Aqueous Phenol Extraction Research Articles

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Cocoa Shell Phenolic Compounds Preserve Mitochondrial Function and Insulin Sensitivity in Adipocytes by Attenuating Their Inflammatory Interplay with Macrophages (FS15-06-19)

ObjectivesThe objective was to evaluate the effect of an aqueous phenolic extract from cocoa shell (CAE) and its main phenolic compounds to prevent the adipogenesis-induced loss of mitochondrial function and insulin sensitivity, targeting the inflammatory interplay macrophages-adipocytes in vitro. MethodsCAE and its main phenolic compounds (protocatechuic acid, procyanidin B2, and epicatechin) were tested in cell culture of RAW264.7 macrophages and 3T3-L1 mature adipocytes. Macrophages were stimulated with lipopolysaccharide (LPS, 1 μg/mL). Adipocytes were treated with the conditioned media (CM) from LPS-stimulated macrophages. Biomarkers for inflammation, mitochondrial function, and insulin sensitivity were determined after 24 h co-treatment with CAE and LPS/CM using chemical and immunochemical techniques. ResultsLPS-stimulated macrophages showed reduced inducible nitric oxide synthase and cyclooxygenase-2 expression and lowered NO and pro-inflammatory cytokines release when treated with CAE and pure phenolics. Inflammatory crosstalk created by stimulating adipocytes with CM was arrested; CAE diminished tumor necrosis factor-α (67%, p < 0.05) and highly promoted adiponectin secretion (12.3-fold, p < 0.05). Mitochondrial function, measured by reactive oxygen species production, mitochondrial content, and activity, was preserved in CM-treated adipocytes through up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-α expression (46%, p < 0.05). Significant (p < 0.05) increases in insulin receptor (9-fold, p < 0.05), phosphoinositide 3-kinase (3-fold, p < 0.05), protein kinase B (4-fold, p < 0.05) phosphorylation and a decrease in insulin receptor substrate 1 serine phosphorylation induced increased glucose uptake (34%, p < 0.05) and glucose transporter 4 translocation (14-fold, p < 0.05) in CM-induced adipocytes. ConclusionsCocoa shell extract polyphenols reduced macrophages-adipocytes inflammatory interaction preventing mitochondrial dysfunction and insulin resistance. For the first time, cocoa shell aqueous extract showed a positive effect on adipogenesis and inflammation-related disorders. Funding SourcesUniversidad Autónoma de Madrid-Banco Santander, United States Department of Agriculture-National Institute of Food and Agriculture-HATCH, and the Spanish Ministry of Science and Innovation.

Front cover: Cocoa Shell Aqueous Phenolic Extract Preserves Mitochondrial Function and Insulin Sensitivity by Attenuating Inflammation between Macrophages and Adipocytes In Vitro

Front cover: Cocoa Shell Aqueous Phenolic Extract Preserves Mitochondrial Function and Insulin Sensitivity by Attenuating Inflammation between Macrophages and Adipocytes In Vitro

Cocoa Shell Aqueous Phenolic Extract Preserves Mitochondrial Function and Insulin Sensitivity by Attenuating Inflammation between Macrophages and Adipocytes In Vitro.

The aim is to assess the action of an aqueous extract from cocoa shell (CAE) and its main phenolic compounds to prevent the loss of obesity-induced mitochondrial function and insulin sensitivity, targeting inflammation between macrophages-adipocytes in vitro. CAE (31-500µgmL-1 ) inhibits 3T3-L1 adipocytes lipid accumulation and induces browning during differentiation. LPS-stimulated RAW264.7 macrophages show reduced inducible nitric oxide synthase and cyclooxygenase-2 expression and lowered pro-inflammatory cytokine production when treated with CAE and pure phenolics. Inflammatory crosstalk created by stimulating adipocytes with macrophage-conditioned media (CM) is arrested; CAE diminishes tumor necrosis factor-α (67%) and promotes adiponectin secretion (12.3-fold). Mitochondrial function, measured by reactive oxygen species production, mitochondrial content, and activity, is preserved in CM-treated adipocytes through up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-α expression. Increases in insulin receptor (9-fold), phosphoinositide 3-kinase (3-fold), protein kinase B (4-fold) phosphorylation, and a decrease in insulin receptor substrate 1 serine phosphorylation induce increased glucose uptake (34%) and glucose transporter 4 translocation (14-fold) in CM-induced adipocytes. CAE phenolics promote a beige phenotype in adipocytes. Macrophages-adipocytes inflammatory interaction is reduced preventing mitochondrial dysfunction and insulin resistance. For the first time, CAE shows a positive effect on adipogenesis and inflammation-related disorders.

Phenolic compounds from Syzygium cumini (L.) Skeels leaves: Extraction and membrane purification

A hybrid process consisting of extraction and microfiltration was proposed in this study for producing purified, clear and stable aqueous phenolic extract from jamun (Syzygium cumini (L.) Skeels) leaf. Response surface methodology was successfully used for optimization of extraction conditions. Pseudo-first order kinetic model successfully described the extraction of total polyphenols from jamun leaves, with the activation energy determined as 9.5 kJ/mol based on the Arrhenius model. The kinetic constants were used to study the kinetic and thermodynamic compensations of extraction of TPC from jamun leaves. Applying the statistical criterion, the kinetic and thermodynamic compensations were concluded to be real and the extraction process was controlled by entropy. A total of ten phenolic compounds including six phenolic acids (tannic acid, gallic acid, ellagic acid, caffeic acid, ferulic acid and p-coumaric acid) and four flavonoids (catechin, epicatechin, quercetin and myricetin 3-O-rhamnoside) were identified and quantified in jamun leaf extract obtained under optimum extraction conditions. The selection of appropriate membrane in the microfiltration step was a critical aspect. To observe the effect of membrane pore size on the permeate flux and permeate quality, leaf extracts were then microfiltered using four different microfiltration membranes (0.1, 0.22, 0.45, and 0.8 μm) under batch concentration mode. The flux decline was successfully described by the Hermia’s cake filtration model. The stability of clarified extract was investigated at 4 °C for 45 days. The 0.45 μm microfiltration membrane was suggested for the clarification of jamun leaf extract in order to achieve high flux, polyphenol recovery, extract purity and improved storage stability.

Phytochemical screening and cytotoxicity studies of Chrysophyllum pruniforme Pierre ex Engl. barks

Background:Chrysophyllum pruniforme of family sapotaceae is a plant used in traditional medicine in Gabon.Materials and Methods:In this study, C. pruniforme barks were subjected to phytochemical screening and cytotoxicity investigations. Different concentrations of aqueous and total phenolic extract were tested on mice and on human erythrocytes.Results:Phytochemical screening of C. pruniforme barks revealed the presence of flavonoids, saponins, and tannins, reducing sugars, polyphenols and traces of anthraquinones. When tested in vitro, aqueous and the phenolic extracts showed hemolytic activities on human erythrocytes with phenolic compounds being more cytotoxic than aqueous extracts. In vivo study of toxicity, allowed to determine the LD50 at 90 mg/kg for the doses of 50, 150 and 250 mg/kg of body weight.Conclusion:These data indicate in one hand that C. pruniforme is rich in phenolic compounds and that the aqueous and total phenolic extracts could be considered as toxic for mice and maybe potentially toxic to humans in the other hand.

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell(1, 2). LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli(3-5), and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence(6-8). For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods(9). LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann(10), is ideal for LPS extraction.

Multiple γ-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

A highly hydrophilic, glutamate-rich protein was identified in the aqueous phenol extract from the cytosolic fraction of brine shrimp (Artemia franciscana) diapausing cysts and termed Artemia phenol soluble protein (PSP). Mass spectrometric analysis revealed the presence of many protein peaks around m/z 11,000, separated by 129 atomic mass units; this value corresponds to that of glutamate, which is strongly suggestive of heterogeneous polyglutamylation. Polyglutamylation has long been known as the functionally important post-translational modification of tubulins, which carry poly(l-glutamic acid) chains of heterogeneous length branching off from the main chain at the γ-carboxy groups of a few specific glutamate residues. In Artemia PSP, however, Edman degradation of enzymatic peptides revealed that at least 13, and presumably 16, glutamate residues were modified by the attachment of a single l-glutamate, representing a hitherto undescribed type of post-translational modification: namely, multiple γ-glutamylation or the addition of a large number of glutamate residues along the polypeptide chain. Although biological significance of PSP and its modification is yet to be established, suppression of in vitro thermal aggregation of lactate dehydrogenase by glutamylated PSP was observed.

An inflammatory polypeptide complex from Staphylococcus epidermidis: isolation and characterization.

Staphylococcus epidermidis releases factors that activate the HIV-1 long terminal repeat, induce cytokine release, and activate nuclear factor B in cells of macrophage lineage. The active material had a mass of 34,500 daltons, was inactivated by proteases and partitioned into the phenol layer on hot aqueous phenol extraction, and thus was termed phenol-soluble modulin (PSM). High performance liquid chromatography (HPLC) of crude PSM yielded two peaks of activity designated PSM peak 1 and peak 2. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy indicated the presence of two components in peak 1, which were designated PSM and PSM. Peak 2 contained a single component, designated PSM. Separation of PSM and PSM in peak 1 could be achieved by a second HPLC procedure. The structure of each component was determined by amino acid sequence analysis and identification and sequencing of their genes. PSM, PSM, and PSM were 22-, 44-, and 25-amino acid, respectively, strongly hydrophobic polypeptides. PSM was identified as Staphylococcus epidermidis delta toxin, whereas PSM and PSM exhibited more distant homology to previously described staphylococcal toxins. They appeared to exist as a complex or aggregate with activity greater than the component parts. The properties of the S. epidermidis PSMs suggest that they may contribute to the systemic manifestations of Gram-positive sepsis.

Lactobacilli and vaginal host defense: activation of the human immunodeficiency virus type 1 long terminal repeat, cytokine production, and NF-kappaB.

Lactobacilli, a component of the normal vaginal flora, can activate the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) in the Jurkat T lymphocyte and THP-1 macrophage cell lines. Activation of the LTR in Jurkat cells was strongly enhanced by vanadate and inhibited by catalase, implicating H2O2. In contrast, activation in THP-1 cells occurred in the absence of vanadate and was unaffected by catalase. The active material partitioned into the phenol layer on hot aqueous phenol extraction. Lactobacilli also increased tumor necrosis factor-alphaand interleukin-1betaproduction and activated NF-kappaB in THP-1 cells and increased tumor necrosis factor-alphaproduction by human monocytes. Human vaginal fluid specimens had comparable properties, which correlated with their bacterial content. These findings suggest the presence in vaginal fluid of agent(s) derived from indigenous bacteria that can activate the HIV-1 LTR, cytokine production, and NF-kappaB in cells of macrophage lineage, with possible influence on vaginal physiology and host defense.

Structure of the O-specific polysaccharide of Acinetobacter baumannii O5 containing 2-acetamido-2-deoxy-D-galacturonic acid.

A polysaccharide containing 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-fucose (FucNAc), and 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid-free, isolated cell walls of the reference strain for Acinetobacter baumannii serogroup O5, by mild acid hydrolysis of the extract and chromatography of the water-soluble products on Sephadex G-50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide: [table: see text]

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O2 containing 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose.

A polysaccharide containing D-galactose, 2-deoxy-2-N-acetylamino-D-galactose and 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose, probably corresponding to the lipopolysaccharide side chain, was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain O2. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched hexasaccharide of the structure shown, where Fuc3N represents 3-amino-3,6-dideoxygalactose and R represents D-3-hydroxybutyryl. Serological tests indicated that the polymer corresponded to the O2 antigen.

Browning-associated mechanisms of resistance to insects in corn callus tissue

Callus of the fungus- and insect-resistant corn inbred, Mp313E, of differing ages and degrees of brownness was tested for effects on neonate larvae of the corn earworm,Helicoverpa zea. Older, browner, 8-week-old callus caused significantly higher mortality ofH. zea after two days compared to larvae fed 5-week-old callus (brown or not) or pinto bean diet. Larvae fed on the 5-week-old callus were significantly smaller after nine days compared to those fed on nonbrown callus of the same age. Ferulic acid,p-coumaric acid, and sinapic acid, corn allelochemicals associated with cell walls, were oxidized (produced a brown product) up to 10-fold more rapidly by peroxidase preparations of the brown callus compared to nonbrown callus. Peroxidase isozymes from both types of brown callus separated by conventional gel electrophoresis and isoelectric focusing showed more intense anodic/acidic bands compared to peroxidase isozymes separated from the nonbrown callus. Some additional peroxidase isozymes were also present in the brown callus and were able to oxidize ferulic acid. Conventional extraction techniques for phenolic acids indicated free ferulic acid levels were similar for same-age callus, but somewhat less for older callus; no freep-coumaric acid was detected. For esterified and insoluble-bound phenolic content, the level ofp-coumaric acid was higher than that of ferulic acid and was somewhat greater in insoluble-bound fractions for the old callus compared to the younger callus. Color ratings of aqueous extracts and phenolic acid extract residues indicated much brown material remained, which was proportional in color to the original material. The differential insect response to, and peroxidase activity of, brown vs. nonbrown callus were similar to results previously noted for brown pericarps of corn compared to nonbrown pericarps. This information suggests the peroxidases are contributing to the browning through oxidation of phenolic acids and thereby enhancing resistance to insects.

Structure of the putative O10 antigen from Acinetobacter baumannii

A polysaccharide containing l-rhamnose, 2-acetamido-2-deoxy- d-glucose, and 2-acetamido-2-deoxy- d-mannose was obtained from an aqueous phenol extract of isolated cell walls from the reference strain for Acinetobacter baumannii serogroup O10. By means of NMR studies and chemical degradations, the repeating unit of the polymer (the putative O10 antigen) was identified as a branched pentasaccharide of the structure shown. ▪

Structure of a surface polysaccharide from Acinetobacter baumannii strain 214

A polysaccharide containing d-galactose, d-glucose, and 2-acetamido-2-deoxy- d-galactose was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain 214. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched trisaccharide of the structure shown. ▪

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide ofEdwardsiella ictalurifrom Channel Catfish

Abstract Lipopolysaccharide (LPS) was purified from 40 isolates of Edwardsiella ictaluri by two methods: (1) enzyme digestion and hot aqueous phenol extraction and (2) enzyme digestion and gel exclusion chromatography. Purified LPS was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblot analysis. Both methods of purification yielded smooth LPS as evidenced by a ladderlike pattern of more than 40 LPS bands. With silver staining, both low- and high-molecular-mass LPS bands were seen. Lower-molecularmass LPS stained more intensely than higher-molecular-mass LPS bands, indicating a preponderance of lower-molecular-mass LPSs. Lipopolysaccharide bands from the 40 isolates migrated similarly within SDS-PAGE gels, indicating a high degree of structural similarity among the isolates examined. The ladderlike array was more easily seen with immunoblot analysis than with silver staining of SDS-PAGE gels. Additionally, immunoblot analysis revealed a high degree of antigen...

Activation of serum complement by polysaccharide‐containing antigens of Porphyromonas gingivalis

We previously reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.

Structural characterization of a novel class of glycophosphosphingolipids from the protozoan Leptomonas samueli.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.

Structure of the O21 antigen from Serratia marcescens

Lipopolysaccharide was isolated from both phases of an aqueous-phenol extraction of defatted cell walls from the reference strain for Serratia marcescens serogroup O21. The product from the aqueous phase was of the R type, lacking a polymeric side-chain. The polymeric fraction of the lipopolysaccharide from the phenolic phase (the O21 antigen) had a disaccharide repeating-unit with the following structure: → 4)-α- d-Glc p-(1 → 4)-β- d-Man pNAc-(1 →.