The glutathione dependence of inorganic sulfate formation from l- or d-cysteine in isolated rat hepatocytes

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added

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-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from

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-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from

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-cysteine was markedly inhibited by the addition of the transaminase inhibitor

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-cycloserine or the γ-cystathionase inhibitor

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-propargylglycine. The major routes of sulfate formation from

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-cysteine therefore seems to involve pathways that do not involve

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-cysteinesulfinate. Similar amounts of sulfate were formed from

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-cysteine as

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-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from

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-cysteine differed from

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-cysteine in that it was inhibited by the

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-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or γ-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from

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-cysteine and involves the oxidation of

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-cysteine by

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-amino acid oxidase to form β-mercaptopyruvate.     

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.     

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.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

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}).     

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.     

Practical synthesis of 2,6-dideoxy-d-lyxo-hexose (“2-deoxy-d-fucose”) from d-galactose

2,6-Dideoxy- -lyxo-hexose was prepared efficiently from -galactose in eight steps (25% overall yield). The synthesis is amenable to multigram scale-up.     

A comparative study of the O-3 reactivity of isomeric N-dimethylmaleoyl-protected d-glucosamine and d-allosamine acceptors

Four isomeric N-dimethylmaleoyl 4,6-O-benzylidene-protected d-hexosamine acceptors (2, 3, 4, and 5) with all possible configurations at C-1 and C-3 (e.g., derived from d-glucosamine and d-allosamine) were prepared, and the assessment of their O-3 relative reactivity through competition experiments using the known per-O-acetylated d-galactopyranosyl trichloroacetimidate donor (15) was then carried out. The reactivities are in the order 4 ? 2 > 5 > 3. The analysis of the NMR spectra of 2–5 at different temperature and modeling experiments carried out on analogs of 2–5 (DFT) and on the acceptors themselves (MM) are coincident, and have helped to establish the stability of the different hydrogen bonds, and of the conformers which carry them. The whole results suggest that the electronic effects (hydrogen bonds) are required to explain the observed trend, in spite of the axial conformation of the most reactive hydroxyl group. The steric effects appear only when hydrogen bonds are weak.     

Efficient conversion of d-glucose into d-sorbitol over MCM-41 supported Ru catalyst prepared by a formaldehyde reduction process

Ru/MCM-41 catalyst prepared by an impregnation–formaldehyde reduction method showed higher catalytic activity and sorbitol selectivity than other catalysts in the hydrogenation of glucose. SEM and XRD indicated the partial surface properties of Ru/MCM-41. Moreover, Ru dispersion and Ru surface area of Ru/MCM-41 were determined by pulse chemisorption, and the result further proved that Ru/MCM-41 had higher catalytic activity. A catalyst recycling experiment demonstrated that Ru/MCM-41 was a better catalyst and it could be reused three or four times. A speculated mechanism was proposed to illustrate the detailed process of d-glucose hydrogenation to produce sorbitol.     

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.     

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.     

基于超声清洗的树木叶面颗粒物粒径分布与吸滞效率研究——以银杏和油松为例

明确在常规叶片清洗方法(泡洗或泡洗+刷洗)上增加超声清洗对叶面各径级颗粒物滞纳量定量评估的影响, 并在此基础上研究叶面颗粒物的粒径分布和吸滞效率, 可进一步提高城市树木大气颗粒物吸滞能力的定量评估精度。该文以城市森林建设常用阔叶树种银杏(Ginkgo biloba)和针叶树种油松(Pinus tabuliformis)为研究对象, 于雨后(降水量>15 mm) 4天(短滞尘时长)和14天(长滞尘时长)分别采集叶样, 并依次对其进行泡洗(WC)、刷洗(BC)、超声清洗(UC)等洗脱程序, 然后对每个清洗步骤下叶片洗脱液中颗粒物的质量和粒径分布进行测试, 并依此估算叶片各径级颗粒物的吸滞效率。结果表明, 以“泡洗+刷洗+超声清洗”清洗流程的测试结果为参照, 若只对叶片进行泡洗, 则银杏和油松对大气颗粒物(PM₁, 粒径d ≤1 µm)、PM_2.5 (d ≤ 2.5 µm)、PM₅ (d ≤ 5 µm)、PM₁₀ (d ≤ 10 µm)吸滞量会分别被低估约一半(54%、53%、53%和53%)和40% (42%、42%、42%和42%); 若只进行“泡洗+刷洗”, 则银杏和油松对相应径级颗粒物的吸滞量仍会分别被低估约15% (17%、16%、15%和15%)和20% (21%、20%、20%和20%)。油松叶面颗粒物粒径分布呈双峰曲线, 而银杏叶面颗粒物粒径则呈单峰分布, 且银杏叶面颗粒物平均粒径在短、长滞尘时长下均大于油松。油松叶片对PM₁、PM_2.5、PM₅、PM₁₀和总悬浮颗粒物的吸滞效率分别为8.96、23.92、23.96、23.96和23.96 mg·m^-2·d^-1, 分别比银杏叶片高112%、73%、34%、37%和42%。     

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.     

Evidence of benzilic rearrangement during the electrochemical oxidation of d-glucose to d-glucaric acid

During the course of the 2,2,6,6-tetramethyl-1-piperidinyloxy free radical-catalyzed electrochemical oxidation of d-glucose to d-glucaric acid a new side-product was observed. This compound was isolated and identified as a tricarboxylic acid of unique structure, which was named maribersonic acid. Its structure was proven by different experiments coupled with several analytical methods, and its appearance during the electrochemical oxidation of d-glucose was rationalized through a thorough study.     

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%.     

Preferred interaction of d-peptidyl-anthraquinones with double-stranded B-DNA

The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone–peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine–glycine dipeptide in the side chains. The d enantiomer binds right handed double stranded DNA more efficiently than the l form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the d-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The d enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development.     

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.     

Determination of d-fenfluramine, d-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.