Search

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

The concentrations of 3,4-dihydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid, epicatechin, gallic acid, p-coumaric acid, quercetin, rutin, ferulic acid, and the major metals in graviola (Annona muricata), atemoya (A. squamosa x A. cherimola), fruta do conde (A. squamosa), biriba (Rollinia mucosa), and the North American pawpaw (Asimina triloba) were determined by UPLC-ESI (−)-MS/MS. It enabled the identification and quantification of phenolic compounds. Catechin was only found in atemoya, at a concentration of 38.6 μg/g-dw. Only 3,4-dihydroxybenzoic acid was found in the fruit pulps of all five fruits analyzed. Atemoya stands out for not only having catechin but also for having much more epicatechin (239 μg/g-dw) than the other fruits. At the same time, graviola had more p-coumaric acid (62.6 μg/g-dw), and the North American pawpaw had more chlorogenic acid (48.1 μg/g-dw) than the other fruits. Metals were determined by ICP equipped with axially viewed plasma. All five fruit pulps had relatively high levels of potassium, with concentrations ranging from 7640 to 15,000 μg/g-dw, with pawpaw being the lowest and atemoya being the highest. The concentrations of other metals ranged from Ca 547 to 1110, Na 14.3 to 123, P 1210 to 1690, Mg 472 to 980, Mn 1.86 to 5.27, and Zn 5.55 to 7.32 μg/g-dw. All five fruits in the Annonaceae family that were analyzed in this study have several phenolic compounds in them and were good sources of potassium, calcium, phosphorus, and magnesium.

Purpose White matter fibre development in childhood involves dynamic changes to microstructural organisation driven by increasing axon diameter, density, and myelination. However, there is a lack of longitudinal studies that have quantified advanced diffusion metrics to identify regions of accelerated fibre maturation, particularly across the early pubertal period. We applied a novel longitudinal fixel-based analysis (FBA) framework, in order to estimate microscopic and macroscopic white matter changes over time. Methods Diffusion-weighted imaging (DWI) data were acquired for 59 typically developing children (27 female) aged 9–13 years at two time-points approximately 16 months apart (time-point 1: 10.4 ± 0.4 years, time-point 2: 11.7 ± 0.5 years). Whole brain FBA was performed using the connectivity-based fixel enhancement method, to assess longitudinal changes in fibre microscopic density and macroscopic morphological measures, and how these changes are related to sex, pubertal stage, and pubertal progression. Follow-up analyses were performed in sub-regions of the corpus callosum to confirm the main findings using a Bayesian repeated measures approach. Results There was a statistically significant increase in fibre density over time localised to medial and posterior commissural and association fibres, including the forceps major and bilateral superior longitudinal fasciculus. Increases in fibre cross-section were substantially more widespread. The rate of fibre development was not associated with age or sex. In addition, there was no significant relationship between pubertal stage or progression and longitudinal fibre development over time. Follow-up Bayesian analyses were performed to confirm the findings, which supported the null effect of the longitudinal pubertal comparison. Conclusion Using a novel longitudinal fixel-based analysis framework, we demonstrate that white matter fibre density and fibre cross-section increased within a 16-month scan rescan period in specific regions. The observed increases might reflect increasing axonal diameter or axon count. Pubertal stage or progression did not influence the rate of fibre development in the early stages of puberty. Future work should focus on quantifying these measures across a wider age range to capture the full spectrum of fibre development across the pubertal period.

The fruits from graviola trees (Annona muricata) are used to make a juice that is quite popular in Puerto Rico in the United States, as well as in Mexico, Brazil, Malaysia, China, India and many other countries. Graviola is also known as soursop and guanabana. The vast majority of the scientific literature on graviola describes its potential health effects—especially its anticancer activity. However, overconsumption of graviola fruit and products made from it has been linked to an atypical form of Parkinson’s disease that does not respond to the standard treatment ( l -3,4-dihydroxyphenylalanine or l -DOPA) ( Caparros-Lefebvre and Elbaz, 1999 ). More recently, it was shown that chronic consumption of graviola juice can trigger and aggravate the phosphorylation of cerebral tau protein, leading to tau pathologies, including Parkinson’s disease ( Rottcholl et al., 2016 ). This is due to the presence of a class of neurotoxic compounds called acetogenins ( Smith et al., 2015 ). They are present in not just graviola fruit, seeds, and leaves, but also the fruit, seeds, and leaves of other plants in the Annonaceae family ( Hollerhage et al., 2015 ). Acetogenins exhibit their neurotoxic and anticancer properties by inhibiting the mitochondrial NADH:ubiquinone oxidoreductase (complex I of the respiratory chain) ( Hollerhage et al., 2009 ). Neurotoxicity is also caused by redistributing the tau protein and modulating the phosphorylation of histone proteins ( Lee et al., 2011 ). There are four goals of this chapter. First, the chemical properties of neurotoxic acetogenins are described. Second, the previously published in vitro, in vivo, and epidemiological data that demonstrated the ability of acetogenins and graviola juice to cause atypical Parkinson’s disease is given. Third, the concentrations of acetogenins that were found in graviola and other fruits in the Annonaceae family is presented, along with a description of the methods used to quantify them. Finally, other important compounds and nutrients that are present in graviola and other fruits in the Annonaceae family are listed. This includes fatty acyl glycosides that act as detergents that suspend lipophilic acetogenins in juices and other aqueous solutions, making them more bioavailable.

BackgroundThe brain’s white matter undergoes profound changes during early childhood, which are believed to underlie the rapid development of cognitive and behavioral skills during this period. Neurite density, and complexity of axonal projections, have been shown to change across the life span, though changes during early childhood are poorly characterized. Here, we utilize neurite orientation dispersion and density imaging (NODDI) to investigate maturational changes in tract-wise neurite density index (NDI) and orientation dispersion index (ODI) during early childhood. Additionally, we assess hemispheric asymmetry of tract-wise NDI and ODI values, and longitudinal changes.MethodsTwo sets of diffusion weighted images (DWI) with different diffusion-weighting were collected from 125 typically developing children scanned at baseline (N=125; age range=4.14-7.29; F/M=73/52), 6-month (N=8; F/M=8/0), and 12-month (N=52; F/M=39/13) timepoints. NODDI and template-based tractography using constrained spherical deconvolution were utilized to calculate NDI and ODI values for major white matter tracts. Mixed-effects models controlling for sex, handedness, and in-scanner head motion were utilized to assess developmental changes in tract-wise NDI and ODI. Paired t-tests were used to assess interhemispheric differences in tract-wise NDI and ODI values and longitudinal changes in cross-sectional and 12-month longitudinal analyses, respectively.ResultsMaturational increases in NDI were seen in all major white matter tracts, though we did not observe the expected tract-wise pattern of maturational rates (e.g. fast commissural/projection and slow frontal/temporal tract change). ODI did not change significantly with age in any tract. We observed higher cross-sectional NDI and ODI values in the right as compared to the left hemisphere for most tracts, but no hemispheric asymmetry for longitudinal changes.ConclusionsThese findings suggest that neurite density, but not orientation dispersion, increases with age during early childhood. In relation to NDI growth trends reported in infancy and late-childhood, our results suggest that early childhood may be a transitional period for neurite density maturation wherein commissural/projection fibers are approaching maturity, maturation in long range association fibers is increasing, and changes in limbic/frontal fibers remain modest. Rightward asymmetry in NDI and ODI values, but not longitudinal changes, suggests that rightward asymmetry of neurite density and orientation dispersion is established prior to age 4.

Early childhood is an important period for cognitive and brain development, though white matter changes specific to this period remain understudied. Here we utilize a novel analytic approach to quantify and track developmental changes in white matter micro- and macro-structure, calculated from individually oriented fiber-bundle populations, termed “fixels”. Fixel-based analysis and mixed-effects models were used to assess tract-wise changes in fiber density and bundle morphology in 73 girls scanned at baseline (ages 4.09-7.02, mean=5.47, SD=0.81), 6-month (N=7), and one-year follow-up (N=42). For comparison, we also assessed changes in commonly utilized diffusion tensor metrics: fractional anisotropy (FA), and mean, radial and axial diffusivity (MD, RD, AD). Maturational increases in fixel-metrics were seen in most major white matter tracts, with the most rapid increases in the corticospinal tract and slowest or non-significant increases in the genu of the corpus callosum and uncinate fasciculi. As expected, we observed developmental increases in FA and decreases in MD, RD and AD, though percentage changes were smaller relative to fixel-metrics. The majority of tracts showed more substantial morphological than microstructural changes. These findings highlight early childhood as a period of dynamic white matter maturation, characterized by large increases in macroscopic fiber bundle size, mild changes in axonal density, and parallel, albeit less substantial, changes in diffusion tensor metrics.

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis., Author summary Enterococcus faecalis is a commensal bacterium colonizing the gastro-intestinal tract of humans. This organism can cause life-threatening opportunistic infections and represents a reservoir for the transmission of antibiotic resistance genes such as resistance to vancomycin. E. faecalis strains responsible for nosocomial infections are also found in healthy individuals and the virulence factors identified so far are not strictly associated with clinical isolates. The molecular basis underpinning E. faecalis infections therefore remains unclear. In this work, we identify several mutations clustered on the chromosome, which play a role in the resistance of E. faecalis to effectors of the innate immune system such as lysozyme and bile salts. We show that the corresponding genes contribute to the decoration of a conserved polysaccharide called the enterococcal polysaccharide antigen and that this decoration is essential for E. faecalis virulence. This mechanism critical for pathogenesis represents an attractive therapeutic target to control enterococcal infections.

Enterococcus faecalisis an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires several genes (oatA, pgdA, dltAandsigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulatingE. faecalisresistance to lysozyme. Random transposon mutagenesis carried out in the quadrupleoatA/pgdA/dltA/sigVmutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conservedepaA-epaRgenes proposed to encode a core synthetic machinery. Theepavariable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promoteE. faecalisinfections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

Differences in brain networks and underlying white matter abnormalities have been suggested to underlie symptoms of autism spectrum disorder (ASD). However, robustly characterizing microstructural white matter differences has been challenging. In the present study, we applied an analytic technique that calculates structural metrics specific to differently-oriented fiber bundles within a voxel, termed “fixels”. Fixel-based analyses were used to compare diffusion-weighted magnetic resonance imaging data from 25 individuals with ASD (mean age = 16.8 years) and 27 typically developing age-matched controls (mean age = 16.9 years). Group comparisons of fiber density (FD) and bundle morphology were run on a fixel-wise, tract-wise, and global white matter (GWM) basis. We found that individuals with ASD had reduced FD, suggestive of decreased axonal count, in several major white matter tracts, including the corpus callosum (CC), bilateral inferior frontal-occipital fasciculus, right arcuate fasciculus, and right uncinate fasciculus, as well as a GWM reduction. Secondary analyses assessed associations with social impairment in participants with ASD, and showed that lower FD in the splenium of the CC was associated with greater social impairment. Our findings suggest that reduced FD could be the primary microstructural white matter abnormality in ASD.

NMR is becoming an important tool for developing new allosteric and allo-network drugs that bind to allosteric sites on enzymes, partially inhibiting them and causing fewer side effects than drugs already developed that target active sites. This is based on systems thinking, in which active enzymes and other proteins are known to be flexible and interact with each other. In other words, proteins can exist in an ensemble of different conformations whose populations are tunable. NMR is being used to find the pathways through which the effects of binding of an allosteric ligand propagate. There are NMR screening assays for studying ligand binding. This includes determining the changes in the spin lattice relaxation due to changes in the mobility of atoms involved in the binding, measuring magnetization transfer from the protein to the ligand by saturation difference transfer NMR (STD-NMR) and the transfer of bulk magnetization to the ligand by water-Ligand Observed via Gradient Spectroscopy, or waterLOGSY. The chemical shifts of (1)H and (15)N of some of the atoms in amino acids change when an allosteric ligand binds to a protein. So, (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra can be used to identify key amino acids and ligand binding sites. The NMR chemical shifts of amino acids affected by ligand binding form a network that can be characterized. Allosteric networks can be identified by chemical shift covariance analysis (CHESCA). This approach has been used recently to study the binding of new molecular entities (NMEs) to potentially therapeutic drug targets.

Dietary supplements containing plant materials of Annonaceae species ( Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae ( A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.

Seeds from the purple and the white fruit produced by the Amazonian palm, Euterpe oleracea Mart., commonly called “acai”, were analyzed by solid state 1 H-decoupled 13 C CPMAS and MAS NMR, solution NMR and LC–MS/MS. The goal was to distinguish the seeds from each colored fruit and determine their spectra for the first time. The seeds of each variety contained primarily glycosidic carbons, due to their cellulose and hemicellulose content. They also contained carbons due to C O, C C, as well as aliphatic carbons. The insoluble fiber found in acai pulp is distinguishable from the seed of the fruit by its unique solid state 1 H-decoupled 13 C CPMAS NMR spectra. The seed also contains fats (0.22–0.33%) not previously reported. The seed contains a mixture of saturated and unsaturated fats. There are also 3.38–4.70% total methanolic extractables, with no detectable cyanidin 3- O -glucoside or cyanidin 3- O -rutinoside, unlike the pulp which contains both cyanidins. When the NMR spectra of the white and purple acai seeds are compared, it was possible to observe differences between these two acai varieties, as well as differences between the composition of pulp of the fruit and its seed.

FCA/UNESP-Faculdade de Ciencias Agronomicas Universidade Estadual Paulista Julio de Mesquita Filho Fazenda Experimental Lageado, 280, C.P237

Hydrophilic and lipophilic extracts of ten cultivars of Highbush and Rabbiteye Brazilian blueberries ( Vaccinium corymbosum L. and V accinium ashei Reade, respectively) that are used for commercial production were analysed for antioxidant activity by the FRAP, ORAC, ABTS and β-carotene–linoleate methods. Results were correlated to the amounts of carotenoids, total phenolics and anthocyanins. Brazilian blueberries had relatively high concentration of total phenolics (1622–3457 mg gallic acid equivalents per 100 g DW) and total anthocyanins (140–318 mg cyanidin-3-glucoside equivalents per 100 g DW), as well as being a good source of carotenoids. There was a higher positive correlation between the amounts of these compounds and the antioxidant activity of hydrophilic compared to lipophilic extracts. There were also significant differences in the level of bioactive compounds and antioxidant activities between different cultivars, production location and year of cultivation.

BACKGROUND. A test for hepatitis C virus (HCV) RNA is essential to identify persons with active, or current, HCV infection. We assessed trends in HCV RNA testing following a positive HCV antibody result among persons in 4 large healthcare organizations. METHODS. Data collected from adults with ≥2 clinical encounters during 2003–2010 were analyzed to determine the frequency of, interval between, and factors associated with having an RNA test after a first positive HCV antibody test. RESULTS. From 2003–2010, 5860 persons had a positive antibody test, of whom 3570 (60.9%) had a follow-up RNA test. During this period, the annual frequency of persons with an eventual RNA test did not change significantly; however, the fraction of persons who had the follow-up RNA test within 6 months improved significantly, from 45% in 2003 to 57% in 2010 (P < .001, for trend). Persons born during 1945–1965, men, and those with annual income

A new paradigm is emerging in modern drug discovery. It is a fusion of traditional and modern medicine, phenotypic and targeted drug discovery, or systems and reductionist thinking. This is exemplified by using a combination of network medicine and high throughput screening. It blends the use of physiologically relevant biological systems with the high throughput and statistical robustness of modern assay technologies. The basic principles of network theory and tools of network medicine are described. Scale-free networks and their organizing principles are discussed. They are emergent properties of living, autopoietic systems. This includes networks of people who do high throughput screening (HTS), and microscopic networks of ions, metabolites, DNA, RNA, proteins, lipids, carbohydrates, viruses, bacteria, fungi, human cells and tissues. Databases have been constructed based on the metabolome, genome, transcriptome, proteome, lipidome, glycocode, virome, bacteriome and many others. Modern HTS can be used to examine the interactions of many parts of the complex human network. High content screening (HCS) can look at perturbations that occur when test compounds are added to single cells. Allo-network drugs can have effects far beyond a single protein and can be transmitted to other cells. Interactions and hidden connections can be revealed, with the goal of developing new drugs that have few, if any harmful side effects and are effective against multi-drug resistant cancer cells or bacteria.

This study evaluated the effects of using passion fruit peel flour together with diet therapy and counseling in 36 patients with HIV lipodystrophy who were in an ambulatory clinic in a university hospital. The patients were divided into two groups. One received 30 g of passion fruit peel flour daily for 90 days and diet therapy counseling. The other group received only diet therapy counseling. The metabolic changes were analyzed before and after the intervention, with a significance level predetermined at p ⿤ 0.05. The use of passion fruit peel flour was effective in reducing total cholesterol and triacylglycerides after 30 days. The concentrations of LDL-C decreased, while HDL-C increased in the blood of lipodystrophy patients after 90 days passion fruit peel flour treatment. No significant differences in food consumption were seen between groups. The use of 30 g of passion fruit peel flour for 90 days together with diet therapy counseling was effective in improving plasma concentrations of total cholesterol, LDL-C, HDL-C and triacylglycerides. Keywords: Passion fruit peel flour, HIV patients, Lipodystrophy syndrome, Dyslipidemia

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemiscals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promotor activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

Graviola leaves (Annona muricata L., Annonaceae) are used by some people to try to treat or even cure cancer, even though over-consumption of the fruit, which contains the neurotoxins annonacin and squamocin has caused an atypical form of Parkinson's disease. In previous analyses, the fruits were extracted with methanol under ambient conditions before analyses. In the present study, UPLC–QTOF–MS and NMR were used to analyze freeze-dried graviola leaves that were extracted using dry methanol and ethanol at 100 °C and 10 MPa (100 atm) pressure in a sealed container. Methanol solubilized 33% of the metabolites in the lyophilized leaves. Ethanol solubilized 41% of metabolites in the lyophilized leaves. The concentrations of total phenolic compounds were 100.3 ± 2.8 and 93.2 ± 2.0 mg gallic acid equivalents per g of sample, for the methanolic and ethanolic extracts, respectively. Moreover, the toxicophore (unsaturated γ-lactone) that is present in neurotoxic acetogenins was found in the lipophilic portion of this extract. The concentrations of the neurotoxins annonacin and squamocin were found by UPLC–QTOF–MS to be 305.6 ± 28.3 and 17.4 ± 0.89 μg/g-dw, respectively, in the dried leaves. Pressurized methanol solubilized more annonacin and squamocin than ethanol. On the other hand, a hot, aqueous infusion solubilized only 0.213% of the annonacin and too little of the squamocin to be detected. So, graviola leaves contain significant amounts of the neurotoxins annonacin and squamocin, as well as some potentially healthy phenolic compounds. Finally, the potential neurotoxicity of whole leaves in dietary supplements could be much higher than that of a tea (hot aqueous infusion) that is made from them. Keywords: Graviola, Annona muricata, UPLC–QTOF–MS, Parkinson's disease, NMR

The concentrations of the neurotoxins, annonacin and squamocin, were determined in a lyophilized sample of the fruit pulp of the North American pawpaw (Asimina triloba) by LC coupled to high resolution mass spectrometry or LC-HRMS. The sample was extracted using dry methanol at 100 °C and 10 MPa pressure in a sealed container. The extraction of annonacin and squamocin was optimal at 100 °C with 7.72 and 0.162 mg/g, respectively, being found. Also, several isomers of annonacin and squamocin were separated and detected but not quantified.

Objective: Recent investigations of schizophrenia have targeted glutamatergic neurotransmission, since phencyclidine, an N-methyl-D-aspartate (NMDA) receptor antagonist, can induce schizophreniform psychosis. The authors previously reported alterations in thalamic NMDA receptor subunit expression in schizophrenia, consistent with the hypothesis that thalamic glutamatergic hypofunction may contribute to the pathophysiology of this illness. In this study they generalized this hypothesis to include other molecules of the glutamate synapse, specifically excitatory amino acid transporters (EAATs), whose normal expression and regulation in the thalamus may also be disrupted in subjects with schizophrenia. Method: In situ hybridization with riboprobes specific for the human excitatory amino acid transporter transcripts EAAT1, EAAT2, and EAAT3 was performed in discrete thalamic nuclei in persons with schizophrenia and comparison subjects. Results: Higher expressions of transcripts encoding EAAT1 and EAAT2, but not EAAT3, were detected in the thalamus of subjects with schizophrenia. Conclusions: These findings support the hypothesis of glutamatergic dysfunction in schizophrenia and suggest that molecules other than glutamate receptors are abnormally expressed in glutamatergic synapses in this illness.

Annonaceous acetogenins (ACGs) make up a large and homogeneous class of natural polyketides, with over 400 representatives. They have pesticidal, antiinfective, and cytotoxic properties. Interestingly, they display activity in multidrug resistant cancer cells and show antitumor potential in several tumor-grafted mouse models. ACGs are strong inhibitors of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and may affect alternative targets, with possible covalent interaction. Their ability to inhibit matrix metalloproteinase and modulate histone H3 phosphorylation, to bind calcium or to target mitochondria was shown, among other issues. However, ACGs are suspected of being environmental neurotoxins, responsible for “Guadeloupean parkinsonism” in the French West Indies and for sporadic atypical parkinsonism/dementia in several tropical communities. ACGs induce tau pathology in cultured neurons, and annonacin, a prototypical ACG, proves to be neurotoxic in several animal models. ACGs were identified in edible fruits such as soursop (Annona muricata L.) and paw paw (Asimina triloba Dunnal). Leaves, bark, and twigs of both species are sold over the Internet as cures for cancer, with undefined risk/benefit ratio. A method based on MALDI-TOF-MS was used for the analysis of ACGs and annonacin content in plant material and dietary supplements.

Although many studies have characterized soluble factors that stimulate or inhibit chemokine secretion, in this review we focus on the event of cellular adhesion as a novel mechanism for stimulating chemokine expression. Recent work has demonstrated chemokine expression following cell-to-cell and cell-to-matrix adhesion. The specificity of this finding was demonstrated utilizing various techniques that illustrate that adhesion, and not a soluble stimulus, is in some cases responsible for initiating or augmenting chemokine expression. For example, co-cultures of peripheral blood monocytes and endothelial cells secreted elevated levels of IL-8 and MCP-1 compared with either cell type alone. When co-cultured in transwells, this effect was significantly attenuated. In other experiments, neutralizing monoclonal antibodies to various adhesion molecules inhibited chemokine expression. The effects of adhesion were not limited to leukocytes. Both immune and non-immune cell types were evaluated as potential sources of adhesion-mediated chemokine expression. Not suprisingly, expression of some chemokines was associated with adhesion, whereas others were not, supporting the notion that adhesion differentially signals chemokine secretion during the inflammatory response. We hypothesize that as a recruited leukocyte encounters different adhesion substrates such as endothelial cells, basement membrane, extracellular matrix, and fibroblasts, the expression of chemokines from both the leukocyte and the substrate may be initiated, inhibited, or augmented. Careful characterization of the contribution of adhesion to regulation of chemokine expression will provide insight into the pathogenesis of many human diseases where chemokines have a central role.

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid (CF3MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF3MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for the in vivo examination of sialidases and for the detection of recombinant sialidase by means of expression cloning.

It is widely believed that consuming foods and beverages that have high concentrations of antioxidants can prevent cardiovascular diseases and many types of cancer. As a result, many articles have been published that give the total antioxidant capacities of foods in vitro. However, many antioxidants behave quite differently in vivo. Some of them, such as resveratrol (in red wine) and epigallocatechin gallate or EGCG (in green tea) can activate the nuclear erythroid-2 like factor-2 (Nrf2) transcription factor. It is a master regulator of endogenous cellular defense mechanisms. Nrf2 controls the expression of many antioxidant and detoxification genes, by binding to antioxidant response elements (AREs) that are commonly found in the promoter region of antioxidant (and other) genes, and that control expression of those genes. The mechanisms by which Nrf2 relieves oxidative stress and limits cardiac injury as well as the progression to heart failure are described. Also, the ability of statins to induce Nrf2 in the heart, brain, lung, and liver is mentioned. However, there is a negative side of Nrf2. When over-activated, it can cause (not prevent) cardiovascular diseases and multi-drug resistance cancer.

Pulmonary fibrosis is the end point of a chronic inflammatory process characterized by leukocyte recruitment and activation, fibroblast proliferation, and increased extracellular matrix production. Previous studies of models of pulmonary fibrosis have investigated the role of cytokines in the evolution of the fibrotic response. The involvement of tumor necrosis factor and interleukin-1 in bleomycin-induced lung injury, a model of idiopathic pulmonary fibrosis, has been well established, suggesting that cytokines mediate the initiation and maintenance of chronic inflammatory lesions. However, the aforementioned cytokines alone cannot account for the recruitment and activation of specific leukocyte populations found in the bleomycin model. Recently, a family of novel proinflammatory cytokines (chemokines) was cloned and characterized, yielding many putative mediators of leukocyte functions. Macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1) belong to the C-C chemotactic cytokine family, a group of low-molecular-weight peptides. These molecules modulate chemotaxis, proliferation, and cytokine expression in leukocyte subsets. Our group has investigated the roles of MCP-1 and MIP-1α in the bleomycin model. Both MCP-1 and MIP-1α are expressed in a time-dependent manner after bleomycin challenge, and passive immunization of these animals with either anti-MIP-1α or anti-MCP-1 antibodies attenuated leukocyte accumulation. In addition, we have identified specific cell types expressing MCP-1 or MIP-1α by in situ hybridization and immunohistochemical localization, respectively. Furthermore, our results indicate that MIP-1α expression is mediated by alveolar macrophage-derived tumor necrosis factor, identifying an important cytokine pathway in the initiation of pulmonary fibrosis. Finally, anti-MIP-1α therapy attenuated fibrosis, providing direct evidence for its involvement in fibrotic pathology. Our work has clearly established that the C-C chemokines MCP-1 and MIP-1α are expressed and contribute to the initiation and maintenance of the bleomycin-induced pulmonary lesion. J. Leukoc. Biol. 57: 782–787; 1995.

SALATRIM is a reduced-calorie fat composed of triacylglycerols containing mixtures of short-chain aliphatic acids and long-chain saturated fatty acids. Studies have been conducted in a clinical environment to confirm the predictable metabolism of ingested SALATRIM in humans. An acute tolerance test (study I) was conducted initially with a single ingestion (45 or 60 g) in a double-blind-crossover design with 10 subjects. The levels selected were chosen to significantly exceed expected exposure levels from consumption of foods containing SALATRIM

Objective. To determine the effects of peptidyl fluoromethyl ketones on the in vitro activity of purified cathepsins B and L, on tissue cysteine proteinase activity, and on cartilage and bone destruction in experimental arthritis. Methods. The effects of the fluoroketones on cathepsins B and L in vitro and the effects of oral administration of fluoroketones on ex vivo cysteine proteinase activity in tissue homogenates were determined by measuring the inhibition of fluorogenic substrate cleavage. To determine the effects on arthritis, animals were injected with adjuvant or type II collagen, treated orally with the fluoroketones, and the severity of arthritis was assessed by clinical, histologic, and radiologic methods. Results. All of the fluoroketones tested were potent inhibitors of purified cathepsins B and L activity. Oral administration of the fluoroketones reduced tissue cysteine proteinase activity by up to 77%. In addition, fluoroketone treatment significantly reduced the severity of clinical joint disease and decreased the destruction of articular cartilage and bone. Quantitative analysis of radiographic images indicated that treatment significantly reduced soft tissue changes, periosteal proliferation, and bone erosion, but only partially reduced juxtaarticular osteoporosis. Conclusion. These studies suggest that cysteine proteinase inhibitors may limit tissue destruction in diseases such as rheumatoid arthritis.

Products labelled as containing extracts from two mushrooms (cordyceps plus reishi) and the juices from acai, goji, mangosteen, noni, pomegranate, and sea buckthorn have been analysed for 174 different pesticides, using the validated QuEChERS method for sample preparation and electrospray LC-MS/MS in the positive ion mode for analysis. Pesticides were found in 10 of the 21 samples analysed. Most pesticides found were below the tolerance levels (1-6 μg/g, depending on the pesticide), but some were not. This included boscalid, dimethomorph, iprovalicarb, pyridaben, pyrimethanil, and imazalil, for which there is no tolerance reported or zero tolerance in any fruit. However, genuine acai that was harvested in the state of Para and lyophilised in Rio de Janeiro had no detectable pesticides, when analysed by both LC-MS/MS and GC-MS/MS, which can detect 213 more pesticides and industrial chemicals. Likewise no pesticides were found in one sample each of cordyceps plus reishi, sea buckthorn and noni.

Previously, we have reported alterations in thalamic NMDA receptor subunit and excitatory amino acid transporter expression in schizophrenia, consistent with the hypothesis that thalamic glutamatergic dysfunction may contribute to the pathophysiology of this illness. We have generalized this hypothesis to include other molecules of the glutamate synapse. Using riboprobes specific for human brain-specific Na+-dependent inorganic phosphate transporter (BNPi) and differentiation-associated Na+/Pi co-transporter (DNPi), both vesicular glutamate transporters, in situ hybridization was performed in the thalami of persons with schizophrenia and comparison subjects. We detected increased expression of DNPi mRNA in the thalamus in schizophrenia, while BNPi mRNA was not expressed in the thalamus in any subjects. These findings support the hypothesis of glutamatergic dysfunction in the thalamus in schizophrenia.

Phosphatidylserine transport in normal and Rhnull red blood cells was determined by measuring characteristic morphologic changes induced by synthetic phospholipids. Treating normal A+ cells with commercial anti- A antisera, anti-Rho(D) antisera, or with saturating concentrations of purified Rho(D) antibodies had no effect on phosphatidylserine transport. Normal B- cells treated with purified anti-B antibodies transported phosphatidylserine at rates equal to those of cells not treated with antibody. Rhnull cells, deficient in the protein bearing the Rho(D) antigen, incorporated dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine at rates and to extents similar to normal cells. Furthermore, incorporated phosphatidylserine, but not phosphatidylcholine, was rapidly transported across the membrane bilayer. Energy depletion or treatment with sulfhydryl reagents inhibited phosphatidylserine transport equally in normal and Rhnull cells. These results indicate that, although Rhnull cells have numerous membrane defects, they are capable of adenosine triphosphate-dependent transport of exogenously added dimyristoylphosphatidylserine. Normal phosphatidylserine transport in the presence of anti-Rho(D) antibodies or in cells deficient in the Rho(D) polypeptide indicates that this protein is not the aminophospholipid transporter.

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5 mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30 min, 1 h and 2 h) after injection. Samples were treated with β-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30 min and 1 h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.

The functionality of DPP-IV, purified from human placenta and isolated from CD4+/CD26+ T cells of noninfected and HIV-1-infected individuals, was investigated as to its ability to bind certain specific peptides. Using isoelectric focusing and the specificity of substrate-impregnated overlay membranes, we found that DPP-IV from term placenta and from T cells of HIV-infected individuals was significantly more sialylated compared with enzyme isozyme patterns of other tissues. We report here that (1) the number of isoforms of DPP-IV and extent of sialylation are critical to function and peptide binding; (2) the number of sialylated isoforms isolated from PBMCs increases significantly with age greater than 40 years; (3) hypersialylation by extreme anionic isoforms is highly associated with HIV infection and pathognomonic to remaining CD4+ cells in overt AIDS; and (4) highly sialylated DPP-IV is more significantly inhibited by Tat and cationic peptides. We conclude that hypersialylation of DPP-IV modifies surface charge of the CD26 antigen, promoting binding of HIV peptides through their cationic domains to the sialic acid residues of DPP-IV, and that certain HIV moieties are likely to engage this phenomenon as an auxiliary adhesion mechanism to fuse with cells. Furthermore, as a consequence of this occurrence, DPP-IV enzymatic activity can be significantly reduced, competitively.

A series of dipeptides which contained phosphonate analogs of proline and piperidine-2-carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite specific. The best inhibitor in the series is Ala-PipP(OPh-4-Cl)2 (13), which has a k(inact) of 0.353 s-1 and KI of 236 microM. The DPP-IV inhibitors Ala-ProP(OPh)2 (6), Ala-ProP(OPh-4-Cl)2 (12), and Ala-PipP(OPh-4-Cl)2 (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds 12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline phosphonate residue (PipP) or a Pro phosphonate residue (ProP) at the P1 site are stable in a pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala-PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of excess inhibitor and incubation in buffer for 1 day. Since the phosphonate inhibitors are specific toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing the biological functions of DPP-IV and may be useful therapeutically in the prevention of the rejection of transplanted tissue.

To assess the efficacy and safety of twice-daily administration of nedocromil sodium 2% ophthalmic solution, we performed a multicenter study involving 140 patients with seasonal allergic conjunctivitis. Subjects had a history of seasonal allergic conjunctivitis and positive results of a skin test to ragweed. The trial coincided with the peak ragweed pollen season at five treatment centers. Patients treated with nedocromil sodium had improvements in symptoms with statistically significant reductions recorded for eye itching (P less than or equal to .04), conjunctival injection (P less than or equal to .001), and overall disease severity (P less than or equal to .001) as compared to the placebo-treated group. Adverse events were minor and transient. We concluded that nedocromil sodium 2% ophthalmic solution administered twice daily is effective in relieving major symptoms associated with seasonal allergic conjunctivitis.

Analysis of the properties of the predominant acid proteinase of rat preputial gland has been carried out on partially purified extracts of lysosomes isolated from this organ, using a fluorometric assay and the synthetic substrate, carbobenzyloxy-alanyl-arginyl-arginyl-4-methoxy-β-naphthylamide. By means of exclusion chromatography on Sephadex G-200, a molecular weight of ∼ 25000 was estimated. The enzymic activity exhibited a pH optimum of 6.2. Calcium ion was required for full activity within a narrow range of concentrations, beyond which inhibition was noted. Substrate specificity of the enzyme toward arginyl and lysyl residues and an absolute requirement for sulfhydryl activation, coupled with the capacity to inactivate aldolase, strongly suggested cathepsin B1-like character. This inference was supported by relative efficiencies of several classes of inhibitors. Powerful constraint of activity was exhibited by the known cathepsin-B1 inhibitors, leupeptin and antipain, while pepstatin, a recognized antagonist of cathepsin D, was without influence. Neither lima bean trypsin inhibitor nor phenylmethylsulfonyl fluoride was active. However, substantial inhibition was seen with tosyl-l-lysyl-chloromethyl ketone, iodoacetate, and ovomucoid. Collectively, these data strongly implicate cathepsin B1 as the predominant lysosomal proteinase of rat preputialgland. In extension of previous work with selected lysosomal hydrolases, estradiol-17β, but not the inactive 17α-epimer, elicited, within 2 min of its administration, a marked reduction in structural latency of cathepsin B1 in lysosome-enriched fractions isolated from the preputial gland of the ovariectomized rat, without evoking concomitant alteration in the total activities in the corresponding unfractionated homogenates. Moreover, administration of the active hormone led to the rapid appearance of this enzymic activity, foreign to the nucleus in the controls, in ultrapurified nuclei isolated froth this organ. The potential significance of such recompartmentation in the subsequent secondary responses initiated by the hormone, including mitogenesis, is discussed.