Synthesis and binding analysis of unique AG2 pentasaccharide to human Siglec-2 using NMR techniques

Siglec-2 is a mammalian sialic acid binding protein expressed on B-cell surfaces and is involved in the modulation of B-cell mediated immune response. We synthesized a unique starfish ganglioside, AG2 pentasaccharide Galfβ(1–3)Galpα(1–4)Neu5Acα(2–3)Galpβ(1–4)Glcp, and found that the synthetic pentasaccharide binds to human Siglec-2 by performing ¹H NMR experiments. Saturation transfer difference NMR experiments indicated that the C7–C9 side-chain and the acetamide moiety of the central sialic acid residue were located in the binding face of human Siglec-2. We determined the binding epitope of AG2 pentasaccharide to human Siglec-2, as the Galpα(1–4)Neu5Acα(2–3)Galp unit.     

Carbonic anhydrase inhibitors. Inhibition of mammalian isoforms I–XIV with a series of natural product polyphenols and phenolic acids

A series of phenolic acids and phenol natural products, such as p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, ferulic acid, gallic acid, syringic acid, quercetin, and ellagic acid, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). All mammalian isozymes of human (h) or murine (m) origin hCA I–hCA XII, mCA XIII and hCA XIV were inhibited in the low micromolar or submicromolar range by these (poly)phenols (K_Is in the range of 0.87–7.79 μM). p-Hydroxybenzoic acid was the best inhibitor of all isozymes (K_Is of 0.87–35.4 μM) and the different isozymes showed very variable inhibition profiles with these derivatives. Phenols like the ones investigated here possess a CA inhibition mechanism distinct of that of the sulfonamides/sulfamates used clinically or the coumarins. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule and bind more externally within the active site cavity, making contacts with various amino acid residues. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs.     

Incorporation of various putative precursors into cuticular 3,4-dihydroxyphenylacetic acid of adult Tenebrio molitor

Post-emergence levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and ketocatechol were determined in cuticle from adult Tenebrio molitor. Possible pathways for biosynthesis of DOPAC were studied by comparing the incorporation of injected [U-¹⁴C]tyrosine, [7-¹⁴C]dopamine, [7-¹⁴C]DOPA, [7-¹⁴C]tyramine, [U-¹⁴C]p-hydroxyphenylpyruvic acid (p-HPPA) and [ring-³H]p-hydroxyphenylacetic acid (p-HPAA) into cuticular DOPAC during its period of maximal increase 1–3 days after adult emergence. Increased incorporation of [U-¹⁴C]tyrosine between days 0 and 3 suggests rapid de novo biosynthesis of DOPAC from this primary precursor. Of the putative intermediates tested, only p-HPPA had a pattern of incorporation similar to that seen with tyrosine. Since p-HPAA was poorly incorporated into both cuticle and DOPAC, a tentative pathway tyrosine → p-HPPA → 3,4-dihydroxyphenylpyruvic acid → DOPAC is proposed.     

Molecular and expression analysis of manganese superoxide dismutase (Mn–SOD) gene under temperature and starvation stress in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species. We cloned cDNA encoding SOD activated with manganese (Mn–SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of Mn–SOD was 1,016 bp and had a 669 bp open reading frame encoding 222 amino acids. The deduced amino acid sequence of B. calyciflorus Mn–SOD showed 89.1, 71.3, and 62.1 % similarity with the Mn–SOD of the marine rotifer Brachionus plicatilis, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, respectively. The phylogenetic tree constructed based on the amino acid sequences of Mn–SODs from B. calyciflorus and other organisms revealed that this rotifer is closely related to nematodes. Analysis of the mRNA expression of Mn–SOD under different conditions revealed that expression was enhanced 5.6-fold (p < 0.001) at 30 °C after 2 h, however, low temperature (15 °C) promoted Mn SOD temporarily (2.5-fold, p < 0.001) and then decreased to normal level (p > 0.05). Moderate starvation promoted Mn–SOD mRNA expression (p ₁₂ < 0.01, p ₃₆ < 0.05), which reached a maximum value (15.3 times higher than control, p ₂₄ < 0.01) at 24 h. SOD and CAT activities also elevated at the 12 h–starved group. These results indicate that induction of Mn–SOD expression by stressors likely plays an important role in aging of B. calyciflorus.     

Protein–DNA photo-crosslinking with a genetically encoded benzophenone-containing amino acid

The photo-crosslinking amino acid, p-benzoyl-l-phenylalanine (pBpa), was genetically incorporated into Escherichia coli catabolite activator protein (CAP) in bacteria in response to an amber nonsense codon using an orthogonal tRNA/aminoacyl-tRNA synthetase pair. The mutant CAP (CAP-K26Bpa) containing pBpa formed a covalent complex with a DNA fragment containing the consensus operator sequence upon UV irradiation. Because this amino acid can be genetically incorporated into any DNA-binding protein in E. coli, yeast or mammalian cells with minimal perturbation of protein structure, this method should be generally useful for investigating DNA–protein interactions.     

Phenolic profiles of untransformed and hairy root cultures of Plantago lanceolata

Untransformed root cultures and Agrobacterium rhizogenes induced root cultures (hairy roots) of Plantago lanceolata were investigated for caffeic acid glycoside esters, i.e. verbascoside (V) and plantamoside (P), by HPLC. Levels of V (6–12 mg.g^–1 DW) and P (30–80 mg.g^–1 DW) from untransformed and hairy root cultures were not modified by 0.1 mM (E)-cinnamic acid addition in Murashige and Skoog's culture medium. A part of the cinnamic acid was converted into (E)-p-coumaroyl-1-O-β-D-glucopyranoside, a phenolic derivative absent from control cultures without cinnamic acid. Maximum levels of this coumaroyl ester (6–8 mg.g^–1 DW) were detected during 10 d and then slightly decreased from both root chemical profiles.     

Soybean root growth inhibition and lignification induced by p-coumaric acid

The effects of 0.25–2 mM p-coumaric acid, a phenylpropanoid metabolite with recognized allelopathic properties, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) activities, soluble and cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H₂O₂) level and lignin content and its monomeric composition in soybean (Glycine max (L.) Merr.) roots. At ≥0.25 mM, exogenously supplied p-coumaric acid induced premature cessation of root growth, increased POD activity and lignin content and decreased the H₂O₂ content. At ≥0.5 mM, the allelochemical decreased the cell viability and PAL activity. When applied jointly with PIP (an inhibitor of the cinnamate 4-hydroxylase, C4H), 1 mM p-coumaric acid increased lignin content. In contrast, the application of MDCA (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with p-coumaric acid did not increase lignin content. The lignin monomeric composition of p-coumaric acid-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and guaiacyl (G) units. Taken together, these results suggest that p-coumaric acid's mode of action is entry via the phenylpropanoid pathway, resulting in an increase of H and G lignin monomers that solidify the cell wall and restrict soybean root growth.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C₁₈ reversed-phase column using isocratic elution at 40°C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r²>0.99) was observed within the calibration ranges studied: 37.4–299.3 μg/ml for PRM, 26.4–211.2 μg/ml for PB, 12.5–100.2 μg/ml for p-HO-PB and 12.1–97.0 μg/ml for PEMA. Repeatability was in the range 3.1–6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.     

A procedure for the synthesis of p-fluorosulfonyl[14C]-benzoyl-5′-adenosine with [14C] in the benzoyl moiety

Carboxy-p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine has been synthesized with the radiolabel ultimately derived from carboxy-p-amino[¹⁴C]benzoic acid by a synthetic route employing four reaction steps. Starting with 1 mmol of p-amino[¹⁴C]benzoic acid, p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine is obtained with an overall yield of 25–30%.     

Reactions of the amino groups of RNAse A: III. The existence of pH-dependent values of pK

The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).     

Biotransformation of o- and p-aminobenzoic acids and N-acetyl p-aminobenzoic acid by cell suspension cultures of Solanum mammosum

Some new biotransformation products, p-aminobenzoic acid 7-O-β-d-glucopyranosyl ester, N-acetyl p-aminobenzoic acid 7-O-β-d-glucopyranosyl ester, o-aminobenzoic acid 7-O-β-d-(β-1,6-O-d-glucopyranosyl)glucopyranosyl ester and o-aminobenzoic acid 7-O-β-d-glucopyranosyl ester were isolated from cell suspension cultures of Solanum mammosum following administration of p-aminobenzoic acid, N-acetyl p-aminobenzoic acid or o-aminobenzoic acid respectively. N-acetyl p-aminobenzoic acid and N-formyl p-aminobenzoic acid were also identified as cell suspension metabolites of p-aminobenzoic acid.     

Simultaneous determination of inulin and p-aminohippuric acid in plasma and urine by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure simultaneously plasma and urine concentrations of both p-aminohippuric acid and inulin. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a C₁₈ reversed-phase column with a flow-rate of 1 ml/min, and monitoring the absorbance at 280 nm. Within the investigated concentration ranges of inulin (0.1–3.2 mg/ml) and p-aminohippuric acid (0.0097–0.3 mg/ml), good linearity (r>0.99) was obtained. Within-run RSD ranged from 2.9 to 6.1% and between-run RSD ranged from 6.4 to 10%. Analytical recoveries were 101–112%, with little differences between plasma and urine samples. The detection limit was 1 μg/ml for all the analytes studied. This method might be ideal for renal function studies where a rapid and reproducible assessment of both renal glomerular filtration rate and blood flow-rate is required.     

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.     

Stimulation of thiamine diphosphate activity by ascorbic acid in rat brain microsomes

The effect of ascorbic acid on microsomal thiamine diphosphate activity in rat brain was examined. Ascorbic acid at 0.02–0.1 mM increased the thiamine diphosphate activity by 20–600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, α,α′-dipyridyl, o-phenanthroline) and an antioxidant (N,N′-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02–0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Identification and simultaneous determination of p-FHA and p-FBA,two metabolites of anti-tumor agent—Fluorapacin in rat urine

Fluorapacin, bis(4-fluorobenzyl)trisulfide, a small molecule natural product derivative of trisulfide, has revealed a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models with excellent safety profile. In the present study, two new metabolites, para-fluorohippuric acid (p-FHA) and para-fluorobenzoic acid (p-FBA), were identified by GC–MS and HPLC as the main metabolites in urine of rats after intravenous administration of fluorapacin. A simple HPLC-UV method for simultaneous determination of these two metabolites in urine has been developed and validated. The newly developed method demonstrated excellent specificity, accuracy, precision, and stability. This method was successfully employed to study the urinary excretion of fluorapacin in rats. The results indicated that p-FHA was the major metabolite in urine, and the total excretion recovery of p-FHA and p-FBA was 67.6 ± 4.9% (mean ± SE, n = 6) of dosage after 48 h of administration.     

The preparation and microbiological evaluation of the inhibitory effects of some acrylic acid derivatives

The following acrylic acid derivatives have been prepared and microbiologically evaluated as possible inhibitors of the growth of lactobacilli; indoleacrylic acid, β-(2-quinolyl)-, β-(3-quinolyl)-, β-(4-quinolyl) acrylic acids, cinnamic acid, p-hydroxycinnamic acid, p-dimethylaminocinnamic acid, p-diethylaminocinnamic acid, thienylacrylic acid, furylacrylic acid, and α-ethylacrylic acid.The utilization of tryptophan by Leuconostoc mesenteroides P-60 and Lactobacillus arabinosus was inhibited by the isomeric quinolylacrylic acid derivatives as well as by indoleacrylic acid. With this latter compound and the β-(3-quinolyl)acrylic acid, competitive inhibition was shown.p-Hydroxycinnamic acid inhibited the utilization of phenylalanine and tyrosine by all the organisms tested. At similar concentrations neither cinnamic acid nor phenol exerted any inhibitory effect.The effects of all inhibitors could be at least partially reversed by the addition of larger quantities of the corresponding amino acids.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.