Pharmacokinetic, metabolic, and antidiarrheal properties of ( and ) heptapeptides of sorbin in rodent

The C-terminal heptapeptide-amide (C7-sorbin) is the minimal biologically active fragment of sorbin inducing an increase in intestinal hydroelectrolytic absorption. An analogue (D7-sorbin), characterized by the replacement of the ultimate C-terminal amino acid -alanine-amide by -alanine-amide, was synthetized. For pharmacokinetic studies, D7-sorbin and C7-sorbin were tritium labeled. After IV injection, clearances were 10.6 and 30.2 ml⁻¹ for D7-sorbin and C7-sorbin, respectively, and MRT were 34 and 18 min. After SC administration, C_max attained 0.41% and 0.12% of the dose/ml, respectively. The IP route showed a 45-min delay before C_max and a 100% bioavailability for both peptides. D7-sorbin was principally excreted in urine, as shown by balance study, and in part in intact form, as controlled by mass spectrometry. D7-sorbin induced a significant decrease of the VIP-induced ileal secretion, previously observed with C7-sorbin. The change of -Ala to -Ala increased the stability of the synthetic C-terminal peptide of sorbin whereas its biological activity, bioavailability, and route of elimination were unchanged.     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

A facile synthesis of d-ribo-C20-phytosphingosine and its C2 epimer from d-ribose

A facile synthetic route to d-ribo-C₂₀-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Hypotensive effects of [d-Tyr,d-Thr]neuropeptide Y(27–36)

An analogue of the 10 C-terminal amino acids of neuropeptide Y (NPY) containing taining three d-isomeric substitutions (27–36-d) has been synthesized and its cardiovascular activity studied in Sprague-Dawley (SD) and spontaneously hypertensive (SHR) rats. Intravenous administration of 1000 nmol/kg 27–36-d decreases MAP in SHR (−59.9 ± 5.0 mmHg) and SD rats (−44.4 ± 4.7 mmHg). The hypotension produced by 1000 nmol/kg 27–36-d diminished by 71.2% following pretreatment with the histamine receptor antagonist diphenhydramine, although histamine depletion with compound does not significantly alter this hypotension. These data suggest that NPY(27–36)-d produces a profound and sustained hypotension in two strains of rat which is partially attributable to activity at histamine receptors.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Determination of l-carnitine, acetyl-l-carnitine and propionyl-l-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene

A new sensitive high-performance liquid chromatographic procedure for the determination of l-carnitine (LC), acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C₁₈). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%–95.4% for all 3 compounds. Good linearity (R≈0.99) was observed within the calibration ranges studied: 5–160 nmol/ml for LC, 1–32 nmol/ml for ALC and 0.25–8 nmol/ml for PLC. Precision was in the range 0.3–16.8% and accuracy was always lower than 10.6%.     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of l-threonine

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD⁺-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD⁺-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

Crystal structures of (2R,4R)-2-(polyhydroxyalkyl)-1,3-thiazolidine-4-carboxylic acids: condensation products of l-cysteine with d-hexoses

We report herein the first crystal structures of (4-carboxy-1,3-thiazolidin-2-yl)pentitols [2-(polyhydroxyalkyl)thiazolidine-4-carboxylic acids], condensation products of l-cysteine with d-galactose and d-mannose: 2-(d-galacto-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Gal-Cys·H₂O (1), and 2-(d-manno-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Man-Cys·H₂O (2). In 1 and 2 the compounds crystallize as zwitterions, with the carboxylic groups deprotonated and the thiazolidine N atoms protonated. The sugar moiety and carboxylate group are in a cis configuration relative to the thiazolidinium ring, which adopts different conformation: twisted (T) on C^β–S in 1, and S-puckered envelope (E) in 2. The carbon chain of the galactosyl/mannosyl moiety remains in an extended zig-zag conformation. The orientation of the sugar O2 atom with respect to the thiazolidinium S and N atoms is trans–gauche in 1 and gauche–gauche in 2. The molecular conformation is stabilized by the intramolecular N–H?O_Cys contacts in both 1 and 2 and by the additional N–H?O_Man interaction in 2. The crystal packing of orthorhombic 1 and monoclinic 2 is determined mainly by N/O/C–H?O hydrogen bonds forming ribbons linked to each other by direct and water-mediated O/C–H?O/S contacts.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Conformation of 4-thio-l-lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio-l-lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.     

Free l-amino acids and d-aspartate content in the nervous system of Cephalopoda. A comparative study

We have determined the content of free l-amino acids and d-aspartate in the nervous tissue of three representative cephalopods: Sepia officinalis, Octopus vulgaris, and Loligo vulgaris, and the optic lobes of adult and embryo Sepia officinalis. Taurine is the most abundant amino acid in the cephalopod nervous tissue. Its content amounts to more than 50% of the total free amino acids. The other most concentrated amino acids are Glu, Ala, Asp, and GABA. High concentrations of d-aspartate were found in the nervous tissue of all cephalopods examined (7–12 μmol/g wet tissue) which represents 50–80% of the total aspartate (d + l), depending on the animal. Among the various regions of the brain of Octopus vulgaris, d-aspartate was found to be evenly distributed in the various regions of the brain. In nerve tissue of Sepia officinalis, there is no significant difference in the pattern of free l-amino acids, in particular of the d-aspartate concentration, between adults and embryos, except for GABA, Gly, His and Thr. This suggests that d-aspartate in nerve tissue of the Cephalopoda is of endogenous origin and not a product of accumulation from exogenous sources. From a comparative study of the content of d-aspartate in the nervous tissue of different animals, we found that protostomia contain a significantly higher amount than deuterostomia. Thus, d-aspartate could be a criterion to distinguish the protostomia phyla from the deuterostomia phyla.     

Modulation of UV-light-induced skin inflammation by d-alpha-tocopherol and l-ascorbic acid: a clinical study using solar simulated radiation

Objective: In this clinical trial we studied whether oral supplementation with d-alpha-tocopherol (α-Toc), l-ascorbic acid (Asc), or α-Toc combined with Asc influenced the solar simulated radiation (SSR) induced skin inflammation in healthy volunteers. Methods: We investigated the following groups in a prospective, randomized and placebo controlled study: Group (1) α-Toc 2 g / day, group (2) Asc 3 g / day, group (3) α-Toc 2 g / day combined with Asc 3 g / day, and group (4) placebo. Before and 50 days after supplementation we analyzed α-Toc and Asc concentrations in keratinocytes. The dose response curve of UV erythema was determined by reflectance spectrophotometry and the minimal erythema dose (MED) by visual grading before and after supplementation. Results: 50 days after supplementation α-Toc keratinocyte levels were increased in groups (1) and (3), Asc concentrations were elevated in groups (2) and (3), and the a/γ-Toc ratio increased in groups (1) and (3). The dose response curve of UVR induced erythema showed a significant flattening and the MED increased from 103 ± 29 mJ/cm² (before supplementation) to 183 ± 35 mJ/cm² (after supplementation) in group (3), while there were no significant changes in groups (1) and (2) after vitamin supplementation. Conclusion: α-Toc and Asc act synergistically in suppression of the sunburn reaction.     

Broadband dielectric spectroscopy and calorimetric investigations of d-lyxose

Using broadband dielectric spectroscopy, we have studied different types of relaxation processes, namely, primary (α), secondary (β), and another sub-T_g process called γ-process, in the supercooled state of d-lyxose, over a wide frequency (10^-2–) and temperature range (120–340 K). In addition, the same sample was analyzed by differential scanning calorimeter. The temperature dependence of the relaxation times as well as the dielectric strength of different processes has been critically examined. It has been observed that the slower secondary relaxation (designated as β-) process shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the observed slower secondary relaxation (β-) is Johari–Goldstein relaxation process and faster one (γ-process) is probably the rotation of hydroxymethyl (–CH₂OH) side group attached to the sugar ring, that is, of intramolecular origin.     

Complete H and C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses ¹H and ¹³C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the ¹H and ¹³C, as well as ³¹P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 ¹H and ¹³C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D ¹H,¹³C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t₁ incremented ¹H,¹³C-HSQC experiment and a 1D ¹H,¹H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3 Hz apart. The ¹H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental ¹H and ¹³C NMR chemical shifts.     

Diastereoselective synthesis of l-threo-3,4-dihydroxyphenylserine by low-specific l-threonine aldolase mutants

Diastereoselectivity-enhanced mutants of l-threonine aldolase (l-TA) for l-threo-3,4-dihydroxyphenylserine (l-threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type l-TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg l-threo-DOPS/ml.