Nordihydroguaiaretic Acid (NDGA) Inhibits CsgA Polymerization, Bacterial Amyloid Biogenesis, and Biofilm Formation.

Escherichia coli and other Enterobacteriaceae thrive in robust biofilm communities through the coproduction of curli amyloid fibers and phosphoethanolamine cellulose. Curli promote adhesion to abiotic surfaces and plant and human host tissues and are associated with pathogenesis in urinary tract infection and food-borne illness. The production of curli in the host has also been implicated in the pathogenesis of neurodegenerative diseases. We report that the natural product nordihydroguaiaretic acid (NDGA) is effective as a curlicide in E. coli. NDGA prevents CsgA polymerization in vitro in a dose-dependent manner. NDGA selectively inhibits cell-associated curli assembly and inhibits uropathogenic E. coli biofilm formation. More broadly, this work emphasizes the ability to evaluate and identify bioactive amyloid assembly inhibitors by using the powerful gene-directed amyloid biogenesis machinery in E. coli.

Distinct subsets of innate lymphoid cells in nasal polyp.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) contribute to the pathogenesis of eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNPs). However, the role of other subsets of ILCs and the differentiation of ILCs in CRSwNPs is not well understood. This study aimed to characterize the ILC subsets and evaluate the differentiation of ILCs from ILC precursors (ILCPs) in NP tissue. METHODS: ILC subsets and ILCPs were evaluated by flow cytometry in fresh sinonasal mucosa from patients with CRSwNPs and control subjects. Subsets were compared based on clinical variables and immunological features of the patients. Sorted ILCPs (Lin-CD127+CD117+CD45RA+IL1R1+) were cultured with cytokines. RESULTS: The frequency of ILC1s and IFN-γ-producing ILC1s increased in non-eosinophilic NPs, whereas that of ILC2s and IL-5-producing ILC2s increased in eosinophilic NPs, particularly in patients with comorbid asthma. The frequency of ILC1s and IFN-γ-producing ILC1s, and frequency of ILC2s and IL-5-producing ILC2s positively correlated with that of neutrophils and eosinophils, respectively. The proportion of IFN-γ-producing ILC1s positively correlated with clinical severity and levels of IFN-γ and IL-8. The proportion of IL-5-producing ILC2s positively correlated with levels of IL-5, CCL24, and total IgE. ILCPs were identified in NP tissue and differentiated into IFN-γ-producing or IL-5-producing ILCs in response to increased IL-12 and IL-18 or IL-25 and IL-33 in non-eosinophilic NPs and eosinophilic NPs, respectively. CONCLUSIONS: ILC1s and ILC2s may be associated with neutrophilic and eosinophilic inflammation in CRSwNPs, respectively. In addition, ILCPs located in the sinus mucosa could differentiate into IFN-γ- or IL-5-producing cells in response to local cytokine stimuli.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.

Development of a mouse model of eosinophilic chronic rhinosinusitis with nasal polyp by nasal instillation of an Aspergillus protease and ovalbumin.

One subtype of chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by the development of a T-helper type 2 (Th2) response and eosinophilic infiltration. Here, we aimed to establish an eosinophilic CRSwNP murine model, which would be essential to understand the underlying pathogenesis and establish a treatment strategy. C57BL/6 mice were challenged intranasally with a mixture of an Aspergillus oryzae-derived protease (AP) and ovalbumin (OVA) for 6, 8, or 12 consecutive weeks (12 mice/group); control mice received the same volume of phosphate-buffered saline for 12 weeks (n = 12). Sinonasal samples were evaluated histologically, and interleukin (IL)-4, IL-5, IL-13, eotaxin, keratinocyte chemoattractant, and macrophage inflammatory protein-2 mRNA levels in sinonasal mucosa were measured by real-time PCR. Protein levels of Th2 cytokines, INF-γ, IL-17A, and chemokines in nasal lavage fluid, and total serum IgE were measured by ELISA. Greater eosinophil infiltration in the subepithelial layer was observed in the challenged groups, compared with the control group. Polypoid mucosal lesions were predominantly observed in the 12-week group, which also exhibited mucosal thickening on micro-CT scans. The IL-4, IL-5, and IL-13 mRNA and protein levels were elevated in the sinonasal mucosa and nasal lavage fluid. INF-γ and IL-17A were undetectable or not elevated relative to the control group levels. In contrast, eotaxin levels were particularly elevated in the sinonasal mucosa and nasal lavage fluid in the 12-week group. In conclusion, intranasal AP and OVA exposure successfully induced Th2-specific CRSwNP in a murine model.

Molecular determinants of mechanical properties of V. cholerae biofilms at the air-liquid interface.

Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.

Plasma proteomic analysis of patients infected with H1N1 influenza virus.

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1-infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano-ultra performance LC-MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1-infected group as compared with the control group. Of 14 up- and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine-rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.

Community behavior and amyloid-associated phenotypes among a panel of uropathogenic E. coli.

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Disruption of Escherichia coli amyloid-integrated biofilm formation at the air-liquid interface by a polysorbate surfactant.

Functional amyloid fibers termed curli contribute to bacterial adhesion and biofilm formation in Escherichia coli . We discovered that the nonionic surfactant Tween 20 inhibits biofilm formation by uropathogenic E. coli at the air-liquid interface, referred to as pellicle formation, and at the solid-liquid interface. At Tween 20 concentrations near and above the critical micelle concentration, the interfacial viscoelastic modulus is reduced to zero as cellular aggregates at the air-liquid interface are locally disconnected and eventually eliminated. Tween 20 does not inhibit the production of curli but prevents curli-integrated film formation. Our results support a model in which the hydrophobic curli fibers associated with bacteria near the air-liquid interface require access to the gas phase to formed strong physical entanglements and to form a network that can support shear stress.

Suppression of LPS-induced inflammatory responses by inflexanin B in BV2 microglial cells.

Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1ß and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 µg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs.

N-methyl-D-aspartate receptors induce M1 polarization of macrophages: Feasibility of targeted imaging in inflammatory response in vivo.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are considered to be involved in several physiological and pathophysiological processes in addition to the progression of neurological disorders. However, how NMDARs are involved in the glycolytic phenotype of M1 macrophage polarization and the possibility of using them as a bio-imaging probe for macrophage-mediated inflammation remain unclear. METHODS: We analyzed cellular responses to NMDAR antagonism and small interfering RNAs using mouse bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS). An NMDAR targeting imaging probe, N-TIP, was produced via the introduction of NMDAR antibody and the infrared fluorescent dye FSD Fluor™ 647. N-TIP binding efficiency was tested in intact and LPS-stimulated BMDMs. N-TIP was intravenously administered to mice with carrageenan (CG)- and LPS-induced paw edema, and in vivo fluorescence imaging was conducted. The anti-inflammatory effects of dexamethasone were evaluated using the N-TIP-mediated macrophage imaging technique. RESULTS: NMDARs were overexpressed in LPS-treated macrophages, subsequently inducing M1 macrophage polarization. Mechanistically, NMDAR-mediated Ca2+ accumulation resulted in LPS-stimulated glycolysis via upregulation of PI3K/AKT/mTORC1 signaling. In vivo fluorescence imaging with N-TIP showed LPS- and CG-induced inflamed lesions at 5 h post-inflammation, and the inflamed lesions could be detected until 24 h. Furthermore, our N-TIP-mediated macrophage imaging technique helped successfully visualize the anti-inflammatory effects of dexamethasone in mice with inflammation. CONCLUSION: This study demonstrates that NMDAR-mediated glycolysis plays a critical role in M1 macrophage-related inflammation. Moreover, our results suggest that NMDAR targeting imaging probe may be useful in research on inflammatory response in vivo.

Quantitative analysis of amyloid-integrated biofilms formed by uropathogenic Escherichia coli at the air-liquid interface.

Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G(s)') and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.

Erythropoietin improves memory function with reducing endothelial dysfunction and amyloid-beta burden in Alzheimer's disease models.

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-ß (Aß). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aß in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.

Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and amyloid-integrated biofilm formation in Escherichia coli.

Escherichia coli directs the assembly of functional amyloid fibers termed "curli" that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [(13)C(2)]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. coli's response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.

Methylalpinumisoflavone inhibits lipopolysaccharide-induced inflammation in microglial cells by the NF-kappaB and MAPK signaling pathway.

Neuroinflammation is chronic inflammation within the brain that is attributed to prolonged activation of microglial cells and results in neurodegenerative events, such as neuronal dysfunction and neuronal loss. Therefore, suppression of neuroinflammation would theoretically slow progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects of 4'-O-methylalpinumisoflavone (methylalpinumisoflavone), isolated from Cudrania tricuspidata, against LPS-induced microglial activation in BV2 cells. Exposure of BV2 cells to LPS (0.5 µg/mL) significantly increased production of pro-inflammatory mediators, including NO, PGE(2), and pro-inflammatory cytokines. Conversely, pre-treatment with methylalpinumisoflavone (10 and 20 µg/mL) prior to treatment with LPS resulted in a significant decrease of LPS-induced production of pro-inflammatory mediators in a dose-dependent manner. In addition, reduction of pro-inflammatory mediators by treatment with methylalpinumisoflavone prior to treatment with LPS was accompanied by a decrease in translocation of NF-κB p50 and p65 from the cytoplasm to the nucleus and by a decrease in activation of mitogen-activated protein kinases (MAPKs), such as ERK1/2 and JNK. Taken together, these results suggest that methylalpinumisoflavone suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by decreasing NF-κB signaling and by phosphorylation of MAPKs. These results suggest the potential of methylalpinumisoflavone as an anti-inflammatory drug candidate.

Nephrotoxicity evaluation and proteomic analysis in kidneys of rats exposed to thioacetamide.

Thioacetamide (TAA) was administered orally at 0, 10, and 30 mg/kg body weight (BW) daily to Sprague-Dawley rats aged 6-7 weeks for 28 consecutive days. Nephrotoxicity and proteomics were evaluated in the kidneys of rats exposed to TAA. The BW decreased, however, the relative kidneys weight increased. No significant histopathologic abnormalities were found in the kidneys. The numbers of monocytes and platelets were significantly increased. However, the mean corpuscular volume and hematocrit values were decreased significantly in rats exposed to 30 mg/kg BW TAA. The expression levels of Kim-1 and NGAL were increased 4 to 5-fold in the kidneys, resulting in significant nephrotoxicity. Proteomic analysis was conducted and a total of 5221 proteins spots were resolved. Of these, 3 and 21 protein spots were up- and downregulated, respectively. The validation of seven proteins was performed by Western blot analysis. The expression level of ASAP2 was significantly upregulated, whereas RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were reduced. Sixteen isoforms of MUP were found by the 2DE immunoblot assay and were significantly downregulated with increasing exposure to TAA. MUP isoforms were compared in the liver, kidneys, and urine of untreated rats and a total of 43 isoforms were found.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 microM) for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 microM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose- and time-dependent fashion. Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size 2-DE revealed the presence of 2776 protein spots. A total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

Influence of plasmid pO157 on Escherichia coli O157:H7 Sakai biofilm formation.

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.

Proteomic analysis of proteins secreted by HepG2 cells treated with butyl benzyl phthalate.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 µM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4-7 and 6-9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

C-methylflavonoids isolated from Callistemon lanceolatus protect PC12 cells against Abeta-induced toxicity.

Increased beta-amyloid (Abeta) production and its aggregation to the oligomeric state is considered to be a major cause of Alzheimer's disease (AD). Therefore, reducing Abeta-induced neurotoxicity could provide a suitable means of prevention or intervention in the disease course of AD. The neuroprotective effects of isolates from Callistemon lanceolatus DC. (Myrtaceae) against Abeta were evaluated using PC12 cells. To evaluate the effects of Abeta on apoptotic cell death and the effects of Bcl-2 family proteins and caspase-3, TUNEL assays and Western blotting were performed, respectively. Substantial fractionation and purification of the EtOAc-soluble extract of the aerial parts of C. lanceolatus afforded six flavonoids, 4',5-dihydroxy-6,8-dimethyl-7-methoxyflavanone (1), eucalyptin (2), 8-demethyleucalyptin (3), sideroxylin (4), syzalterin (5), and quercetin (6). Compounds 1, 5, and 6 were found to protect PC12 cells effectively against Abeta-induced toxicity. In particular, compound 1 showed the most promising neuroprotective effect with an ED (50) value of 6.7 microM in terms of decreasing Abeta-induced apoptotic cell death, and this was accompanied by a decrease in caspase-3 activation and an increase in Bcl-2/Bax ratio. These results suggest that compound 1 could be developed as a candidate anti-AD agent due to its attenuation of Abeta-induced apoptotic cell death.