Phase High Pressure Liquid Chromatography Research Articles

Analysis of the antimalarial drug halofantrine and its major metabolite N-desbutylhalofantrine in human plasma by high performance liquid chromatography

The determination of halofantrine and its major metabolite N-desbutylhalofantrine in human plasma by reversed phase high-pressure liquid chromatography is described. The method involves protein precipitation of plasma samples by acetonitrile followed by basification with sodium hydroxide and subsequent liquid–liquid extraction using hexane-diethyl ether (1:1, v/v). The chromatographic separation was carried out on a C-18 column with a mobile phase consisting of methanol/0.05 M KH 2PO 4 (78:22, v/v) containing 55 mM perchloric acid. Chlorprothixen was used as internal standard. The relative standard deviations of intraday and interday precision for both compounds were less than 7%, the relative standard deviation of the accuracy did not exceed 7.1% at concentrations of 50 and 300 ng/ml. This method is simple, rapid, sensitive and cost effective and was applied to the determination of the pharmacokinetics of halofantrine and N-desbutylhalofantrine in two healthy male volunteers after an oral administration of 500 mg halofantrine. Moreover, the influence of the frequently consumed kolanut on the pharmacokinetics of halofantrine was investigated.

Accommodation and Repair of a UV Photoproduct in DNA at Different Rotational Settings on the Nucleosome Surface

Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30 degrees bending and 9 degrees unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.

Subcelluar Distribution of Myeloid-Related Protein 8 (MRP8) and MRP14 in Human Neutrophils.

Myeloid-related protein 8 (MRP8) and MRP14 are S100 family calcium binding proteins that form a heterodimer known as MRP8/14 that is present in the cytosol of neutrophils and monocytes. MRP8/14 becomes associated with endothelium at sites of monocyte and neutrophil adhesion and transmigration and induces a thrombogenic and inflammatory response by increasing the endothelial transcription of proinflamatory chemokines and adhesion molecules. MPR8/14 is the most abundant protein in neutrophil cytosol making up 30 to 60% of all cytosolic protein. However, the distribution of MPR8/MRP14 among neutrophil granules and plasma membranes in unclear and was investigated to better understand the role or MPR8/14 in acute inflammation. Three monoclonal antibodies specific for MRP8 and MPR14 were characterized, AHN-17, ANH-17.1, and 15H9, and were used to investigate the subcellar distribution of MPR8/14. The 10 and 14 kDa proteins recognized by the three antibodies were isolated by affinity chromatography with 15H9 and were separated by reverse phase high pressure liquid chromatography. N-terminal amino acid sequencing of the 10 kDa protein yielded a sequence of 29 amino acids that was identical to MRP8. The N-terminus of the 14 kDa protein was blocked, however, the amino acid sequence of two tryptic peptides were found to be identical to MRP14 at 25 of 27 amino acids. Nitrogen cavitation and density gradient separation was used to isolate neutrophil cytosol, plasma membranes, primary granules, and secondary granules. The secondary granule fraction also contains a distinct granule population termed tertiary or secretory granules. The proteins recognized by AHN-17 were isolated from each of these fractions by affinity chromatography and analyzed by SDS-PAGE under reducing conditions. MRP8 and MRP14 were located in the cytosol, plasma membrane, and secondary granule, and to a lesser degree primary granule fraction. To further determine the location of MPR-8/14 in the cellular granules, primary and secondary granules were washed and disrupted by freeze-thawing and sonication and the membranes were separated from the granule contents by centrifugation and were analyzed by immunoblotting with 15H9. Both MRP8 and MRP14 were detected in the soluble portion of the secondary granules. MPR14 was also detected in the soluble portion of primary granules and MRP8 in the plasma membrane fraction. The largest quantities of the MRP8 and MRP14 were present in cytosol and in the soluble portion of the secondary granules. MRP8 and MRP14 had a loose calcium-dependent adherence to neutrophil primary granules and plasma membranes and they were removed by washing with EGTA in a high ionic strength buffer. In conclusion MRP8/14 is located in neutrophil cytosol and in the contents of secondary granules and is loosely associated with plasma membranes and primary granules. MRP8/14 released with secondary granules by activated neutrophils likely binds to endothelium and plays an important role in acute inflammation.

Contribution of unburned lubricating oil and diesel fuel to particulate emission from passenger cars

In this study we determined particle-bound paraffins in the exhaust of six light-duty diesel vehicles on a chassis dynamometer for different driving cycles and ambient temperatures. The filters containing particulate matter were extracted with dichloromethane in a Soxhlet apparatus, and the paraffin analysis was performed using two-dimensional normal phase high-pressure liquid chromatography (HPLC) coupled on-line to gas chromatography-flame ionization detection (GC–FID). The different molecular mass of lubricant and diesel paraffins facilitated the distinction between diesel and lubricant contribution to the emission. Although all vehicles were certified according to the same emission class, there were considerable variations between vehicles. The study showed that under cold-start conditions the organic mass fraction ranged from 10% to 30% with respect to particle mass and the paraffins from 30% to 60% with respect to the organic mass. With cold engine, falling ambient temperature increased the emission of unburned diesel fuel, whereas that from unburned lubricating oil was less affected. Under warm-start conditions, the ambient temperature had less impact on the emission of paraffins. The emissions were also affected by the operating conditions of the engine: driving cycles with higher mean load tend towards higher emissions of lubricant. The operating conditions also affected the distribution of paraffins: the emission of light paraffins seemed to be lower with higher load in the driving cycle. With an urban and a highway cycle, roughly 40% and 80% w/w, respectively, of unburned paraffins were contributed by the lubricant. Measurements of polycyclic aromatic hydrocarbons (PAH) in lubricating oil showed lubricant to be a sink for PAHs. As lubricant significantly contributes to the organic emission, as shown in this study, it can be assumed that it is also a significant source of PAH emissions.

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

Discovery, Structural Determination, and Putative Processing of the Precursor Protein That Produces the Cyclic Trypsin Inhibitor Sunflower Trypsin Inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

The evaluation of printability and non-enzymatic NADH reduction of 4′-chlorophenylazo-2-naphthol for use as a printing pigment

The printability of 4′-chlorophenylazo-2-naphthol (CPAN) was evaluated on gloss-coated paper. The preliminary results indicated that the proposed pigment could be used in offset and oil-based ink production. The pigment and its potential reduction product, 4-chloroaniline, were then separated by a reverse phase high-pressure liquid chromatography system. The pigment was incubated with NADH at 37°C for half an hour at different pH values to investigate its possible reduction into the corresponding primary amines (ie 4-chloroaniline and 2-amino-1-naphthol). However, no reduction was observed. This finding indicates a desirable evidence for the pigment in terms of its stability against NADH (nicotinamide adenine dinucleotide), a known biochemical reducing agent in living organisms when the potential toxicity from aromatic primary amines is considered.

3-Hydroxykynurenine-mediated Modification of Human Lens Proteins: STRUCTURE DETERMINATION OF A MAJOR MODIFICATION USING A MONOCLONAL ANTIBODY

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.

Mutation of Cys105 Inhibits Dimerization of p12CDK2-AP1 and Its Growth Suppressor Effect

p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.

C-Jun N-terminal Kinase (JNK) Positively Regulates NFATc2 Transactivation through Phosphorylation within the N-terminal Regulatory Domain

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr(116) and Ser(170) are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr(116). Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members.

Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Metastasis-associated Protein S100A4 Induces Angiogenesis through Interaction with Annexin II and Accelerated Plasmin Formation

Many advanced tumors overexpress and secrete the S100A4 protein that is known to promote angiogenesis and metastasis development. The mechanisms of this effect and the endothelial receptor for S100A4 are both still unknown. Here we report that extracellular S100A4 interacts with annexin II, an endothelial plasminogen co-receptor. Co-localization and direct binding of S100A4 and annexin II were demonstrated, and the binding site was identified in the N-terminal region of annexin II. S100A4 alone or in a complex with annexin II accelerated tissue plasminogen activator-mediated plasminogen activation in solution and on the endothelial cell surface through interaction of the S100A4 C-terminal lysines with the lysine-binding domains of plasminogen. A synthetic peptide corresponding to the N terminus of annexin II prevented S100A4-induced plasmin formation in the endothelial cell culture. Local plasmin formation induced by circulating S100A4 could contribute to tumor-induced angiogenesis and metastasis formation that makes this protein an attractive target for new anti-cancer and anti-angiogenic therapies.

Molecular Determinants for the Complex Binding Specificity of the PDZ Domain in PICK1

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.

Analytical method development and validation of related substances by rp-hplc of emtricitabine and tenofovir disoproxil fumarate tablets

The developed method was a simple, accurate, precise, specific and robust method for the validation of Emtricitabine and Tenofovir isoproxil Fumarate Tablets by reverse phase high pressure liquid chromatography. For Emtricitabine and Tenofovir Disoproxil Fumarate Chromatography was performed on Agilent 1200 series, UV and PDA Detector, Waters X-bridge C18 (250 mm x 4.6 mm, 5 μm) by preparing Buffer solution: Dissolve 0.63 g of ammonium formate in 1000 mL of purified water and mix. Adjust to pH of 3.90 +0.05 with diluted formic acid. And used it as mobile phase A. Mobile Phase B: mixture of buffer solution and methanol in the ratio of (20 : 80) % v/v at a flow rate of 1.0 mL/min and at 254 nm wavelength. The retention times of Emtricitabine and Tenofovir Disoproxil Fumarate are approx. 29 min and 70 min. respectively. 5-Fluorocytosinc, Sulfoxide Impurity Isomer 1, Sulfoxide Impurity isomer 2, 5-Fluorouracil analogue, Tenofovir…

Redirection of Eicosanoid Metabolism in mPGES-1-deficient Macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.

Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

Quantitative determination of bovine κ-casein macropeptide in dairy products by Liquid chromatography/Electrospray coupled to mass spectrometry (LC-ESI/MS) and Liquid chromatography/Electrospray coupled to tamdem mass spectrometry (LC-ESI/MS/MS)

κ-Casein macropeptide (CMP) is a polypeptide of 64 amino acid residues, derived from the C–terminal part of bovine κ-casein. It is a complex mixture of non-glycosylated and variously glycosylated forms. Using reversed phase—high pressure liquid chromatography (RP-HPLC) coupled to a mass spectrometer fitted with a electrospray ionisation source (ESI-MS) sensitive, reliable and specific methods were developed to enable unequivocal determination of aglyco-CMPA, aglyco-CMPB and total CMPAB. Several selective detection methods were proposed for their quantification. Aglyco-CMPA and aglyco-CMPB were quantified, respectively, from 13595+, 16984+, 22643+ and 13525+, 16904+, 22533+ m/z specific ions from multiple ion monitoring (MIM) mode. Within-day repeatability and between-day repeatability were 1% and 5%, respectively, with a standard deviation lower than 1%. Total CMPAB was quantified from peptide κ-CN (162–169) released from pepsin hydrolysis using UV, single ion monitoring (SIM) and multiple reaction monitoring (MRM) detection. A high degree of selectivity was obtained using MRM detection; the limit of quantification was 10 pmol within the standard curve range of 10–1000 pmol. Several commercial products were analysed and data obtained were in agreement with those indicated in the literature.

A Novel Insulin-releasing Substance, Phanoside, from the Plant Gynostemma pentaphyllum

Extracts from Gynostemma pentaphyllum Makino (Cucurbitaceae), a Southeast Asian herb, has been reported to affect numerous activities resulting in antitumor, cholesterol-lowering, immunopotentiating, antioxidant, and hypoglycemic effects. We have isolated one active compound by ethanol extraction, distribution in n-butyl alcohol/water, solid phase extraction/separation, and several rounds of reverse phase high pressure liquid chromatography. We have shown by NMR and mass spectrometry that this active compound is a novel saponin, a gypenoside, which we have named phanoside (21-,23-epoxy-,3beta-,20-,21-trihydroxydammar-24-ene-3-O-([alpha-d-rhamnopyranosyl(1-->2)]-[beta-d-glycopyranosyl(1-->3)]-beta-d-lyxopyranoside)), with a molecular mass of 914.5 Da. Phanoside is a dammarane-type saponin, and four stereoisomers differing in configurations at positions 21 and 23 were identified, each of which were found to stimulate insulin release from isolated rat pancreatic islets. We have also found that the stereoisomers are interconvertible. Dose-dependent insulin-releasing activities at 3.3 and 16.7 mM glucose levels were determined for the racemic mixture containing all four stereoisomers. Phanoside at 500 microM stimulates insulin release in vitro 10-fold at 3.3 mM glucose and potentiates the release almost 4-fold at 16.7 mM glucose. At these glucose levels, 2 microm glibenclamide stimulates insulin release only 2-fold. Interestingly, beta-cell sensitivity to phanoside is higher at 16.7 mM than at 3.3 mM glucose, although insulin responses were significantly increased by phanoside below 125 microM only at high glucose levels. Also when given orally to rats, phanoside (40 and 80 mg/ml) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia.

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, α-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 °C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C 18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a p I value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys 10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.

Cultured rat microglial cells synthesize the endocannabinoid 2-arachidonylglycerol, which increases proliferation via a CB2 receptor-dependent mechanism.

Microglia, as phagocytes and antigen-presenting cells in the central nervous system, are activated in such disease processes as stroke and multiple sclerosis. Because peripheral macrophages are capable of producing endocannabinoids, we have examined endocannabinoid production in a macrophage-colony stimulating factor (M-CSF)-dependent rat microglial cell line (RTMGL1) using reversed phase high-pressure liquid chromatography and liquid chromatography-mass spectroscopy. We determined that cultured microglial cells produce the endocannabinoid 2-arachidonylglycerol (2-AG) as well as anandamide in smaller quantities. When 2-AG, but not anandamide, is added exogenously, RTMGL1 microglia increase their proliferation. This increased proliferation is blocked by an antagonist of the CB(2) receptor N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and mimicked by the CB(2) receptor-specific agonist 1,1-dimethylbutyl-1-deoxy-Delta(9)-tetrahydrocannabinol (JWH133). Accompanying the increase in proliferation seen with 2-AG is an increase in active ERK1 that is also blocked with SR144528. The RTMGL1 microglial cells, which exist in a primed state, express the CB(1) and CB(2) receptors as demonstrated by reverse transcription-polymerase chain reaction and immunostaining. The CB(2) receptor in untreated cells is expressed both at the cell surface and internally, and exposure of the cells to 2-AG significantly increases receptor internalization. These data suggest that 2-AG activation of CB(2) receptors may contribute to the proliferative response of microglial cells, as occurs in neurodegenerative disorders.