Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

We have prepared, selected and cloned four mouse hybridomas that secreted monoclonal antibodies against human plasma apolipoprotein A-I. These antibodies are all of the IgG-I subclass, and were named anti-A-I 6B8, 5G6, 3D4 and 5A6. We characterized the specificity of the antibodies, finding that all four of them reacted similarly, and with only the major proteins having the molecular weight and isoelectric focusing characteristics of apolipoprotein A-I. The antibodies reacted with all known charge-polymorphs of apolipoprotein A-I and pro apolipoprotein A-I. Thus, the polymorphs of apolipoprotein A-I are alike in that they all contain the antigenic sites of these four antibodies. In a solid-phase, antibody competition radioimmunoassay we found inhibition or enhancement of antibody binding to apolipoprotein A-I, according to the pair of antibodies tested. Antibodies 6B8, 5G6 and 3D4 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. We compared the reactions of the four antibodies with CNBr-cleaved fragments of apolipoprotein A-I separated by polyacrylamide gel electrophoresis. We found three different patterns of reaction with the apolipoprotein A-I fragments; 6B8, 5G6 and 3D4 were different, but 5A6 resembled 3D4. Thus, the four antibodies reacted with at least three different antigenic sites in apolipoprotein A-I, which were present in different CNBr fragments of apolipoprotein A-I, but not on fragment 4 which forms the carboxy-terminal segment.     

Solid-phase synthesis of 4,8-disubstituted-8,9-dihydropyrazino[2,3-g]quinazolin-7(6H)-ones

An approach for the solid-phase synthesis of 4,8-disubstituted-8,9-dihydropyrazino[2,3-g]quinazolin-7(6H)-ones from 4-chloro-7-fluoro-6-nitroquinazoline scaffold is described. A chemoselective reaction of resin-bound arylamines (3) with 4-chloro-7-fluoro-6-nitroquinazoline (8) yielded resin-bound 4-arylamino-7-fluoro-6-nitroquinazolines (4), which were reacted with amino acid methyl esters to afford the corresponding resin-bound compound (5). Following the reduction of nitro group and intramolecular cyclization of 5, resin-bound 4,8-disubstituted-8,9-dihydropyrazino[2,3-g]quinazolin-7(6H)-one (6) was yielded. The desired products (7) were obtained in good yields and purities after cleavage from the resin.     

New carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; Synthesis,X-ray crystallography and anticancer activity

An amine group was synthesized starting from an optically active bicyclo[2.2.1]heptane compound, which was then used to build the 5 atoms ring of a key 6-chloropurine intermediate. This was then reacted with ammonia and selected amines obtaining new adenine- and 6-substituted adenine conformationally constrained carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane skeleton in the sugar moiety. X-ray crystallography confirmed an exo-coupling of base to the ring and a L configuration of the nucleoside analogues. The compounds were tested for anticancer activity.     

Synthesis and structure-activity relationships of taxuyunnanine C derivatives as multidrug resistance modulator in MDR cancer cells

A series of new generation taxoids bearing a bulky group on different positions such as C-2, C-5, C-7, C-9, C-10 or C-14 were obtained by chemical modifications and biotransformation of taxuyunnanine C (1) and its analogs, 4, 5, and 10. Compounds 3, 5, 6, 8, and 9a showed significant activity toward calcein accumulation in MDR 2780AD cells. The most effective compound 9a with a cinnamoyloxy group at C-14 and a hydroxyl group at C-10 was actually efficient for the cellular accumulation of the anticancer agent, vincristine, in MDR 2780AD cells. The enhancing effects of 6 and 9a for taxol, adriamycin, and vincristine were at the same levels as those of verapamil toward MDR 2780AD cells. Thus, compounds 6 and 9a can modulate the multidrug resistance of cancer cells. The cytotoxicity (IC(50)) of the compounds was examined against human normal cell line, WI-38, and cancer model cell lines, VA-13 and HepG2. Since compounds 6 and 8 had no cytotoxicity, they were expected to be lead compounds of MDR cancer reversal agents. On the contrary, compounds 3, 5, and 9a showed cell growth inhibitory activity toward VA-13 and/or HepG2 as well as accumulation activity of calcein and/or vincristine in MDR 2780AD and they were expected to be lead compounds of new-type anticancer agents.     

A novel method for the incorporation of glycoprotein-derived oligosaccharides into neoglycopeptides.

We describe a new method for the transfer of carbohydrate moieties to polypeptides in which complex carbohydrate, in the form of glycosyl amino acid, is removed from an available glycoprotein, derivatized, and reacted with a polypeptide via an iodoacetylated alpha-amino group. A family of oligomannose chains, N-linked to the side chain of Asn, was obtained from ovalbumin by pronase digestion and purified as previously described. A reactive sulfhydryl group was specifically placed on these molecules by reaction of 2-iminothiolane with the Asn alpha-amino group. Separately, the alpha-amino group of the peptide GGYR was specifically iodoacetylated by reaction with iodoacetic anhydride at pH 6. Reaction of the thiol-containing carbohydrate with iodoacetylated peptide at pH 8 gave in high yield the corresponding oligomannosyl-peptides, whose structures were confirmed by mass spectrometry. A peptide inhibitor of HIV protease was also oligomannosylated by this procedure. The principle advantage of this method is the efficiency of the reaction even when performed with stoichiometric amounts of the two molecules at low concentration. It should be feasible to extend this chemistry to larger polypeptides.     

Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains.

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis--381, ATCC 33277, BH18/10, OMZ314, OMZ406, 6/26 and HW24D-1--by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost identical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.     

Bile acids. 39. Metabolism of 5 -cholestane-3 ,26-diol and 5 -cholestane-3 ,7 ,26-triol in the rat with a bile fistula

25r-5alpha-[5alpha,6alpha-(3)H(2)]Cholestane-3beta,7alpha,26-triol was prepared from 3beta,26-diacetoxy-5alpha[5alpha,6alpha-(3)H(2)]cholestan-7-one that was obtained from kryptogenin. Huang-Minlon reduction of the ketone provided 25r-5alpha-[5alpha-(3)H]cholestane-3beta,26-diol. Results from mass spectrometry, molecular rotation, and several types of chromatography are consonant with the assigned structures. Bile was collected for 8 days from adult male rats, with cannulated bile ducts, that had received approximately 0.8 mg of the triol or diol intraperitoneally. Bile from the first 12 hr was hydrolyzed, and the bile acids were separated by partition chromatography. The chromatographic pattern of separated bile acids was much simpler for the triol than the diol. Approximately 50% of the bile acids derived from the triol were trihydroxy allo acids (allocholic acid, 44%, and its 3beta isomer, 5.3%); only 16.4% allocholic acid was obtained from the diol. Comparable amounts of allochenodeoxycholic acid were derived from the diol and triol (21.2% and 28.2%, respectively). Unidentified metabolites in the dihydroxy acid fraction derived from the diol constitute 15.8% of chromatographed material.     

Synthesis and cytotoxic evaluation of a series of resveratrol derivatives

A total of 17 resveratrol (=(E)-5-[2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol) derivatives were synthesized from resveratrol (RES) through a facile approach. Among them, 13 compounds, 2 and 6-17, were reported for the first time, while 1 and 3-5 had already been reported several years ago. The cytotoxic activities of these compounds were evaluated against human nasopharyngeal epidermoid tumor cell line KB, and compounds 1 and 8-11 showed strong anticancer activities in vitro, comparable with that of 5-fluorouracil, an anticancer drug. On the basis of the experimental data obtained, structure-activity relationships are discussed.     

Laser flash photolysis studies of electron transfer between ferredoxin-NADP+ reductase and several high-potential redox proteins

Complex formation and the kinetics of electron transfer between ferredoxin-NADP+ reductase (FNR) and two structurally homologous acidic 4Fe-4S high-potential ferredoxins (HiPIP's) from Ectothiorhodospira halophila (HP1 and HP2) and two structurally homologous cytochromes c2 from Paracoccus denitrificans and Rhodospirillum rubrum (PC2, and RC2, respectively) have been investigated by gel filtration and laser flash photolysis techniques. Gel filtration studies indicated that complex formation occurred between FNRox and HP1ox or HP2ox at low ionic strength (10 mM) and that the complexes were completely dissociated at high ionic strength (310 mM). Laser flash photolysis using lumiflavin as the reductant demonstrated that both free HP1ox and HP2ox reacted primarily with the anionic form of fully reduced lumiflavin (LFH-), whereas FNR was unreactive. Second-order rate constants of 1 X 10(6) and 0.8 X 10(6) M-1 s-1 were obtained for these reactions at 10 mM ionic strength. Increasing the ionic strength to 310 mM resulted in an approximately 1.5-fold increase in the rate constant. Inclusion of stoichiometric amounts of FNRox into the reaction mixture at low ionic strength led to a 2.5-fold increase in the rate constants. The reaction of 5-deazariboflavin semiquinone (5-dRf.) with the oxidized HiPIP's was also investigated by laser flash photolysis. Second-order rate constants of 3.0 X 10(8) M-1 s-1 (HP1) and 2.5 X 10(8) M-1 s-1 (HP2) were obtained for the free proteins at 10 mM ionic strength. Under the same conditions, 5-dRf. reacted with free FNRox, resulting in the formation of the neutral protein-bound semiquinone (FNR.), with a second-order rate constant of 6 X 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypochaerin: a new sesquiterpene lactone from Hypochaeris setosus

Hypochaeris setosus contains desacetoxymatricarin, achillin, 1-hydroxy-6β,7α,11β-H-eudesm-4-en-6,12 olide, jacquinelin and hypochaerin, a new guaianolide, established as 3-oxo-4β,5α,6β,7α,11β-H-guai-1(2)-en-6,12 olide.     

Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Method for the synthesis of uric acid derivatives

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO2 and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O6-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).     

Antigenic analysis of potato virus A particles and coat protein

Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.     

Probing the surface of Z-DNA with anti-nucleoside antibodies

Antibodies specific for cytidine (C) and guanosine (G) were used to probe the surface of two Z-DNA conformers. When tested by ELISA, anti-G reacted with poly(dG-dC).poly(dG-dC) treated with bromine water [Br-poly(dG-dC).poly(dG-dC)] but anti-C did not. A weak reaction with anti-C was detected by dot immunobinding. In contrast, anti-C reacted strongly with poly(dG-dC).poly(dG-dC) treated with N-acetoxy-2-(acetylamino)fluorene [AAF-poly(dG-dC).poly(dG-dC)]; anti-G reacted weakly, despite the fact that most G residues had not been substituted with AAF. Neither antinucleoside bound to the B conformation of poly(dG-dC).poly(dG-dC). In competition experiments, GMP was the most efficient competitor of the reaction of anti-G with Br-poly(dG-dC).poly(dG-dC); AMP and TMP were 100-fold less efficient, and CMP did not compete to a significant extent. In contrast, the reaction of anti-Z with Br-poly(dG-dC).poly(dG-dC) was not inhibited by nucleotides. Of five possible sites recognized on guanosine by anti-G antibodies (N1, C6, O6, N7, and C8), AMP and TMP share three or their equivalent and CMP only one. The binding of anti-C to AAF-poly(dG-dC).poly(dG-dC) was inhibited best by CMP; AMP was 8 times less efficient; GMP and TMP were about 35-fold less efficient than CMP. Thus, although the amino group on the C4 position of CMP appears to be immunodominant, the capacity of GMP and TMP to inhibit the reaction indicates that other sites are also recognized in AAF-poly(dG-dC).poly(dG-dC), e.g., the exposed C5 position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Synthesis and in vitro and in vivo anticancer activity of novel phenylmethylene bis-isoxazolo[4,5-b]azepines

A series of novel phenylmethylene bis-isoxazolo[4,5-b]azepine derivatives (10) have been synthesized from 3-methyl-4-nitro-5-styrylisoxazoles 6. The reaction of 6 with 3,5-dimethyl-4-nitroisoxazole (7) in piperidine afforded the Michael type adducts 8, which on treatment with different substituted chalcones in the presence of piperidine gave the Michael adducts 9. Compounds 9 underwent reductive cyclization on treatment with SnCl(2)-MeOH to afford the title compounds 10. Structure of these compounds was established on the basis of IR, (1)H NMR, (13)C NMR and Mass spectral data. The title compounds 10a-j were evaluated for in vitro and in vivo anticancer activity. Compound 10j exhibited good anticancer activity as that of standard drug Cisplatin.     

IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

Antigenic components in the crude extracts of Spirometra mansoni plerocercoid were analyzed in early experimental infections and in IgG subclass observed in clinical sparganosis. By IgG immunoblot, sera obtained serially from experimental mice, fed 5 spargana each, were reacted with the crude extracts. Protein bands at 36-26 kDa and 103 kDa showed positive reactions since two weeks after infection. In a differential immunoblot, in which a monospecific antibody against sparganum chymase at 36 kDa was pre-treated, the reactions at 36-26 kDa disappeared, indicating that the sparganum chymase and its degradation products invoked IgG antibody reactions. When 69 patients sera of human sparganosis were examined for their IgG subclass responses, IgG4 levels showed the highest reaction which was followed by IgG1. The IgG4 antibody also reacted mainly with 36-31 kDa protease. These results indicate that 36 kDa chymase of S. mansoni plerocercoid is the main antigenic component inducing IgG antibody response in early stage of experimental sparganosis and for specific IgG subclass reactions in human sparganosis.     

Topography of Escherichia coli ribosomal proteins. The order of reactivity of thiol groups

1. 30S and 50S ribosomal subunits of Escherichia coli were treated with N-[2,3-(14)C]-ethylmaleimide and iodo[(14)C]acetamide. 2. The proteins in the native subunits which reacted with the reagents were S1,double dagger S2, S12, S13, S18, S21, L2, L5, L6, L10, L11, L15, L17, L20, L26+28 and L27. 3. Several proteins, such as S1, S12, S14, S18, L2, L6, L10, L11 and either L26 or 28, had thiol groups in an oxidized form and reacted to a greater extent after reduction with beta-mercaptoethanol or dithiothreitol. 4. The total number of thiol groups in 30S and 50S subunits was determined as 16-17 and 26-27 respectively. The total number of thiol groups in each ribosomal protein was also determined. 5. The reaction of 30S and 50S subunits with iodoacetamide under several different conditions established the order of reactivity of thiol groups.     

Tissue origin of soluble component proteins in saline extract of adult Paragonimus westermani.

Tissue origin of individual component proteins in crude extract of adult Paragonimus westermani was investigated. Major soluble component proteins were separated by disc-PAGE in 8% slab gel. By predefined Rf values, strips of gel containing each band protein was cut out. Each band protein was eluted by electrophoresis. Monospecific antibodies were prepared by immunizing rabbits with each band protein. When peroxidase-antiperoxidase (PAP) staining was done, antiserum to Band 1 reacted to content of eggs both in the worm and in the infected lung tissue. Antiserum to Band 2 reacted to parenchymal tissue of the worm. Antiserum to Band 4 showed the positive reaction at intestinal content while that to Band 5 reacted to the intestinal epithelial border. Antiserum to combined proteins of Bands 6/7 and that to Band 8 reacted to parenchymal tissue of the worm respectively. From the results, the origin of individual proteins in crude extract of adult P. westermani could be differentiated.