Pressure Liquid Chromatography System Research Articles

Conformational Features of a Natural Break in the Type IV Collagen Gly-X-Y Repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.

Substrate Specificity and Activity Regulation of Protein Kinase MELK

Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function.

The Crystal Structure of Mitochondrial (Type 1A) Peptide Deformylase Provides Clear Guidelines for the Design of Inhibitors Specific for the Bacterial Forms

Peptide deformylase (PDF) inhibitors have a strong potential to be used as a new class of antibiotics. However, recent studies have shown that the mitochondria of most eukaryotes, including humans, contain an essential PDF, PDF1A. The crystal structure of the Arabidopsis thaliana PDF1A (AtPDF1A), considered representative of PDF1As in general, has been determined. This structure displays several similarities to that of known bacterial PDFs. AtPDF1A behaves as a dimer, with the C-terminal residues responsible for linking the two subunits. This arrangement is similar to that of Leptospira interrogans PDF, the only other dimeric PDF identified to date. AtPDF1A is the first PDF for which zinc has been identified as the catalytic ion. However, the zinc binding pocket does not differ from the binding pockets of PDFs with iron rather than zinc. The crystal structure of AtPDF1A in complex with a substrate analog revealed that the substrate binding pocket of PDF1A displays strong modifications. The S1' binding pocket is significantly narrower, due to the creation of a floor from residues present in all PDF1As but not in bacterial PDFs. A true S3' pocket is created by the residues of a helical CD-loop, which is very long in PDF1As. Finally, these modified substrate binding pockets modify the position of the substrate in the active site. These differences provide guidelines for the design of bacterial PDF inhibitors that will not target mitochondrial PDFs.

Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

The Vitamin K-dependent Carboxylase Has Been Acquired by Leptospira Pathogens and Shows Altered Activity That Suggests a Role Other than Protein Carboxylation

Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation.

Hepatocyte Nuclear Factor 4α Is a Central Regulator of Bile Acid Conjugation

Hepatocyte nuclear factor 4alpha (HNF4alpha) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4alpha expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4alpha was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression. In conclusion, HNF4alpha plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.

Bioreversion and Oral Bioavailability of thel‐Valine Dipeptide Ester Prodrug of Acyclovir, Val‐Valacyclovir, in Sprague‐Dawley Rats

The overall objective of this study was to compare bioavailability of acyclovir (ACV) in Sprague‐Dawley (SD) rats following oral administration of the ACV prodrugs valacyclovir and val‐valacyclovir (10 mg/kg body weight ACV equivalent). ACV, valacyclovir and val‐valacyclovir were estimated using a high pressure liquid chromatography system equipped with a fluorescence detector. Intact prodrugs, valacyclovir and val‐valacyclovir could not be detected in the plasma. Differences in Cmax (4.47 ± 1.68 µg/ml and 2.92 ± 0.90 µg/ml), Tmax (51.66 ± 27.14 and 27.14 and 25.83 ± 11.14 min) and AUC (366.02 ± 146.78 and 231.3 ± 90.88 min. µg/ml) achieved for ACV derived from valacyclovir and val‐valacyclovir, respectively, were not statistically significant. Val‐valacyclovir was rapidly hydrolyzed in rat enterocyte and whole intestinal homogenates (half‐lives of 6.5 and 15 min respectively) and plasma (half‐life of 8.4 min). Valacyclovir demonstrated comparatively longer half‐lives in plasma (half‐life 466.9 min) and w...

Proteomic Consequences of a Human Mitochondrial tRNA Mutation beyond the Frame of Mitochondrial Translation

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.

Comparative study of properties and acceptability of Haitian and US honey‐roasted peanuts

AbstractA honey‐roasted peanut product, flavoured with ginger and anise, was recently developed in Haiti in an effort to increase the consumption of locally grown peanuts. The objective of this study was to compare quality and consumer acceptability of the Haiti product with a traditional US‐manufactured brand. Haitian panellists (199) evaluated colour, appearance (dry/oily), crunchiness, peanut flavour and sweetness of the products using five‐point just‐about‐right scales. Colour (Minolta chromameter), hardness (TA.XT2i texture analyser fitted with a TA42 knife blade) and moisture (Denver IR‐30 moisture analyser), lipid (ethyl ether extraction) and sugar (Dionex DX‐500 high pressure liquid chromatography system) contents of the products were also determined. The Haiti product was significantly (P < 0.05) darker brown, harder and contained more (P < 0.05) sucrose and glucose but less (P < 0.05) lipid and fructose than the US product. Panellists indicated that crunchiness, peanut flavour and sweetness of both products were just right. The US product had acceptable appearance but was rated too light (hue angle = 69.6 ± 0.70, L‐value = 58.2 ± 0.47) in colour, whereas the Haiti product appeared too dry and dark (hue angle = 62.6 ± 2.79, L‐value = 44.6 ± 2.22). The Haiti peanuts were not deskinned, and their testae may have decreased the acceptability of the product's appearance and colour. The Haiti product was significantly (P < 0.05) just right to the female panellists and to the 21‐year plus panellists from all income groups. Deskinning of the peanuts and increased oil addition during roasting may improve the acceptability of the Haiti product, especially to 14–20 year olds. Decrease in sugar content may also make the Haiti product more appealing to this age group and to male Haitians.

Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

8-(3-Chlorostyryl)caffeine May Attenuate MPTP Neurotoxicity through Dual Actions of Monoamine Oxidase Inhibition and A2A Receptor Antagonism

Caffeine and more specific antagonists of the adenosine A(2A) receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A(2A) antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP(+), we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP(+) and of MPP(+), suggesting that CSC blocks the conversion of MPTP to MPDP(+) in vivo. In assessing the direct effects of CSC and A(2A) receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with a K(i) value of 100 nm, whereas caffeine and another relatively specific A(2A) antagonist produced little or no inhibition. The A(2A) receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A(2A) receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A(2A) receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A(2A) receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.

Isolation of protein subpopulations undergoing protein-protein interactions.

A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.

The Human Protein HSPC021 Interacts with Int-6 and Is Associated with Eukaryotic Translation Initiation Factor 3

The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H(2)O(2) triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

Very high pressure gradient LC/MS/MS.

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.

Characterization of Stimulatory Effect of Wasabi Leafstalk(Wasabia japonica MATSUM.) Extract on Bone Calcification in Mouse Calvaria Tissue Culture.

The stimulatory effect of wasabi leafstalk (Wasabia japonica MATSUM.) extract on bone calcification in mouse calvaria tissue culture in vitro was characterized. The extract had an appreciable effect on increasing bone calcium content; the effect was great at 10 and 50 μg/ml, but at higher concentrations (100-300 μg/ml) the effect was weaker. Sinigrin (5-20 μg/ml) had no an effect. Wasabi leafstalk extract (10 μg/ml) did not inhibit the effect of parathyroid hormone (PTH; 10-6 M), a bone-resorbing hormone, in decreasing bone calcium content, and the effect of the wasabi extract on bone calcification was stable when heated (100°C) and in acid or alkaline. It was subjected to gel filtration chromatography with a superose 12 column connected with a high pressure liquid chromatography system. The presence of fraction IV (retention time of 160-200 min) in the tissue culture caused a remarkable elevation of the bone calcium content; the effect was great at a concentration of 100 μl/ml of medium. The present study suggests that the small molecular components in wasabi leafstalk extract have a stimulatory effect on bone calcification in vitro.

Separation and clean-up effects in medium pressure liquid extraction

A new concept for extraction of polluted soil is described. The idea is to modify a given soil by grinding it with silica gel or aluminium oxide to destroy the interaction between the soil matrix and the pollutant. The produced homogeneous phase can be used as a stationary phase of a medium pressure liquid chromatographic system. The elution profiles of the substances are shown to depend on the nature of the modifier. However, the aspired separation of the PAH, PCB and the halogenated aliphatics could not completely be achieved. Using aluminium oxide the extraction of the selected pollutants showed moderate recoveries only, but no clean-up of the eluates was necessary prior to measurement.

The synthesis of 17 α-ethynylestradiol glucuronides by mammalian microsomes immobilised in hollow-fibres

Uridine 5′-diphosphate glucuronosyl transferases (UGTs, E.C.2.4.1.17) catalyse the glucuronidation of xenobiotics. The oral contraceptive 17 α-ethynylestradiol is partly cleared by glucuronidation on the 3 and 17 positions. These glucuronides are not commercially available and were synthesised as standards for characterisation of the metabolic profile of the drug. (1) Rat and dog microsomal suspensions were investigated, and the rates of glucuronide formation were found to be <0.09 and 0.49 nmol min −1 mg −1 protein, respectively. Therefore, dog microsomes were selected and immobilised in hollow-fibre membrane bioreactors. (2) Two bioreactors (15-ml capacity) were run at 20°C and fed with a saturated solution (3.3 pmol/mg protein) of the sparingly soluble drug and co-factor UDPGA (0.15 mmol/mg protein) in recycle and single pass modes, at a flow rate of 20 ml/h. The recycle mode produced more glucuronide than the single pass up to 46 h (substrate to product peak area ratios of 1.6 and 5.9, respectively). The recycle mode was adopted and the bioreactors run continuously for 88 h. (3) A second batch using the same bioreactor was run for 120 h. The estimated conversion reduced from 50% on the first run to 25% on the second run. (4) A primary clean up was effected by adsorption on octadecyl silyl resin. Separation of the glucuronides from 17 α-ethynylestradiol was on a normal phase high pressure liquid chromatography (HPLC) system. After collection of the glucuronide fraction, the solvents were evaporated off providing 1 mg of the two glucuronides (recovery efficiency>90%).

Palmitic acid is the major fatty acid responsible for significant anti- N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) activity in yogurt

We describe here the isolation and identification of palmitic acid as being responsible for significant anti- N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) activity in yogurt. The Ames test ( Salmonella typhimurium TA 100) was used to direct fractionation of activity. Yogurt was freeze-dried and extracted with acetone to yield a crude extract. The crude extract was purified by normal phase silica gel, Sephadex LH-20, and reversed phase medium pressure liquid chromatographies. The major compound in the active medium pressure liquid chromatographic fractions was determined to be palmitic acid on the basis of gas chromatography-mass spectrometry (GC/MS), MS/MS, by co-elution with authentic palmitic acid on GC and high pressure liquid chromatography (HPLC) systems, and by nuclear magnetic resonance (NMR) analysis. Other saturated straight chain and methyl branched fatty acids were detected by GC/MS and were later shown to possess anti-MNNG activity. Of the straight chain fatty acids, palmitic acid had the highest anti-MNNG activity. All ω-1 methyl branched fatty acids tested were more active than their straight chain counterparts. A trace amount of isopalmitic acid (14-methyl pentadecanoic acid), a minor milk lipid, was detected in one of the active fractions, and was later shown to be five times more active than palmitic acid. Isopalmitic acid also inhibited mutagenesis induced by 4-nitroquinoline- N-oxide (4NQO), and 7,12-dimethyl benz[ a]anthracene (DMBA), and was found to inhibit the metabolic activation of DMBA.