Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

Determination of 3′-azido-2′,3′-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3′-azido-3′-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150×4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 μg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

Quantification of a potent mutagenic 4-amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) and the related chemicals in water from the Waka River in Wakayama, Japan

4-Amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) is a novel chemical exerting strong mutagenicity, especially in the absence of metabolic activation. In addition to mutagenicity, ADDB may also disrupt the endocrine system in vitro. ADDB may be discharged from chemical plants near the Waka River and could be unintentionally formed via post-emission modification of drainage water containing 3,3′-dichlorobenzidine (DCB), which is a precursor in the manufacture of polymers and dye intermediates in chemical plants. The main purpose of this study was to make a comprehensive survey of the behaviour and levels of ADDB and suspected starting material or intermediates of ADDB, i.e., DCB, 3,3′-dichloro-4,4′-dinitrobiphenyl (DDB), and 4-amino-3,3′-dichloro-4′-nitrobipheny (ADNB) in Waka River water samples. We also postulated the formation pathway of ADDB. Water samples were collected at five sampling sites from the Waka River four times between March 2003 and December 2004. Samples were passed through Supelpak2 columns, and adsorbed materials were then extracted with methanol. Extracts were used for quantification of ADDB and the related chemicals by HPLC on reverse-phase columns; mutagenicity was evaluated in the Salmonella assay using the O-acetyltransferase-overexpressing strain YG1024. High levels of ADDB, DCB, DDB, and ADNB (12.0, 20,400, 134.8, and 149.4 ng/L-equivalent) were detected in the samples collected at the site where wastewater was discharged from chemical plants into the river. These water samples also showed stronger mutagenicity in YG1024 both with and without S9 mix than the other water samples collected from upstream and downstream sites. The results suggest that ADDB is unintentionally formed from DCB via ADNB in the process of wastewater treatment of drainage water containing DCB from chemical plants.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Hydroxylation of naringin by Trichoderma harzianum to dramatically improve its antioxidative activity

Transforming naringin using the mycelium of Trichoderma harzianum CGMCC 1523 produces two metabolites, 3′,4′,5,7-tetrahydroxy flavanone-7-rhamnoglucoside (3′-OHN) and 3′,4′,5′,5,7-pentahydroxy flavanone-7-rhamnoglucoside (3′,5′-DOHN), both of which were characterized by ESI–MS, ¹H NMR and ¹³C NMR analyses. The time course of the biotransformation by T. harzianum showed that 3′-OHN and 3′,5′-DOHN appeared simultaneously at 6 h, and the conversion yield (32.6%) of 3′,5′-DOHN was higher (10.6%) than that of 3′-OHN at 56 h. The optimal biotransformation temperature was 30 °C, the optimal pH was 5.0, and the optimal concentration of naringin was 400 mg/l. The bigger volume of biotransformation mixture and lower shaking speed did not favor hydroxylation reactions. The radical scavenging activity of naringin at 2000 μM was 11.1%, whereas activity of 3′-OHN at 100 μM could reach 38.4%, which is 68.6 times more than naringin. Antioxidative activity of 3′,5′-DOHN was increased 13.5% at 100 μM compared to 3′-OHN.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates inhibit the repair of DNA in rat liver chromatin

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis.     

Furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis

Synthesis and anti-inflammatory effects of certain furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives 12–18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydoxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3′,2′:3,4]naphtho[1,2-d]imidazole (12) was inactive (IC₅₀ value of >30 μM) while its 5-phenyl derivative 13, with an IC₅₀ value of 16.3 and 11.4 μM against lysozyme and β-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC₅₀ value of 19.5 and 11.3 μM against lysozyme and β-glucuronidase release, respectively. An electron-withdrawing NO₂ substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC₅₀ value of 7.4 and 5.0 μM against lysozyme and β-glucuronidase release, respectively.For the LPS-induced NO production, the phenyl derivatives 12–15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC₅₀ value of 1.5 and 1.3 μM, respectively, which are more active than that of the positive 1400 W. Compounds 16–18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC₅₀ of 0.52 μM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.     

High-performance liquid chromatographic assay for the determination of 2′-deoxy-3′-thiacytidine (lamivudine) in human plasma

A method for the quantification of 2′-deoxy-3′-thiacytidine (lamivudine, 3-TC), which incorporated the use of 3-isobutyl-methylxanthine as internal standard (I.S.) was developed and validated in human plasma, using HPLC with UV absorbance detection. Using solid-phase extraction, 3-TC and I.S. were selectively extracted from human plasma. Subsequently, chromatographic separation was performed using a YMC phenyl column with ion-pair chromatography and detection at 270 nm. The method was validated over a concentration range of 10 to 5000 ng/ml using 0.5 ml of human plasma. The extraction recovery for both 3-TC and I.S. was greater than 95%. The determination of inter- and intra-day precision (RSD) was less than 10% at all concentration levels, while the inter- and intra-day accuracy (% difference) was less than 6%.     

Syntheses of daidzein-7-yl β- -glucopyranosiduronic acid and daidzein-4′,7-yl di-β- -glucopyranosiduronic acid

Syntheses of the title compounds — commonly known as ‘daidzein 7-glucuronide’ and ‘daidzein 4′,7-diglucuronide’ — are described. Selective 7-deacetylation of 4′,7-di-O-acetyldaidzein is employed.     

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4′-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4′-hydrazino-2-stilbazole (4′H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis–trans isomerism at the proline–pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4′H2S is performed in a water–acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2–100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4′H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.     

In vivo determination of 5-bromo-2′-deoxyuridine incorporation into DNA tumor tissue by a new P-postlabelling thin-layer chromatographic method

The halopyrimidine 5-bromo-2′-deoxyuridine (BUDR) can serve as one of many indicators of tumor malignity, complementary to histologic grade. We have developed a thin-layer chromatographic (TLC) technique that can assess tumor DNA base composition and analogue (BUDR) incorporation which vies with immunochemistry for BUDR. This requires post-labeling DNA by nick-translation and radioactive 5′-phosphorylation of representative ³²P-α-dNMPs (deoxynucleotide monophosphates). Subsequent 3′-monophosphate digest exchanges a radioactive ³²PO₄ for the neighboring cold nucleotide. Separation in two dimensional PEI-cellulose TLC is carried out in acetic acid, (NH₄)₂SO₄, and (NH₄)HS0₄. TLC of dNMPs was applied to control HeLa DNA, and HeLa cells receiving BUDR. BUDR is detected in 10⁶ HeLa cells after 12–72 h incubations. Findings in HeLa DNA demonstrate normal TLC retention factors for all ³²P-dNMPs. Two dimensional R_F (x,y axes in cm) demonstrate: dAMP=1.4, 9.4; dCMP=10.0, 13.5; dGMP=4.6, 4.4; dTMP=9.0, 7.4; and BUDRMP 6.4, 6.6. This technique quantifies BUDR-which parallels tumor S phase, and serves as an indicator of labelling index (LI).     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Synthesis of Neu5Ac- and Neu5Gc-α-(2→6′)-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-α-(2→6′)-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-α-(2→6′)-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4′,6′-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

2′-hydroxymethyl abscisic acid: A major catabolite of (±)-abscisic acid in leaves of Hordeum vulgare L.

Radioactive (±)-abscisic acid (ABA), supplied via the transpiration stream to light-grown leaves of Hordeum vulgare was catabolized to 2′-hydroxymethyl ABA. Identification was made by capillary gas chromatography-mass spectrometry (GC-MS).     

Comparative biochemical studies of carotenoids in sea cucumbers

The results of an investigation of the carotenoids in the seven species of sea cucumber (Stichopus japonicus, Holothuria leucospilota, H. moebi and H. pervicax of the order Aspidochirotida, Cucumaria japonica, C. echinata and Pentacta australis of the order Dendrochirotida), from the comparative biochemical point of view, are reported. β-Carotene, β-echinenone, canthaxanthin, phoenicoxanthin and astaxanthin were common in all the sea cucumbers examined. A series of novel marine carotenoids (cucumariaxanthin A, B and C) was obtained from the sea cucumbers of the order Dendrochirotida, while they could not be found from those of the order Aspidochirotida. Significant differences in the carotenoid patterns of the two orders were also observed. The structures of cucumariaxanthin A, B and C have been determined, by chemical and spectroscopic investigations, to be (9Z,9′Z)-5,6,5′,6′-tetrahydro-β,β-carotene-4,4′-dione, (9Z,9′Z)-4′-hydroxy-5,6,5′,6′-tetrahydro-β,β-caroten-4-one, and (9Z,9′Z)-5,6,5′,6′-tetrahydro-β,β-carotene-4,4'-diol, respectively. From the experimental results of carotenoids in the sea cucumbers examined, an oxidative metabolic pathway for β-carotene to astaxanthin, and a new reductive and isomeric metabolic pathway for canthaxanthin to cucumariaxanthin C (via cucumariaxanthin A and B) are proposed.