Soluble Preparations Research Articles

Killer Ig-like Receptor 2DL4 Does Not Mediate NK Cell IFN-γ Responses to Soluble HLA-G Preparations

The MHC class Ib molecule HLA-G has previously been reported to be the ligand for the NK cell receptor killer Ig-like receptor (KIR)2DL4, but this remains controversial. In this study, we investigated IFN-γ production by freshly isolated NK cells in response to both soluble and solid-phase ligands, including anti-KIR2DL4 mAbs and rHLA-G. Although freshly isolated CD56(bright) NK cells produced IFN-γ in response to soluble HLA-G preparations, the response was found to be absolutely dependent on the presence of small numbers of contaminating CD56(-), CD14(-), CD11c(+) myeloid dendritic cells (mDCs). HLA-G tetramers bound only to the contaminating mDCs in the NK preparations, and Abs to KIR2DL4 and HLA-G did not block NK cell IFN-γ production. NK cells did not respond to plate-bound HLA-G. Freshly isolated NK cells also produced IFN-γ in response to unpurified soluble anti-KIR2DL4 mAb but not to low endotoxin affinity-purified Ab. The data suggest that previous reports of functional interactions between KIR2DL4 and HLA-G may have resulted from the use of purified NK cells that were contaminated with mDCs and HLA-G preparations that were contaminated with material capable of stimulating mDCs to produce cytokines that stimulate NK cells to produce IFN-γ.

Tolerance and the effect of high doses of wheat bran extract, containing arabinoxylan-oligosaccharides, and oligofructose on faecal output: a double-blind, randomised, placebo-controlled, cross-over trial.

Wheat bran extract (WBE) is a food-grade soluble fibre preparation that is highly enriched in arabinoxylan-oligosaccharides. In this placebo-controlled cross-over human intervention trial, tolerance to WBE as well as the effects of WBE on faecal parameters, including faecal output and bowel habits, were studied. After a 2-week run-in period, twenty healthy volunteers consumed WBE (15g/d in the first week, 30g/d in the second week), oligofructose (15g/d in the first week, 30g/d in the second week) and placebo (for 2 weeks) in a random order, with 2-week washout periods between each treatment period. Subjects collected a 72h stool sample for analysis of faecal output, stool pH and stool moisture concentration. Additionally, the volunteers completed questionnaires scoring occurrence frequency and distress severity of eighteen gastrointestinal (GI) symptoms. An overall GI symptom measure was calculated to analyse the overall effect of WBE and oligofructose on GI symptoms. Intake of both 30g/d WBE and 30g/d oligofructose lowered stool pH, indicative of increased colonic fermentation, and increased stool moisture concentration as compared with placebo intake. Intake of 30g/d oligofructose increased the overall GI symptom measure by 1·9-fold as compared with placebo intake. Intake of WBE at doses up to 30g/d did not affect the overall GI symptom measure. WBE exerts beneficial effects on stool characteristics and is well tolerated at up to 30g/d. Oligofructose exerts comparable beneficial effects on stool characteristics. However, intake of 30g/d oligofructose appears to cause GI discomfort to some extent.

Platelet-activating factor antagonists enhance intracellular degradation of amyloid-β42 in neurons via regulation of cholesterol ester hydrolases.

IntroductionThe progressive dementia that is characteristic of Alzheimer’s disease is associated with the accumulation of amyloid-beta (Aβ) peptides in extracellular plaques and within neurons. Aβ peptides are targeted to cholesterol-rich membrane micro-domains called lipid rafts. Observations that many raft proteins undertake recycling pathways that avoid the lysosomes suggest that the accumulation of Aβ in neurons may be related to Aβ targeting lipid rafts. Here we tested the hypothesis that the degradation of Aβ by neurons could be increased by drugs affecting raft formation.MethodsPrimary neurons were incubated with soluble Aβ preparations. The amounts of Aβ42 in neurons or specific cellular compartments were measured by enzyme-linked immunosorbent assay. The effects of drugs on the degradation of Aβ42 were studied.ResultsAβ42 was targeted to detergent-resistant, low-density membranes (lipid rafts), trafficked via a pathway that avoided the lysosomes, and was slowly degraded by neurons (half-life was greater than 5 days). The metabolism of Aβ42 was sensitive to pharmacological manipulation. In neurons treated with the cholesterol synthesis inhibitor squalestatin, less Aβ42 was found within rafts, greater amounts of Aβ42 were found in lysosomes, and the half-life of Aβ42 was reduced to less than 24 hours. Treatment with phospholipase A2 inhibitors or platelet-activating factor (PAF) antagonists had the same effects on Aβ42 metabolism in neurons as squalestatin. PAF receptors were concentrated in the endoplasmic reticulum (ER) along with enzymes that constitute the cholesterol ester cycle. The addition of PAF to ER membranes triggered activation of cholesterol ester hydrolases and the release of cholesterol from stores of cholesterol esters. An inhibitor of cholesterol ester hydrolases (diethylumbelliferyl phosphate) also increased the degradation of Aβ42 in neurons.ConclusionsWe conclude that the targeting of Aβ42 to rafts in normal cells is a factor that affects its degradation. Critically, pharmacological manipulation of neurons can significantly increase Aβ42 degradation. These results are consistent with the hypothesis that the Aβ-induced production of PAF controls a cholesterol-sensitive pathway that affects the cellular localization and hence the fate of Aβ42 in neurons.

Improvement of P. aeruginosa 42A2 lipase preparations for FAMEs production, both in immobilized and soluble form

Abstract Lipase application in industrial biocatalytic processes is a topic of great interest due to their versatility and mild conditions of use. Many lipases from different sources have been studied, improved and used in a variety of chemical reactions. One of the most recent applications is the transesterification of vegetal triglycerides with methanol to produce fatty acid methyl esters (FAMEs), aimed at the synthesis of enzymatic biodiesel. In general, this process has been performed using immobilized lipases as biocatalysts. However, last tendencies are promoting the use of soluble enzyme preparations to reduce the cost of the enzymatic preparations and to make the whole process more competitive with respect to the chemical systems. In this work, we evaluated both, the soluble and immobilized preparations of two cold-adapted lipases: LipC from Pseudomonas aeruginosa 42A2, and its thermo stable variant LipCmut, which were over expressed and produced using a low cost protocol. Several conditions of soybean oil transesterification with such lipase preparations were tested to evaluate the enzymatic FAMEs production process.

Heparin Modulates the Mitogenic Activity of Fibroblast Growth Factor by Inducing Dimerization of its Receptor. A 3D View by Using NMR

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.

Aroma enhancement in wines using co-immobilized Aspergillus niger glycosidases

A major fraction of monoterpenes and norisoprenoids in young wines is conjugated to sugars representing a significant reservoir of aromatic precursors. To promote their release, β-glucosidase, α-arabinosidase, and α-rhamnosidase from a commercial Aspergillus niger preparation, were immobilized onto acrylic beads. The aim of this work was the development and application of an immobilized biocatalyst, due to the well-known advantages over soluble enzyme preparations: control of the reaction progress and preparation of enzyme-free products. In addition, the obtained derivative showed increased stability in simile wine conditions. After the treatment of Muscat wine with the biocatalyst for 20days, free monoterpenes increased significantly (from 1119 to 2132μg/L, p<0.01) with respect to the control wine. Geraniol was increased 3,4-fold over its flavor thresholds, and accordingly its impact on sensorial properties was very relevant: nine of ten judges considered treated wine more intense in fruit and floral notes.

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection.

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.

Proteomic analysis of schistosomiasis japonica vaccine candidate antigens recognized by UV-attenuated cercariae-immunized porcine serum IgG2

Many studies have showed that the radiation-attenuated cercariae (RAC) vaccine could induce the high protection of laboratory animals to resist the schistosoma infection by cellular and humoral mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG2 antibody from RAC-vaccinated pigs or vaccination and challenge pigs. The antigens from the schistosomal soluble worm antigen preparation (SWAP) recognized by the porcine IgG2 antibody were obtained using immunoprecipitation technique. These antigens were separated by 2-D electrophoresis, and 116 spots were successfully identified by MALDI-TOF MS from about 400 putative spots in gels. Among these spots, 113 spots could match to the Schistosoma japonicum. These identified proteins in four groups were classified by Gene Ontology (Go) database, and the mainly functions of these proteins were involved in binding, catalytic activity (thioredoxin peroxidase-2, et al.), signal transduction class (MAP Kinase, et al.), cell process (the heat shock 70-kDa protein 9B, et al.), and the intracellular component (tektin, et al.). Our methods suggested that it was possible to pull-down the interesting proteins recognized by specific antibodies. Our results may provide new clues for exploring the mechanism of high protection induced by RAC and shed some light on the research for anti-schistosomiasis japonica vaccine.

Schistosoma mansoni in a low-prevalence area in Brazil: the importance of additional methods for the diagnosis of hard-to-detect individual carriers by low-cost immunological assays

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni

The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group. We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings. ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg. Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples*[S

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-β (Aβ) levels. Evidence suggests physical interactions between apoE and Aβ are partially responsible for these functional effects. However, the apoE/Aβ complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aβ in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aβ and an increase in soluble Aβ, specifically oligomeric Aβ (oAβ), are associated with APOE4 and AD. Previously, soluble Aβ42 and oAβ levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aβ levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aβ levels isoform-specifically modulate soluble oAβ clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aβ levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aβ levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aβ42 levels decreased in AD patients compared with controls, oAβ levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aβ modulates oAβ levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

Efficacy of L-carnosine against Schistosoma mansoni antigens in liver, heart, kidney and brain of rabbits

Article history: The present investigation was an attempt to study the enhancememt effect of L-carnosine (beta alanyl-l-histidine) on the influence of vaccination on healthy (non-infected) rabbits treated with Schistosoma mansoni egg antigens, cercariae antigens, and worms antigens as protective agents against infection. This study involved individual injection of three Schistosoma mansoni antigens: soluble egg antigen (SEA), cercarial antigen preparation (CAP) and soluble worm antigen preparation (SWAP), in three rabbit groups, respectively. Three other groups each received the same specific antigen in conjunction with the administration of L-carnosine, biochemical parameters including DNA, RNA, DNA/RNA ratio concentrations in addition ATPase, and acetyl cholinesterase activities were measured in liver, heart, kidney and brain in all groups. Elevation in most parameters was observed in the immunized groups. Carnosine treatment of rabbit groups immunized with SEA, CAP and SWAP in comparison to the non-carnosine-treated immunized groups resulted in amelioration the changes of DNA, RNA, ATPase and acetyl cholinesterase activities in most groups. L-carnosine has a beneficial effect in the amelioration of the changes in biochemical parameters as a result of S. mansoni antigen immunization.

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay. The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora β-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

Oral Administration of Soluble β-Glucan Preparation from the Cauliflower Mushroom, Sparassis crispa (Higher Basidiomycetes) Modulated Cytokine Production in Mice

Soluble β-glucan preparation from the cold NaOH extract of Sparassis crispa (SCG) is a six-branched 1,3-β-D-glucan that is a major cell-wall structural component in fungi. Leukocytes from DBA/2 mice are highly sensitive to SCG, producing cytokines in vitro. We previously reported that the intraperitoneal (i.p.) administration of β-glucan decreased cytokine induction by SCG in vitro in DBA/2 mice. In this study, we examined the effects of the oral (p.o.) administration of polysaccharide fractions extracted from S. crispa, using hot water (SCHWE), a β-glucan from S. crispa, to DBA/2 mice on cytokine induction by SCG in the spleen in vitro. The level of induction of IFN-γ and GM-CSF by SCG was significantly increased in SCHWE-treated mice. This activity was more clearly observed when chlorpromazine was administered as a pretreatment in SCHWE-treated mice. The production of GM-CSF, IFN-γ, and IL-6 by immune cells in Peyer's patches was higher in SCHWE-treated mice than in control mice. These results suggest that orally administered β-glucan may modulate cytokine induction by SCG in the spleen through the activation of Peyer's patches.

Genital Melanocytic Nevus Arising in a Background of Lichen Sclerosus in a 7-Year-Old Female

Genital melanocytic nevus represents a distinct form of melanocytic proliferation, which can exhibit significant atypia, both clinically and histologically. In a background of lichen sclerosus (LS), the histologic changes could be misconstrued as indicative of malignant melanoma. We present herein a case of the atypical genital nevus of childhood complicated by LS, and a review of the literature is performed. Tissue was available for routine light microscopy and immunohistochemical evaluation to assess the expression of soluble adenylyl cyclase. Fluorescent in situ hybridization studies were conducted to assess for abnormalities in Myb1, CCND1, RREB1 and CEP6. The specimen showed an atypical compound melanocytic proliferation arising in a background of LS. The lesion exhibited significant architectural atypia based on the high-density confluent nature of the junctional melanocytic proliferation with epidermal effacement, rare areas of pagetoid ascent, and the heavily pigmented epithelioid quality of the melanocytes. Fluorescent in situ hybridization studies were normal. The soluble adenylyl cyclase antibody preparation demonstrated a benign nevus-like pattern. The lesion was felt to represent an atypical genital melanocytic nevus, which can resemble a partially regressed melanoma in a background of LS. It is very important for the pathologist to be aware of this entity to avoid misdiagnosis.

Adult worm‐specific IgE/IgG4 balance is associated with low infection levels of Schistosoma mansoni in an endemic area

Field studies have suggested an immune-mediated mechanism associated with resistance to Schistosoma mansoni infection. Overall, levels of specific IgE have been correlated with resistance to infection, whereas levels of IgG4 have been associated with susceptibility. This study aimed to evaluate serum levels of soluble adult worm antigen preparation (SWAP)-specific IgE and IgG4 in relation to current infection in a large casuistic of individuals living in an endemic area of schistosomiasis in Bahia, Brazil. The prevalence of S.mansoni infection was 37·7% and the mean parasite burden was 55·4 (0-2100) epg/faeces. There was no significant difference in the levels of SWAP-specific IgE in individuals with different parasite burden, whereas high producers of parasite-specific IgG4 presented higher parasite burden when compared to low IgG4 producers. Additionally, S.mansoni parasite load was positively correlated with the levels of specific IgG4 or total IgE. No significant correlation was observed between parasite burden and SWAP-specific IgE. Nevertheless, SWAP-specific IgE/IgG4 ratio was higher in uninfected or lightly infected individuals (1-99 epg/faeces) than in heavily infected ones (≥400 epg/feces). These findings highlight the important role of IgE/IgG4 ratio in the resistance to infection, which could be useful for further studies in schistosomiasis vaccine candidates.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum

BackgroundThe complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.Methodology/Principal FindingsTwo monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.Conclusions/SignificanceIn this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.

P4‐034: Alzheimer's disease human brain beta‐amyloid‐containing extracts inhibit hippocampal long‐term potentiation in rats in vivo: Relationship between soluble and insoluble preparations

P4‐034: Alzheimer's disease human brain beta‐amyloid‐containing extracts inhibit hippocampal long‐term potentiation in rats <i>in vivo</i>: Relationship between soluble and insoluble preparations

Benzaldehyde is a precursor of phenylpropylamino alkaloids as revealed by targeted metabolic profiling and comparative biochemical analyses in Ephedra spp.

Ephedrine and pseudoephedrine are phenylpropylamino alkaloids widely used in modern medicine. Some Ephedra species such as E. sinica Stapf (Ephedraceae), a widely used Chinese medicinal plant (Chinese name: Ma Huang), accumulate ephedrine alkaloids as active constituents. Other Ephedra species, such as E. foeminea Forssk. (syn. E. campylopoda C.A. Mey) lack ephedrine alkaloids and their postulated metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone. Solid-phase microextraction analysis of freshly picked young E. sinica and E. foeminea stems revealed the presence of increased benzaldehyde levels in E. foeminea, whereas 1-phenylpropane-1,2-dione was detected only in E. sinica. Soluble protein preparations from E. sinica and E. foeminea stems catalyzed the conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione. The activity, termed benzaldehyde carboxyligase (BCL) required the presence of magnesium and thiamine pyrophosphate and was 40 times higher in E. sinica as compared to E. foeminea. The distribution patterns of BCL activity in E. sinica tissues correlates well with the distribution pattern of the ephedrine alkaloids. (S)-Cathinone reductase enzymatic activities generating (1R,2S)-norephedrine and (1S,1R)-norephedrine were significantly higher in E. sinica relative to the levels displayed by E. foeminea. Surprisingly, (1R,2S)-norephedrine N-methyltransferase activity which is a downstream enzyme in ephedrine biosynthesis was significantly higher in E. foeminea than in E. sinica. Our studies further support that benzaldehyde is the metabolic precursor to phenylpropylamino alkaloids in E. sinica.