Synthesis and structure–activity relationships of N-aryl(indol-3-yl)glyoxamides as antitumor agents

The synthesis and study of the structure–activity relationships of cytotoxic compounds based on N-pyridinyl or N-aryl-2-(1-benzylindol-3-yl)glyoxamide skeleton, represented by the lead structures D-24241 and D-24851, are described. The presence of N-(pyridin-4-yl) moiety was crucial for activity and 2-[1-(4-chloro-3-nitrobenzyl)-1H-indol-3-yl]-2-oxo-N-(pyridin-4-yl)acetamide (55), the most potent derivative, showed IC₅₀ = 39 nM, 51 nM and 11 nM against HeLa/KB (human cervix carcinoma), L1210 (murine leukemia) and SKOV3 (human ovarian carcinoma) cell lines proliferation assay, respectively, as active as the lead compounds.     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Kinetic properties and substrate specificities of two recombinant human N-acetylglucosaminyltransferase-IV isozymes

N-acetylglucosaminyltransferase (GnT)-IV catalyzes the formation of the GlcNAcβ1-4 branch on the GlcNAcβ1-2Manα1-3 arm of the core structure of N-glycans. Two human GnT-IV isozymes (GnT-IVa and GnT-IVb) had been identified, which exhibit different expression profiles among human tissues and cancer cell lines. To clarify the enzymatic properties of the respective enzymes, their kinetic parameters were determined using recombinant full-length enzymes expressed in COS7 cells. The K _m of human GnT-IVb for UDP-GlcNAc was estimated to be 0.24 mM, which is 2-fold higher than that of human GnT-IVa. The K _m values of GnT-IVb for pyridylaminated (PA) acceptor sugar chains with different branch numbers were 3- to 6-fold higher than those of GnT-IVa. To compare substrate specificities more precisely, we generated recombinant soluble enzymes of human GnT-IVa and GnT-IVb with N-terminal flag tags. Both enzymes showed similar substrate specificities as determined using fourteen PA-sugar chains. They preferred complex-type N-glycans over hybrid-types. Among the complex-type N-glycans tested, the relative activities of both enzymes were increased in proportion to the number of GlcNAc branches on the Man α1-6 arm. The Man α1-6 arm of the acceptors was not essential for their activities because a linear pentasaccharide lacking this arm, GlcNAcβ1-2Manα1-3Manβ1-4GlcNAcβ1-4 GlcNAc-PA, was a substrate for both enzymes. These results indicate that human GnT-IVb exhibits the same acceptor substrate specificities as human GnT-IVa, although GnT-IVb has lower affinities for donors or acceptors than GnT-IVa. This suggests that GnT-IVa is more active than GnT-IVb under physiological conditions and that it primarily contributes to the biosynthesis of N-glycans.     

32P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10⁴ nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10⁴ nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10⁸ nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10⁸ nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.     

Discovery and in vitro and in vivo profiles of 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as novel class of an orally active metabotropic glutamate receptor 1 (mGluR1) antagonist

We identified 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide 27 as a potent mGluR1 antagonist. The compound possessed excellent subtype selectivity and good PK profile in rats. It also demonstrated relatively potent antipsychotic-like effects in several animal models. Suitable for development as a PET tracer, compound 27 would have great potential for elucidation of mGluR1 functions in human.     

Design,Synthesis and Bioevaluation of Two Series of 3‐[(1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐ones and N‐(1‐Benzylpiperidin‐4‐yl)quinazolin‐4‐amines

Two series of 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐ones and N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines were designed initially as potential acetylcholine esterase inhibitors. Biological evaluation demonstrated that N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines significantly inhibited AChE activity. Especially, two compounds of them were found to be the most potent with relative AChE inhibition percentages of 87 % in comparison to donepezil. The docking studies with AChE showed similar interactions between donepezil and four derivatives. N‐(1‐Benzylpiperidin‐4‐yl)quinazolin‐4‐amines also exhibited significant DPPH scavenging effects. The two series of compound also exerted moderate to good cytotoxicity against three human cancer cell lines, including SW620 (human colon cancer), PC‐3 (prostate cancer), and NCI?H23 (lung cancer), with 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one being the most cytotoxic agent. 3‐[(1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one significantly induced early apoptosis and arrested the SW620 cells at G2/M phase. From this study, two compounds of N‐(1‐benzylpiperidin‐4‐yl)quinazolin‐4‐amines could serve as new leads for further design and AChE inhibitors, while 3‐[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]quinazolin‐4(3H)‐one could serve as a new lead for the design and development of more potent anticancer agents.     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.     

Metabolism of <Emphasis Type="Italic">N</Emphasis>-Methyl, <Emphasis Type="Italic">N</Emphasis>-propargylphenylethylamine: Studies with Human Liver Microsomes and cDNA Expressed Cytochrome P450 (CYP) Enzymes

Summary 1. We used an in vitro screening procedure and studies with individual human liver microsomes and cDNA-expressed CYP enzymes to investigate the metabolism of the putative neuroprotective drug N-methyl,N-propargyl-2-phenylethylamine (MPPE) to N-methylphenylethylamine (N-methylPEA) and N-propargylphenylethylamine (N-propargylPEA). 2. An electron-capture gas chromatographic procedure previously developed in our laboratories was used to measure the quantities of N-methylPEA and N-propargylPEA formed in the experiments with a single donor human liver microsome panel and cDNA expressed single CYP enzyme systems. The data were fitted to nonlinear regressions using Prism to determine kinetic constants. The results from a fluorogenic screen determined which cDNA-expressed single CYP enzymes were investigated. 3. CYP2B6, CYP2C19, and CYP2D6 all contributed to the formation of N-methylPEA, while only CYP2B6 catalyzed the formation of N-propargylPEA. The K _M and V _max values for N-propargylPEA formation were 290 ± 70 μM and 139±16 ng/mL/min. The values for formation of N-methylPEA were not determined from these experiments due to the complexity of fitting the data to a three-variable equation, but data on the time course of N-methylPEA formation are presented. 4. Catabolism of MPPE to N-methylPEA and N-propargylPEA is catalyzed by CYP enzymes. CYP2B6, 2C19 and 2D6 all contribute to the depropargylation of the parent compound, but only CYP2B6 also catalyzes demethylation. CYP2C19 was found to be the most active with respect to generation of N-methylPEA.     

Design,Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A₃ adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N ⁶-(3-iodobenzyl)- and 2-chloro-N ⁶-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K _i = 0.38 nM and 0.28 nM, respectively) at the human A₃AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.     

Alteration of lacto-series glycolipid glycosyltransferase activities in human colonic adenocarcinoma DLD-1 cells after culture in N,N-dimethylformamide-containing medium

Human colonic adenocarcinoma DLD-1 cells were grown under conditions which induce characteristics of differentiated cells using medium containing 0.8% N,N-dimethylformamide in order to study alterations in glycosphingolipid glycosyltransferase activities during this process. Analysis of biosynthetic reactions involved in lacto-series antigen synthesis revealed no changes in the specific activities of either β1→4galactosyltransferase or α1→3/4fucosyltransferase with N,N-dimethylformamide treatment. However, a dramatic decrease of from 14- to 20-fold in the β1→3N-acetylglucosaminyltransferase activity was observed in the treated cells. This enzyme catalyzes the rate-limiting step in lacto-series core chain synthesis. This is consistent with the pattern of regulation of lacto-series antigen expression found to occur during oncogenesis in human colonic mucosa (Holmes EH, Hakomori S, Ostrander GK: J Biol Chem 262:15649, 1987). Total glycolipids from untreated and N,N-dimethylformamide-treated cells were isolated and subjected to TLC immunostain analysis and solid phase radioimmunoassay with a series of monoclonal antibodies specific for lacto-series-based carbohydrate antigens. A decrease of about 2-fold or less in the quantity of lacto-series antigens was observed as a consequence of N,N-dimethylformamide treatment in both neutral glycolipid and ganglioside fractions. The results suggest that only very low levels of β1→3N-acetylglucosaminyltransferase activity are required for the steady state expression of significant levels of lacto-series based glycolipids and that modulation of its activity levels by N,N-dimethylformamide treatment in DLD-1 cells represents a convenient in vitro system for studying aspects of regulation of lacto-series antigen expression.     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

Interactions of a few azole derivatives with a transport protein: role of heteroatoms

The interaction of a few azole derivatives, 2‐(4′‐N,N‐dimethylaminophenyl)benzimidazole, 2‐(4′‐N,N‐dimethylaminophenyl)benzoxazole, 2‐(4′‐N,N‐dimethylaminophenyl)oxazolo[4,5‐b]pyridine with bovine serum albumin (BSA) were examined by absorption and fluorescence spectroscopy. The results were compared with the previously studied imidazopyridine derivative 2‐(4′‐N,N‐dimethylaminophenyl)imidazo[4,5‐b]pyridine. Displacement studies were carried out with site selective probes to locate the binding site of these ligands. The spectral shifts and the binding constant vary depending on the nature of the ligand. The fluorescence intensity of both oxazole derivatives 2‐(4′‐N,N‐dimethylaminophenyl)benzoxazole and 2‐(4′‐N,N‐dimethylaminophenyl) oxazolo[4,5‐b]pyridine increases substantially in the presence of BSA, whereas the intensity of 2‐(4′‐N,N‐dimethylaminophenyl)benzimidazole decreases. However, hypsochromic shift is observed in presence of BSA. The results obtained from the docking studies are also in good agreement with the experimental results. The location and orientation of binding depend upon the nature of the ligand. The studies revealed that apart from hydrophobic interaction, hydrogen bonding also plays a vital role in the molecular binding. Oxazoles have higher binding affinity than imidazoles and substitution of extra nitrogen further increases the binding affinity. Copyright © 2015 John Wiley & Sons, Ltd.     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ～2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.     

Theoretical,in Vitro Antiproliferative,and in Silico Molecular Docking and Pharmacokinetics Studies of Heteroleptic Nickel(II) and Copper(II) Complexes of Thiosemicarbazone-Based Ligands and Pefloxacin

Twelve new heteroleptic nickel(II) and copper(II) complexes of the type [M(L^1–6)(Pfx)₂] ( 1 – 12 ), where L^1–6=2-benzylidenehydrazinecarbothioamide (L¹), 2-benzylidene-N-methylhydrazinecarbothioamide (L²), 2-benzylidene-N-phenylhydrazinecarbothioamide (L³), 2-(4-methylbenzylidene)hydrazinecarbothioamide (L⁴), 2-(4-methylbenzylidene)-N-methylhydrazinecarbothioamide (L⁵) and 2-(4-methylbenzylidene)-N-phenylhydrazinecarbothioamide (L⁶), Pfx=pefloxacin and M=Ni(II) or Cu(II) have been synthesised, and their structures were confirmed by different spectral techniques. The spectral data and density functional theory (DFT) calculations supported the bonding of pefloxacin drug molecule via one of the carboxylate oxygen atoms and the pyridone oxygen atom, and the thiosemicarbazone ligand via the imine nitrogen and the thione sulfur atoms with the metal(II) ion, forming distorted octahedral geometry. In vitro antiproliferative activity of the synthesized complexes was evaluated against three human breast cancer (T47D, estrogen negative (MDA-MB-231) and estrogen positive (MCF-7)) as well as non-tumorigenic human breast epithelial (MCF-10a) cell lines, which showed the higher activity for the copper(II) complexes. The interaction of the synthesized complexes with an oncogenic protein H-ras (121 p) was explored by in silico molecular docking studies. Further, in silico pharmacokinetics and ADMET parameters were also analysed to predict the drug-likeness as well as non-toxic and non-carcinogenic behavior, and safe oral administration of the complexes.     

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N⁶-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N⁶-(3-aminopropyl)adenine, N⁶-(4-aminobutyl)adenine, N⁶-(4-amino-trans-but-2-enyl)adenine, N⁶-(4-amino-cis-but-2-enyl)adenine and N⁶-(4-aminobut-2-ynyl)adenine. From these, N⁶-(4-aminobutyl)adenine and N⁶-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (K_m～10^?4?M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N⁶-(3-aminopropyl)adenine, N⁶-(4-amino-cis-but-2-enyl)adenine and N⁶-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N⁶-(3-aminopropyl)adenine and N⁶-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (K_i～10^?5–10^?4?M) whereas, N⁶-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

Methylated N-aryl chitosan derivative/DNA complex nanoparticles for gene delivery: Synthesis and structure–activity relationships

In this study, three kinds of methylated chitosan containing different aromatic moieties were synthesized by two steps, reductive amination and methylation, respectively. The chemical structures of all methylated derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) were characterized by ATR–FTIR and ¹H NMR spectroscopy. The complexes between the chitosan derivatives and plasmid DNA at different N/P ratios were characterized by gel electrophoresis, dynamic light scattering, and atomic force microscopic techniques. The smallest particle sizes of these complexes were obtained at N/P ratio of 5 and ranged from 95 to 124 nm while the zeta-potentials were in the range of 18–27 mV. Transfection efficiencies of these complexes were investigated by expression of the plasmid DNA encoding green fluorescence protein (pEGFP-C2) on human hepatoma cells (Huh 7 cells) compared to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efficiency was MPyMeChC > MDMBzChC > TMChC > MDMCMChC, respectively. The cytotoxicity of these complexes was also studied by MTT assay where the MPyMeChC complex exhibited less toxicity than other derivatives even at high N/P ratios. Therefore, MPyMeChC demonstrated potential as its safe and efficient gene carrier.     

Inhibitory effect of ursolic acid derivatives on recombinant human aldose reductase

Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives with ursane skeleton were tested for recombinant human aldose reductase (rhAR) inhibitory activity for development of diabetic complications. Among them, N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) showed most potent rhAR inhibitory activity in vitro. Inhibition mode of N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) was tested uncompetitively by kinetic analysis using the Lineweaver-Burk plots. Ursolic acid derivative N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid is able to inhibit rhAR uncompetitively and could be offered as a lead compound for AR inhibition.     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.