-40's binding activity with heme. TMB assay also revealed that the peroxidase activity of heme-A?1-40Y10(3N)T (tyrosine 10 was replaced with 3-nitrtotyrosine in A?1-40) complex was moderately increased compared with that of heme-A?1-40 complex. Furthermore, Thioflavin T fluorescence and transmission electron microscopic characterization indicated that tyrosine nitration significantly decreased the aggregation of A?1-40. In addition, cytotoxicity test verified that the cytotoxicity of wild type A?1-40 was more toxic than A?1-40Y10(3N)T. These results suggested that nitration of A?1-40 might be an A? detoxicant process and a compensatory reaction to nitrative stress. Our findings may lead to a detailed understanding of the function of A?1-40 and would be helpful in preventing and curing AD.
Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10?40 cm), which indicated that the pollution is more serious in recent decades.
Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71.
Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent for tomato gray mold. The genome of B. subtilis strain BAB-1 was fully sequenced and annotated, genes encoding the antifungal active compound were identified, and multiple sets of regulatory systems were found in the genome.
Arsenic (As) is ubiquitous in the environment in the carcinogenic inorganic forms, posing risks to human health in many parts of the world. Many microorganisms have evolved a series of mechanisms to cope with inorganic arsenic in their growth media such as transforming As compounds into volatile derivatives. Bio-volatilization of As has been suggested to play an important role in global As biogeochemical cycling, and can also be explored as a potential method for arsenic bioremediation. This review aims to provide an overview of the quality and quantity of As volatilization by fungi, bacteria, microalga and protozoans. Arsenic bio-volatilization is influenced by both biotic and abiotic factors that can be manipulated/elucidated for the purpose of As bioremediation. Since As bio-volatilization is a resurgent topic for both biogeochemistry and environmental health, our review serves as a concept paper for future research directions.
Misfolded protein amyloid-beta protein (A?) and tau protein are two high hallmarks of Alzheimer's disease (AD), representing significant targets in treating AD. Researches on mechanisms of the two proteins inducing neuron dysfunctions provide therapeutic strategies of AD, including inhibition of A? production and aggregation, acceleration of A? clearance as well as reduction of tau hyperphosphorylation. Proteoglycans (PGs) consist of a core protein and glycosaminoglycans (GAGs) chains, with enormous structural diversity due to variation in the core protein, the number of GAGs chains as well as extent and position of sulfation. Considerable evidences have indicated that PGs and GAGs play important roles in A? and tau processing. Numbers of GAGs and analogues have potential therapeutic function in AD. In this Review, we focus on the relationship of PGs and GAGs with misfolded proteins in AD and their potential therapeutic implications.
High-fat, low-carbohydrate diets (HFLCD) are often eaten by humans for a variety of reasons, but the effects of such diets on the heart are incompletely understood. We evaluated the impact of HFLCD on myocardial ischemia/reperfusion (I/R) using an in vivo model of left anterior descending coronary artery ligation. Sprague-Dawley rats (300 g) were fed HFLCD (60% calories fat, 30% protein, 10% carbohydrate) or control (CONT; 16% fat, 19% protein, 65% carbohydrate) diet for 2 wk and then underwent open chest I/R. At baseline (preischemia), diet did not affect left ventricular (LV) systolic and diastolic function. Oil red O staining revealed presence of lipid in the heart with HFLCD but not in CONT. Following I/R, recovery of LV function was decreased in HFLCD. HFLCD hearts exhibited decreased ATP synthase and increased uncoupling protein-3 gene and protein expression. HFLCD downregulated mitochondrial fusion proteins and upregulated fission proteins and store-operated Ca(2+) channel proteins. HFLCD led to increased death during I/R; 6 of 22 CONT rats and 16 of 26 HFLCD rats died due to ventricular arrhythmias and hemodynamic shock. In surviving rats, HFLCD led to larger infarct size. We concluded that in vivo HFLCD does not affect nonischemic LV function but leads to greater myocardial injury during I/R, with increased risk of death by pump failure and ventricular arrhythmias, which might be associated with altered cardiac energetics, mitochondrial fission/fusion dynamics, and store-operated Ca(2+) channel expression.
Urocortin-2 (UCN2) is cardioprotective in ischemia/reperfusion injury (I/R) through short-lived activation of ERK1/2. Key factors involved in I/R, e.g. apoptosis, mitochondrial damage, p38 kinase, and Bcl-2 family, have not been well-investigated in UCN2-induced cardioprotection. We assessed the role of p38-MAPK in anti-apoptotic Bcl-2 signaling and mitochondrial stabilization as a putative mechanisms in UCN2-induced cardioprotection. Isolated hearts from adult Sprague-Dawley rats and cultured H9c2 cells were subjected to I/R protocols with or without 10 nM UCN2 treatment. The effect of a specific p38 inhibitor SB202190 was tested in H9c2 cells. Cardiac function, LDH release, and mitochondrial membrane potential (MMP) were used to assess the degree of myocardial injury in hearts and H9c2 cells. Post-perfusion, hearts were collected for Western blot analyses or mitochondria/cytosol isolation to analyze p38 activation and Bcl-2 family members. UCN2 treatment improved rate-pressure product (58 ± 5 vs. 31 ± 4 % of Baseline; P < 0.05) and decreased LDH release (20 ± 9 vs. 90 ± 40 mU/ml LDH, P < 0.01) at the end of 60 min reperfusion. UCN2 reduced phospho-p38 levels and Bax activation. UCN2 increased the expression of Bcl-2 and inhibited the accumulation of p-Bim. With additional experiments, it was confirmed that UCN2 increases the phosphorylation of ERK1/2 in the early phase of UCN2 treatment and increases the overshot recovery of ERK1/2 phosphorylation during reperfusion. UCN2 and SB202190 partially prevented the loss of MMP induced by I/R. However, combined treatment with UCN2 and SB202190 did not provide additive benefit. UCN2 is cardioprotective in I/R in association with reduced phosphorylation of p38 together with the increased ERK1/2 activation and increased Bcl-2 family member pro-survival signaling. These changes may stabilize cardiac mitochondria, similar to p38 inhibitors, as part of a pro-survival mechanism during I/R.
The use of self-assembled biomacromolecules in the development of functional bionanocomposite foams is one of the best lessons learned from nature. Here, we show that monolithic, flexible and porous zinc oxide bionanocomposite foams with a hierarchical architecture can be assembled through the mediation of bacterial cellulose. The assembly is achieved by controlled hydrolysis and solvothermal crystallization using a bacterial cellulose aerogel as a template in a non-aqueous polar medium. The bionanocomposite foam with a maximum zinc oxide loading of 70 wt% is constructed of intimately packed spheres of aggregated zinc oxide nanocrystals exhibiting a BET surface area of 92 m(2) g(-1). The zinc oxide bionanocomposite foams show excellent antibacterial activity, which give them potential value as self-supporting wound dressing and water sterilization materials.
Inhibition of A? aggregation and attenuation of its cytotoxicity are considered to valuable therapeutics for Alzheimer's disease (AD). Here, a glucan named as LJW0F2 was purified from flowers of Lonicera japonica Thunb. Using monosaccharides composition analysis, methylation analysis, IR and NMR spectroscopy, this polysaccharide was elucidated to be an ?-D-(1?4)-glucan with an ?-(1?4) linked branch attached to the C-6 position. Its inhibitory effect on A?42 aggregation was measured by fluorescence spectroscopic analysis with thioflavine T (ThT) and atomic force microscopy (AFM). We showed that polysaccharide LJW0F2 could inhibit A?42 aggregation in a dose-dependent-manner. Besides, LJW0F2 could attenuate the cytotoxicity induced by A?42 aggregation in SH-SY5Y neuroblastoma cells. To the best of our knowledge, this was the first report that the exogenous plant-derived polysaccharide might block A?42 aggregation directly and reduce its toxicity in SH-SY5Y cells.
Insulin-like growth factor-1 (IGF-1) is a brain-specific multifunctional protein involved in neuronal polarity and axonal guidance. Mature IGF-1 triggers three enzymes, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphoinositide phospholipase C-? (PLC-?), which are its predominant downstream regulators. The PI3K-AKT signaling pathway is upstream of the mammalian target of rapamycin (mTOR), which is of great importance in the induction of autophagy. However, whether the neuroprotective effect of IGF-1 against excitotoxicity is mediated by autophagy through the PI3K/AKT/mTOR pathway remains to be elucidated. The induction of autophagy following NMDA treatment was determined by microtubule-associated protein light chain 3 (LC3) conversion and the result of this autophagy was assessed by monitoring the cleavage of caspase 3 in cultured hippocampal neurons. Cell viability was determined using MTT and LDH assay, and PI-staining was used to estimate the fate of autophagy and the protective effect of IGF-1. In addition, IGF-1 was found to decrease autophagy induced by NMDA using transmission electron microscopy and MDC staining. The protective effect of IGF-1 against autophagy was accompanied with up-regulation of phospho-AKT (p-AKT) and phospho-mTOR (p-mTOR), which was blocked by the inhibitor of PI3K. At the same time, the activation of NR2B resulting in the down-regulation of p-AKT and p-mTOR was blocked by IGF-1. Together, these data suggest that NMDA induces the autophagy, followed by apoptosis in cultured hippocampal neurons, and that IGF-1 can block this effect via inhibition of NR2B receptors and activation of the PI3K-AKT-mTOR pathway.
Metastasis and thrombosis are serious threats to cancer patients and generally associated with poor prognosis. The elusive mechanisms underlying the pathogenesis of metastasis and thrombosis have been subjects of extensive investigations. The presence of circulating tumor cells (CTCs) is closely related to tumor metastasis, and these cells play an important role in thrombosis in cancer patients. In this review, we describe the latest findings on the role of CTCs in tumor metastasis and cancer-related thrombosis and the regulatory role of microRNAs in CTCs and thrombosis. Additionally, we discuss anticoagulant-based strategies for the prevention of thrombosis and reduction of cancer metastasis and the potential to translate current knowledge on these strategies to the treatment of cancer.
The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is recognized as a key mediator of tumorigenesis in many human epithelial tumors. Erlotinib is tyrosine kinase inhibitor approved by FDA for use in oncology. It inhibits the intracellular phosphorylation of tyrosine kinase associated with the EGFR to restrain the development of the tumor. To investigate the antitumor effect of erlotinib at different dosing times and the underlying molecular mechanism via the PI3K/AKT pathway, we established a mouse model of Lewis lung cancer xenografts. The tumor-bearing mice were housed four or five per cage under standardized light-dark cycle conditions (light on at 7:00 AM, 500 Lux, off at 7:00 PM, 0 Lux) with food and water provided ad libitum. The mice were observed for quality of life, their body weight and tumor volume measured, and the tumor growth curves drawn. After being bled, the mice were sacrificed by cervical dislocation. The tumor masses were removed at different time points and weighed. The mRNA expression of EGFR, AKT, Cyclin D1 and CDK-4 were assayed by quantitative real-time PCR (qRT-PCR). Protein expression levels of AKT, P-AKT and Cyclin D1 were determined by Western blot analysis. The results suggest that erlotinib has a significant antitumor effect on xenografts of non-small cell lung cancer in mice, and its efficacy and toxicity is dependent on the time of day of administration. Its molecular mechanism of action might be related to the EGFR-AKT-Cyclin D1-CDK-4 pathway which plays a crucial role in the development of pathology. Therefore, our findings suggest that the time of day of administration of Erlotinib may be a clinically important variable.
Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3) vs. 10±3×10(3) mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data suggest mTORC2 phosphorylation of Akt is a key mediator of H2S-induced cardioprotection in I/R.
Bacillus subtilis strain NCD-2 is strongly antagonistic toward phytopathogenic fungi, and functions as an excellent biocontrol agent for cotton soil-borne diseases. The aims of this study were to characterize the main active antifungal compound from strain NCD-2 and clarify its role in suppressing cotton damping-off disease. Strain NCD-2 and lipopeptide extract prepared from an NCD-2 culture strongly inhibited the growth of Rhizoctonia solani in vitro. The lipopeptides of strain NCD-2 were separated by fast protein liquid chromatography (FPLC) and the antifungal compound was identified as a cluster of fengycin homologs analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A fengycin-deficient mutant was obtained by in-frame deletion of the fengycin synthetase gene in B. subtilis NCD-2. Compared with the wild-type strain, this mutant showed decreased abilities to inhibit the growth of R. solani in vitro and to suppress cotton damping-off disease in vivo. Studies showed that the population of fengycin-deficient mutant was almost same as that of the wild-type NCD-2 strain in the cotton rhizosphere. However, the population of R. solani in the cotton rhizosphere colonized by the fengycin-deficient mutant was twice that in the cotton rhizosphere colonized by the NCD-2 wild-type strain. This study demonstrated that fengycin-type lipopeptides are the main antifungal active compounds produced by B. subtilis NCD-2. These compounds play a major role in restricting the population of R. solani in the cotton rhizosphere and in suppressing cotton damping-off disease.
Pancreatic ductal adenocarcinoma (PDAC) has one of the poorest prognoses among all cancers. Over the past several decades, investigators have made great advances in the research of PDAC pathogenesis. Importantly, identification of pancreatic cancer stem cells (PCSCs) in pancreatic cancer cases has increased our understanding of PDAC biology and therapy. PCSCs are responsible for pancreatic tumorigenesis and tumor progression via a number of mechanisms, including extensive proliferation, self-renewal, high tumorigenic ability, high propensity for invasiveness and metastasis, and resistance to conventional treatment. Furthermore, emerging evidence suggests that PCSCs are involved in the malignant transformation of pancreatic intraepithelial neoplasia. The molecular mechanisms that control PCSCs are related to alterations of various signaling pathways, for instance, Hedgehog, Notch, Wnt, B-cell-specific Moloney murine leukemia virus insertion site 1, phosphoinositide 3-kinase/AKT, and Nodal/Activin. Also, authors have reported that the proliferation-specific transcriptional factor Forkhead box protein M1 is involved in PCSC self-renewal and proliferation. In this review, we describe the current knowledge about the signaling pathways related to PCSCs and the early stages of PDAC development, highlighting the pivotal roles of Forkhead box protein M1 in PCSCs and their impacts on the development and progression of pancreatic intraepithelial neoplasia.
The La-related protein 1 (LARP1) has been found to be a RNA binding protein and was related to spermatogenesis, embryogenesis and cell-cycle progression. The aim of this study was to investigate the prognostic value of LARP1 in hepatocellular carcinoma (HCC).
Cyclopropyl Grignard reagents react with carbonyl compounds in the presence of diethyl phosphite to give homoallylic bromides. The reaction is effectively carried out under mild conditions in a one-pot fashion with moderate to good yields.
A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the hosts nitrogen use efficiency.
Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.
Cystathionine ?-lyase (CSE) is one of the major enzymes producing hydrogen sulfide (H2S) in lungs, participating in the regulation of respiratory functions. The role of CSE-derived H2S in eosinophil-dominant inflammation in allergic diseases has been unclear. The objective of this study was to explore the protective role of H2S against allergen-induced airway hyperresponsiveness (AHR) and inflammation. CSE expression and H2S production rate were assessed in mouse lung tissues with ovalbumin (OVA)-induced acute asthma. AHR, airway inflammation, and Th2 response in wild-type (WT) mice were compared with those in CSE gene knockout (KO) mice. The effect of NaHS, an exogenous H2S donor, was also evaluated on these parameters. CSE expression was absent and H2S production rate was significantly lower in the lungs of CSE KO mice when compared with WT littermates. OVA challenge decreased lung CSE expression and H2S production in WT mice. CSE deficiency resulted in aggravated AHR, increased airway inflammation, and elevated levels of Th2 cytokines such as IL-5, IL-13, and eotaxin-1 in bronchoalveolar lavage fluid after OVA challenge. The aforementioned alterations were reversed by exogenous H2S treatment. More importantly, NaHS supplement rescued CSE KO mice from the aggravated pathological process of asthma. The CSE/H2S system plays a critical protective role in the development of asthma. A new therapeutic potential for asthma via targeting CSE/H2S metabolism is indicated.
Asthma is a chronic inflammatory disease, with hyper-responsive bronchoconstriction and airway remodelling, leading to extensive airway narrowing. The regulation of airway responsiveness and inflammation by endogenous hydrogen sulfide (H(2)S) during the pathogenic development of asthma has been suggested. Hydrogen sulfide can be produced in the lung and airway tissues via the actions of two H(2)S-generating enzymes, cystathionine ?-synthase (CBS) and/or cystathionine ?-lyase (CSE). The abnormal metabolism and function of H(2)S have been reported in experimental animals with asthma, especially ovalbumin-induced rat or mouse models. In patients with asthma, serum H(2)S levels are significantly reduced. Supplementation with exogenous H(2)S has been shown to mitigate the severity of asthma in experimental animals. It is hypothesized that decreased H(2)S production in the lung and airway tissues may be used as an early detection biomarker, and H(2)S-based therapy would represent a new treatment strategy for asthma. Major challenges for establishing the diagnostic and treatment values of H(2)S include the differential expression of CSE and CBS along the airway and their changes during asthma, the effects of H(2)S on bronchoconstriction and airway remodelling, as well as the underlying mechanisms, and the detection of the changes in H(2)S levels in airway tissues and in exhaled air.
Ammonium-based alkali-catalyzed ?-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with ?1,3-linked cores. In contrast to the previously studied ?1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the ?1,3-linkage is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages (?1,2-, ?1,2-, ?1,3-, ?1,3-, ?1,4-, ?1,4-, ?1,6-, and ?1,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O- and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O- and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glycan release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies.
The glycosylation profile of a recombinant protein is important because glycan moieties can play a significant role in the biological properties of the glycoprotein. Here we determined the site-specific N-glycosylation profile of human lactoferrin (hLF) and recombinant human lactoferrin (rhLF) expressed in the milk of transgenic cloned cattle. We used combined approaches of monosaccharide composition analysis, lectin blot, glycan permethylation and sequential exoglycosidase digestion and analyzed samples using high-performance ion chromatography and mass spectrometry (MS). N-glycans from hLF are comprised entirely of highly branched, highly sialylated and highly fucosylated complex-type structures, and many contain Lewis(x) epitopes. Six of these structures are reported here for the first time. However, N-glycans from rhLF are of the high mannose-, hybrid- and complex-type structures, with less N-acetylneuraminic acid and fucose. Some contain a terminal N-acetylgalactosamine-N-acetylglucosamine (LacdiNAc) disaccharide sequence. Monosaccharide composition analysis of rhLF revealed small amounts of N-glycolylneuraminic acid, which were not detected by MS. hLF and rhLF appear to be glycosylated at the same two sites: Asn138 and Asn479. The third putative glycosylation site, at Asn624, is unglycosylated in both hLF and rhLF. The relative abundance of each N-glycan at each site was also determined. The different N-glycosylation profile of rhLF when compared with that of hLF is in consistent with the widely held view that glycosylation is species- and tissue/cell-specific. These data provide an important foundation for further studies of glycan structure/function relationships for hLF and rhLF and help to better understand the glycosylation mechanism in bovine mammary epithelial cells.
The present study tested the hypothesis that the effects of mental imagery on subsequent perception occur at a later matching stage in perceptual identification, but not in the early perceptual stage as in perceptual detection. The behavioral results suggested that the effect of visual imagery on visual identification is content-specific, i.e., imagining a congruent face facilitates face identification, whereas a mismatch between imagery and perception leads to an interference effect. More importantly, the ERP results revealed that a more negative N2 response to the subsequent visual face stimuli was elicited over fronto-central sites in the mismatch and no-imagery conditions as compared to that in the match condition, with the early P1 and N170 components independent of manipulations. The latency and distribution of the neural effects demonstrate that the matching step, but not the earlier perceptual process, is affected by the preceding visual imagery in the context of face identification. We discuss these results in a broader context that the imagery-perception interaction may depend on task demand.
Chemoresistance is the main obstacle encountered in cancer treatment and is frequently associated with multidrug resistance (MDR). Astragaloside is a saponin which is widely used in traditional Chinese medicine. It has been reported that Astragaloside has antitumour effects on hepatocellular carcinoma Bel-7402 cells in vitro and in vivo. The purpose of this study was to examine the effects of Astragaloside II on the reversal of MDR and its molecular mechanism in vitro.
Hyperglycemia-induced vascular cell apoptosis is a seminal early event in diabetic retinopathy. Prolonged hyperglycemia is known to increase intracellular cytosolic free calcium ([Ca(2+)]i) in retinal vascular endothelial cells (RECs), suggesting that [Ca(2+)]i is a critical trigger for microvascular degeneration. This study aims to elucidate Ca(2+)-dependent signaling mechanisms that mediate hyperglycemia-induced apoptosis in RECs.
Existing spatial independent component analysis (ICA) methods for multi-subject fMRI datasets have mainly focused on detecting common components across subjects, under the assumption that all the subjects in a group share the same (identical) components. However, as a data-driven approach, ICA could potentially serve as an exploratory tool at multi-subject level, and help us uncover inter-subject differences in patterns of connectivity (e.g., find subtypes in patient populations). In this work, we propose a methodology named gRAICAR that exploits the data-driven nature of ICA to allow discovery of sub-groupings of subjects based on reproducibility of their ICA components. This technique allows us not only to find highly reproducible common components across subjects but also to explore (without a priori subject groupings) components that could classify all subjects into sub-groups. gRAICAR generalizes the reproducibility framework previously developed for single subjects (Ranking and averaging independent component analysis by reproducibility-RAICAR-Yang et al., Hum Brain Mapp, 2008) to multiple-subject analysis. For each group-level component, gRAICAR generates its reproducibility matrix and further computes two metrics, inter-subject consistency and intra-subject reliability, to characterize inter-subject variability and reflect contributions from individual subjects. Nonparametric tests are employed to examine the significance of both the inter-subject consistency and the separation of subject groups reflected in the component. Our validations based on simulated and experimental resting-state fMRI datasets demonstrated the advantage of gRAICAR in extracting features reflecting potential subject groupings. It may facilitate discovery of the underlying brain functional networks with substantial potential to inform our understandings of development, neurodegenerative conditions, and psychiatric disorders.
We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 ?M 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 ?M 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 ?M 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 ?M 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.