Berberine, A Phytoalkaloid, Inhibits Inflammatory Response Induced by LPS through NF-Kappaß Pathway: Possible Involvement of the IKKα.

Berberine (BBR), a plant alkaloid, is known for its therapeutic properties of anticancer, cardioprotective, antidiabetic, hypolipidemic, neuroprotective, and hepatoprotective activities. The present study was to determine the molecular mechanism of BBR's pharmacological activity in human monocytic (THP-1) cells induced by arachidonic acid (AA) or lipopolysaccharide (LPS). The effect of BBR on AA/LPS activated proinflammatory markers including TNF-α, MCP-1, IL-8 and COX-2 was measured by ELISA or quantitative real-time PCR. Furthermore, the effect of BBR on LPS-induced NF-κB translocation was determined by immunoblotting and confocal microscopy. AA/ LPS-induced TNF-α, MCP-1, IL-6, IL-8, and COX-2 markers were markedly attenuated by BBR treatment in THP-1 cells by inhibiting NF-κB translocation into the nucleus. Molecular modeling studies suggested the direct interaction of BBR to IKKα at its ligand binding site, which led to the inhibition of the LPS-induced NF-κB translocation to the nucleus. Thus, the present study demonstrated the anti-inflammatory potential of BBR via NF-κB in activated monocytes, whose interplay is key in health and in the pathophysiology of atherosclerotic development in blood vessel walls. The present study findings suggest that BBR has the potential for treating various chronic inflammatory disorders.

Variable Secondary Metabolite Profiles Across Cultivars of Curcuma longa L. and C. aromatica Salisb.

Background: Curcuma spp. (Zingiberaceae) are used as a spice and coloring agent. Their rhizomes and essential oils are known for medicinal properties, besides their use in the flavoring and cosmetic industry. Most of these biological activities were attributed to volatile and nonvolatile secondary metabolites present in the rhizomes of Curcuma spp. The metabolite variations among the species and even cultivars need to be established for optimized use of Curcuma spp. Objectives: We compared the phytochemical profiles of rhizomes and their essential oils to establish the variability among seven cultivars: five of Curcuma longa L. (Alleppey Supreme, Duggirala Red, Prathibha, Salem, Suguna) and two of C. aromatica Salisb. (Kasturi Araku, Kasturi Avidi). The GC-MS and LC-MS-based analyses were employed to profile secondary metabolites of these selected cultivars. Methods: Rhizomes of Curcuma spp. were subjected to hydro-distillation to collect essential oil and analyzed by GC-MS. The methanol extracts of fresh rhizomes were subjected to LC-MS analyses. The compounds were identified by using the relevant MS library databases as many compounds as possible. Results: The essential oil content of the cultivars was in the range of 0.74-1.62%. Several compounds were detected from the essential oils and rhizome extracts by GC-MS and LC-MS, respectively. Of these, 28 compounds (13 from GCMS and 15 from LCMS) were common in all seven cultivars, e.g., α-thujene, and diarylheptanoids like curcumin. Furthermore, a total of 39 new compounds were identified from C. longa L. and/or C. aromatica Salisb., most of them being cultivar-specific. Of these compounds, 35 were detected by GC-MS analyses of essential oils, 1,2-cyclohexanediol, 1-methyl-4-(1-methylethyl)-, and santolina alcohol, to name a few. The other four compounds were detected by LC-MS of the methanolic extracts of the rhizomes, e.g., kaempferol-3,7-O-dimethyl ether and 5,7,8-trihydroxy-2',5'-dimethoxy-3',4'-methylene dioxyisoflavanone. Conclusions: We identified and recorded the variability in the metabolite profiles of essential oils and whole rhizome extracts from the seven cultivars of Curcuma longa L. and C. aromatica Salisb. As many as 39 new metabolites were detected in these seven Indian cultivars of Curcuma spp. Many of these compounds have health benefits.

Interactions of different lipoproteins with supported phospholipid raft membrane (SPRM) patterns to understand similar in-vivo processes.

To better understand how lipoproteins interact and enter endothelium and participate in cellular processes, we investigated preferential lipid partitioning of triglyceride rich lipoproteins (TGRL), chylomicrons (CM), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and their lipolysis products using supported phospholipid raft membrane (SPRM) patterns. We prepared SPRM patterns with Texas red labeled phospholipid patterns and Marina blue labeled raft patterns and added Atto-520 labeled lipoproteins (TGRL, CM, VLDL, LDL) and their lipolysis products in separate experiments and characterized these interactions using fluorescence microscopy. We observed that VLDL and LDL preferentially interacted with raft patterns. In contrast the TGRL and lipolysed products of TGRL interacted with both the patterns, slightly elevated preference for raft patterns and CM and its lipolysis products showed greater affinity to phospholipid patterns. The clear preference of VLDL and LDL for raft patterns suggests that these lipoproteins associate with cholesterol and sphingomyelin rich lipid micro-domains during their early interactions with endothelial cells, leading to atherosclerosis.

Metabolomics of Withania somnifera (L.) Dunal: Advances and applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera L. (Solanaceae), commonly known as Ashwagandha or Indian ginseng, is used in Ayurveda (Indian system of traditional medicine) for vitality, cardio-protection and treating other ailments, such as neurological disorders, gout, and skin diseases. AIM OF THE REVIEW: We present a critical overview of the information on the metabolomics of W. somnifera and highlight the significance of the technique for use in quality control of medicinal products. We have also pointed out the use of metabolomics to distinguish varieties and to identify best methods of cultivation, collection, as well as extraction. MATERIAL AND METHODS: The relevant information on medicinal value, phytochemical studies, metabolomics of W. somnifera, and their applications were collected from a rigorous electronic search through scientific databases, including Scopus, PubMed, Web of Science and Google Scholar. Structures of selected metabolites were from the PubChem. RESULTS: The pharmacological activities of W. somnifera were well documented. Roots are the most important parts of the plant used in Ayurvedic preparations. Stem and leaves also have a rich content of bioactive phytochemicals like steroidal lactones, alkaloids, and phenolic acids. Metabolomic studies revealed that metabolite profiles of W. somnifera depended on plant parts collected and the developmental stage of the plant, besides the season of sample collection and geographical location. The levels of withanolides were variable, depending on the morpho/chemotypes within the species of W. somnifera. Although studies on W. somnifera were initiated several years ago, the complexity of secondary metabolites was not realized due to the lack of adequate and fool-proof technology for phytochemical fingerprinting. Sophistications in chromatography coupled to mass spectrometry facilitated the discovery of several new metabolites. Mutually complementary techniques like LC-MS, GC-MS, HPTLC, and NMR were employed to obtain a comprehensive metabolomic profile. Subsequent data analyses and searches against spectral databases enabled the annotation of signals and dereplication of metabolites in several numbers without isolating them individually. CONCLUSIONS: The present review provides a critical update of metabolomic data and the diverse application of the technique. The identification of parameters for standardization and quality control of herbal products is essential to facilitate mandatory checks for the purity of formulation. Such studies would enable us to identify the best geographical location of plants and the time of collection. We recommend the use of metabolomic analysis of herbal products based on W. somnifera for quality control as well as the discovery of novel bioactive compounds.

Dry leaf extracts of Tinospora cordifolia (Willd.) Miers attenuate oxidative stress and inflammatory condition in human monocytic (THP-1) cells.

BACKGROUND: Tinospora cordifolia (Willd.) Miers is known for its therapeutic value in Indian traditional medicine for treating diabetes, rheumatoid arthritis, jaundice and cardiac diseases. However, information regarding its protective role against inflammatory diseases at the molecular level is limited. PURPOSE: The objective of the present work is to study the antioxidant and anti-inflammatory effect of alcoholic and water extracts of T. cordifolia (Willd.) Miers leaves in activated human monocytic THP-1 cells. STUDY DESIGN/METHODS: Phytochemical analyses of the dry leaf extracts of T. cordifolia (Willd.) Miers prepared using the solvents alcohol (TCAE) or water (TCWE) are performed employing spectrophotometric methods for estimating total phenolic and flavonoid content, and the plant material was authenticated by detecting T. cordifolia (Willd.) Miers metabolite biomarkers using LC-MS/MS. Arachidonic acid (AA)- and lipopolysaccharide (LPS)-activated human monocytic (THP-1) cells were used as experimental models to investigate the antioxidant and anti-inflammatory activities of the plant extracts. Arachidonic acid (AA)-induced reactive oxygen species (ROS) in THP-1 cells were monitored by confocal microscopy/spectrofluorimetry and transcript of antioxidant enzyme catalase (CAT), by quantitative real time PCR. Lipopolysaccharide (LPS)-induced proinflammatory marker like TNF-α at transcription and protein levels in THP-1 cells were measured by quantitative real-time PCR or ELISA respectively. Further, the effect of T. cordifolia (Willd.) Miers extracts on LPS-induced NF-κB translocation, and IκB and P-IκB protein levels, were studied by immunoblotting and confocal microscopy. RESULTS: T. cordifolia (Willd.) Miers extracts exhibited significant amounts of total phenolic and flavonoid content, and LC-MS/MS analyses detected tinosponone, a TC-specific clerodane-derived diterpene. Both types of extracts attenuated AA-induced ROS generation via enhancing catalase enzyme activity in THP-1 cells. Real time PCR and ELISA experiments revealed that the elevated levels of LPS-induced TNF-α was remarkably attenuated in THP-1 cells pretreated with T. cordifolia (Willd.) Miers extracts. Western blot and confocal microscopy showed that the alcoholic extract's anti-inflammatory activity by attenuating NF-κB translocation into the nucleus in LPS-activated THP-1 cells via the inhibition of IκB degradation in the cytosol. CONCLUSION: Our findings suggest that T. cordifolia (Willd.) Miers dry leaf extracts possess antioxidant and anti-inflammatory properties via upregulation of antioxidant enzymes and attenuation of NF- κB nuclear translocation in activated human monocytic (THP-1) cells, therefore the present study supports our proposed molecular basis for the traditional use of T. cordifolia (Willd.) Miers for treating various inflammatory diseases.

Terpenes and isoprenoids: a wealth of compounds for global use.

MAIN CONCLUSION: Role of terpenes and isoprenoids has been pivotal in the survival and evolution of higher plants in various ecoregions. These products find application in the pharmaceutical, flavor fragrance, and biofuel industries. Fitness of plants in a wide range of environmental conditions entailed (i) evolution of secondary metabolic pathways enabling utilization of photosynthate for the synthesis of a variety of biomolecules, thereby facilitating diverse eco-interactive functions, and (ii) evolution of structural features for the sequestration of such compounds away from the mainstream primary metabolism to prevent autotoxicity. This review summarizes features and applications of terpene and isoprenoid compounds, comprising the largest class of secondary metabolites. Many of these terpene and isoprenoid biomolecules happen to be high-value bioproducts. They are essential components of all living organisms that are chemically highly variant. They are constituents of primary (quinones, chlorophylls, carotenoids, steroids) as well as secondary metabolism compounds with roles in signal transduction, reproduction, communication, climatic acclimation, defense mechanisms and more. They comprise single to several hundreds of repetitive five-carbon units of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, there are two pathways that lead to the synthesis of terpene and isoprenoid precursors, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. The diversity of terpenoids can be attributed to differential enzyme and substrate specificities and to secondary modifications acquired by terpene synthases. The biological role of secondary metabolites has been recognized as pivotal in the survival and evolution of higher plants. Terpenes and isoprenoids find application in pharmaceutical, nutraceutical, synthetic chemistry, flavor fragrance, and possibly biofuel industries.

Effects of extracts prepared from modified porous poly(ether imide) microparticulate absorbers on cytotoxicity, macrophage differentiation and proinflammatory behavior of human monocytic (THP-1) cells.

Remaining uremic toxins in the blood of chronic renal failure patients represent one central challenge in hemodialysis therapies. Highly porous poly(ether imide) (PEI) microparticles have been recently introduced as candidate absorber materials, which show a high absorption capacity for uremic toxins and allow hydrophilic surface modification suitable for minimization of serum protein absorption. In this work, the effects of extracts prepared from PEI microparticles modified by nucleophilic reaction with low molecular weight polyethylene imine (Pei) or potassium hydroxide (KOH), on human monocytic (THP-1) cells are studied. The obtained results suggested that the extracts of Pei and KOH modified PEI absorbers have no negative effect on THP-1 cell viability and do not initiate the critical differentiation towards macrophages. The extracts did not enhance transcript or protein levels of investigated proinflammatory markers in THP-1 cells, namely, TNFµ, MCP1, IL6 and IL8. Based on these findings such modified PEI microparticles should be qualified for further pre-clinical evaluation i.e. in an in vivo animal experiment.

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata.

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest - Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ±â€¯11 nM) and chymotrypsin (Ki of 446 ±â€¯9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 µg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

Influence of nanoporous poly(ether imide) particle extracts on human aortic endothelial cells (HAECs).

Accumulated uremic toxins like indoxyl sulphate, hippuric acid and p-cresyl sulphates in renal failure patients stimulate proinflammatory effects, and consequently kidney and cardiovascular diseases. Low clearance rate of these uremic toxins from the blood of uremic patients by conventional techniques like hemodialysis is due to their strong covalent albumin binding (greater than 95%) and hydrophobic nature, which led to alternatives like usage of hydrophobic adsorber's in removing these toxins from the plasma of kidney patients. Polymers like polyethylene, polyurethane, polymethylmethacrylate, cellophane and polytetrafluoroethylene were already in use as substitutes for metal devices as dialysis membranes. Among new synthetic polymers, one such ideal adsorber material are highly porous microparticles of poly(ether imide) (PEI) with diameters in the range from 50-180µm and a porosity around 88±2% prepared by a spraying and coagulation process.It is essential to make sure that these synthetic polymers should not evoke any inflammatory or apoptotic response during dialysis. Therefore in our study we evaluated in vitro effect of PEI microparticle extracts in human aortic endothelial cells (HEACs) concerning toxicity, inflammation and apoptosis. No cell toxicity was observed when HAECs were treated with PEI extracts and inflammatory/apoptotic markers were not upregulated in presence of PEI extracts. Our results ensure biocompatibility of PEI particles and further hemocompatibility of particles will be tested.

Adsorption capacity of poly(ether imide) microparticles to uremic toxins.

Uremia is a phenomenon caused by retention of uremic toxins in the plasma due to functional impairment of kidneys in the elimination of urinary waste products. Uremia is presently treated by dialysis techniques like hemofiltration, dialysis or hemodiafiltration. However, these techniques in use are more favorable towards removing hydrophilic than hydrophobic uremic toxins. Hydrophobic uremic toxins, such as hydroxy hipuric acid (OH-HPA), phenylacetic acid (PAA), indoxyl sulfate (IDS) and p-cresylsulfate (pCRS), contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease. Therefore, objective of the present study is to test adsorption capacity of highly porous microparticles prepared from poly(ether imide) (PEI) as an alternative technique for the removal of uremic toxins. Two types of nanoporous, spherically shaped microparticles were prepared from PEI by a spraying/coagulation process.PEI particles were packed into a preparative HPLC column to which a mixture of the four types of uremic toxins was injected and eluted with ethanol. Eluted toxins were quantified by analytical HPLC. PEI particles were able to adsorb all four toxins, with the highest affinity for PAA and pCR. IDS and OH-HPA showed a partially non-reversible binding. In summary, PEI particles are interesting candidates to be explored for future application in CKD.

Effect of extracts of poly(ether imide) microparticles on cytotoxicity, ROS generation and proinflammatory effects on human monocytic (THP-1) cells.

Current haemodialysis techniques are not capable to remove efficiently low molecular weight hydrophobic uremic toxins from the blood of patients suffering from chronic renal failure. With respect to the hydrophobic characteristics and the high level of protein binding of these uremic toxins, hydrophobic adsorber materials might be an alternative to remove these substances from the plasma of the chronic kidney disease (CKD) patients. Here nanoporous microparticles prepared from poly(ether imide) (PEI) with an average diameter of 90 ± 30 µm and a porosity around 88 ± 2% prepared by a spraying/coagulation process are considered as candidate adsorber materials. A prerequisite for the clinical application of such particles is their biocompatibility, which can be examined i.e. indirectly in cell culture experiments with the particles' extracts. In this work we studied the effects of aqueous extracts of PEI microparticles on the viability of THP-1 cells, a human leukemia monocytic cell line, as well as their macrophage differentiation, reactive oxygen species (ROS) generation and inflammation.A high cell viability of around 99 ± 18% and 99 ± 5% was observed when THP-1 cells were cultured in the presence of aqueous extracts of the PEI microparticles in medium A and medium B respectively. The obtained microscopic data suggested that PEI particle extracts have no significant effect on cell death, oxidative stress or differentiation to macrophages. It was further found that the investigated proinflammatory markers in THP-1 cells were not up-regulated. These results are promising with regard to the biocompatibility of PEI microparticles and in a next step the hemocompatibility of the microparticles will be examined.

Ocimum sanctum leaf extracts attenuate human monocytic (THP-1) cell activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ocimum sanctum (OS), commonly known as Holy basil/Tulsi, has been traditionally used to treat cardiovascular diseases (CVD) and manage general cardiac health. The present study is designed to evaluate the antiinflammatory effect of O. sanctum and its phenolic compound and eugenol (EUG) in human monocytic (THP-1) cells and validate its traditional use for treating cardiovascular diseases. MATERIALS AND METHODS: The phytochemical analysis of alcoholic and water extracts of OS-dry leaves (OSAE and OSWE) was done using LC-QTOF-MS. A phenolic compound, EUG was quantified in both OSAE and OSWE by an LC-MS technique using a mass hunter work station software quantitative analysis system. The effect of both OSAE, OSWE, pure compound EUG and positive control imatinib (IMT) was investigated in THP-1 cells by studying the following markers: lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF-α) secretion by ELISA, gene expression of inflammatory markers (TNF-α, IL-6, MIP-1α and MCP-1) by real time PCR and translocation of nuclear factor kappa B (NF-κB) by confocol microscopy. Furthermore, the effect of the extracts, EUG and IMT, was studied on phorbol-12-myristate-13-acetate (PMA) induced monocyte to macrophage differentiation and gene expression of CD14, TLR2 and TLR4. RESULTS: The LC-MS analysis of OSAE and OSWE revealed the presence of several bioactive compounds including eugenol. Quantitative analysis revealed that OSAE and OSWE had EUG of 12 ng/mgdwt and 19 ng/mgdwt respectively. OSAE, OSWE (1 mg dwt/mL) pure compound EUG (60 µg/mL) and positive control IMT (20 µg/mL) showed marked inhibition on LPS induced TNF-α secretion by THP-1 cells. At the selected concentration, the plant extracts, EUG and IMT inhibited gene expression of cytokines and chemokines (IL-6, TNF-α, MIP-1α, MCP-1) and translocation of NF-κB-p65 to the nuclei. In addition, they showed significant inhibition on PMA induced monocyte to macrophage differentiation and the gene expression of CD14, TLR2 and TLR4 markers. CONCLUSION: The result of the present study validated traditional use of Ocimum sanctum for treating cardiovascular disease for the first time by testing antiinflammatory activity of Ocimum sanctum in acute inflammatory model, LPS induced THP-1 cells. The plant extracts showed significant antiinflammatory activity, however, further to be evaluated using chronic inflammatory animal models like diabetic or apolipoprotein E-deficient mice to make it evidence based medicine. The phenolic compound eugenol (60 µg/mL) showed significant antiinflammatory activity. However the amount of eugenol present in 1mg of OSAE and OSWE (12 ng/mg and 19 ng/mg dwt respectively) used for cell based assays was very low. It suggests that several other metabolites along with eugenol are responsible for the efficacy of the extracts.

Stem-bark of Terminalia arjuna attenuates human monocytic (THP-1) and aortic endothelial cell activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia arjuna - stem bark extract is traditionally used as cardiotonic in Ayurvedic medicine. AIM OF THE STUDY: The present study was aimed to evaluate the molecular basis for cardioprotective potential of Terminalia arjuna (TA) stem bark, using cell cultures of human monocytic (THP-1) and human aortic endothelial cells (HAECs). MATERIALS AND METHODS: Inhibitory effect of alcoholic (TAAE) and aqueous (TAWE) extracts of TA-stem bark was assessed on human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LpL) and lipid peroxidation in rat (wistar) liver and heart homogenates. The patterns of H2O2 induced reactive oxygen species (ROS) generation were observed by confocal microscopy. The activities of antioxidant enzymes and reducing power of the cells were measured in a microplate reader. Gene transcripts of proinflammatory markers in THP-1 and HAECs were assayed by real time PCR and levels of inflammatory protein markers by ELISA or flow cytometry. Phytochemical analyses of TAAE and TAWE were done using liquid chromatography, coupled to mass spectrometry (LC-MS). RESULTS: TAAE and TAWE inhibited the lipid peroxidation and HMG-CoA reductase but had no effect on LpL. Both the extracts attenuated H2O2 mediated ROS generation in THP-1 cells by promoting catalase (CAT), glutathione peroxidase (GPx) activities, and by sustaining cellular reducing power. TAAE was highly effective in attenuating proinflammatory gene transcripts in THP-1 cells and HAECs, whereas the response to TAWE depended on the type of transcript and cell type. Both extracts decreased the levels of typical inflammatory marker proteins, viz. LPS induced tumor necrosis factor (TNF)-α secreted by THP-1 cells and TNF-α induced cell surface adhesion molecules on HAECs, namely vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Phytochemical analyses indicated the richness in phenolic compounds and terpenes of TAAE and TAWE, while revealing variability in their metabolite profile. CONCLUSION: Our study scientifically validates the antioxidative and antiinflammatory properties of Terminalia arjuna stem bark. The marked effects on cultured human monocytic and aortic endothelial cells (HAEC) provide the biochemical and molecular basis for therapeutic potential of TA-stem bark against cardiovascular diseases (CVD).

Postprandial apoE isoform and conformational changes associated with VLDL lipolysis products modulate monocyte inflammation.

OBJECTIVE: Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. METHODS AND RESULTS: We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. CONCLUSION: Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

Gum resin of Boswellia serrata inhibited human monocytic (THP-1) cell activation and platelet aggregation.

ETHNOPHARMACOLOGICAL RELEVANCE: Stem bark gum resin extract of Boswellia serrata is traditionally used in India for its hemostatic, antiinflammatory and cardiovascular health effects and it is named as Sallaki in Ayurvedic medicine. AIM OF THE STUDY: This study was conducted to evaluate the antioxidative and antithrombotic properties of stem bark gum resin extracts of Boswellia serrata (BS). MATERIALS AND METHODS: The inhibitory activity of the BSWE and BSAE on FeCl(3) induced lipid peroxidation (in vitro) in rat liver and heart homogenates was measured spectrophotometrically. Their effect on H(2)O(2) induced reactive oxygen species (ROS) generation in human monocytic (THP-1) cells was investigated by tracking intensity of a cell permeable fluorescent dye, H(2)DCFDA and subjecting the cell samples to confocal microscopy. Further, the effect of BSAE and BSWE on ADP-induced platelet aggregation was assessed using a multimode detection plate reader, plasma coagulation times using an automated blood coagulation analyzer and on human blood clotting factors Xa and XIa using chromogenic substrate. Phytomarker analysis of the water (BSWE) and hydroalcoholic (BSAE) extracts of BS-gum resin was done through HPLC using a standard compound AKßBA. RESULTS: BSAE and BSWE inhibited, to varied extents, the lipid peroxidation in liver (80%) and heart (50%) tissue homogenates of male Wistar rats. Further, BSAE (30 µg dwt/mL) and BSWE (300 µg dwt/mL) attenuated ≥ 60% of H(2)O(2) mediated ROS generation in THP-1 cells. In case of standard compounds, ascorbate (20 µg dwt/mL) and butylated hydroxytoluene (BHT) (10 µg dwt/mL) completely scavenged ROS in the cells. BSAE and BSWE at 3 mg dwt/mL completely inhibited ADP induced platelet aggregation and activities were comparable to 20 µg/mL of heparin. The extracts also showed very high activity in prolonging coagulation time periods. Both types of extracts extended prothrombin time (PT) from ∼13 to >60s and activated partial thromboplastin time (APTT) from ∼32s to >90s. BSAE inhibited clotting factors Xa and XIa remarkably at 6 µg of dwt where as BSWE did not show much effect on FXa and showed 30% inhibition on FXIa at 120 µg. 10 µg of heparin was required to inhibit about 30% activity of the above factors. HPLC analyses suggested that BSAE and BSWE had AKßBA of 9% (w/w) and 7.8% (w/w) respectively. CONCLUSION: Present study demonstrated antioxidant and antithrombotic anticoagulant activities of water and hydroalcoholic extracts of Boswellia serrata's gum resin. We suggest that BS-gum resin as a good source for lead/therapeutic compounds possessing antioxidant, antiplatelet and anticoagulant activities.

VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation.

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL

Development of the light-harvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii is regulated by the novel Tla1 gene.

The Chlamydomonas reinhardtii tla1 (truncated light-harvesting chlorophyll antenna size) mutant was generated upon DNA insertional mutagenesis and shown to specifically possess a smaller than wild type (WT) chlorophyll antenna size in both photosystems. Molecular and genetic analysis revealed that the exogenous plasmid DNA was inserted at the end of the 5' UTR and just prior to the ATG start codon of a hitherto unknown nuclear gene (termed Tla1), which encodes a protein of 213 amino acids. The Tla1 gene in the mutant is transcribed with a new 5' UTR sequence, derived from the 3' end of the transforming plasmid. This replacement of the native 5' UTR and promoter regions resulted in enhanced transcription of the tla1 gene in the mutant but inhibition in the translation of the respective tla1 mRNA. Transformation of the tla1 mutant with WT Tla1 genomic DNA successfully rescued the mutant. These results are evidence that polymorphism in the 5' UTR of the Tla1 transcripts resulted in the tla1 phenotype and that expression of the Tla1 gene is a prerequisite for the development/assembly of the Chl antenna in C. reinhardtii. A blast search with the Tla1 deduced amino acid sequence

Apolipoprotein E3- and nitric oxide-dependent modulation of endothelial cell inflammatory responses.

OBJECTIVE: Although apolipoprotein E3 (apoE3) is known to be atheroprotective, its mechanisms of protection in endothelial cells remain unclear. METHODS AND RESULTS: Cultured human aortic endothelial cells were stimulated with tumor necrosis factor (TNF)-alpha in the presence of human recombinant apoE3 solubilized in dimyristoyl phosphatidylcholine liposomes. Using flow cytometry and real-time polymerase chain reaction, a significant increase of inflammatory cell adhesion proteins (vascular cell adhesion molecule-1 and E-Selectin), and MCP-1, interleukin-8, and intercellular adhesion molecule-1 gene expression was observed within 5 hours of TNF-alpha exposure, which was markedly attenuated in cells coincubated with apoE3. Treatment with apoE4 resulted in increased inflammatory gene expression relative to either TNF treatment alone or TNF + apoE3 treatment. NO synthase inhibition experiments demonstrated NO to be an active participant in the actions of both TNF and apoE. To clarify the role of NO, dose-response experiments were performed with 0.03 to 300 micromol/L DEA-NONOate. Using flow cytometry and real-time polymerase chain reaction, a modulatory role of NO in TNF-induced endothelial cell activation was observed. CONCLUSIONS: These data suggest a role of vascular wall apoE3 to balance the intracellular redox state in injured endothelial cells via NO-dependent pathways.

C-terminal interactions of apolipoprotein E4 respond to the postprandial state.

Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state.