Synthesis of glycosides in which the aglycon is an N-(hydroxymethyl)amino-1,3,5-triazine derivative

The synthesis of analogues of the anti-tumour drug 2-[N-(hydroxymethyl)methylamino]-4,6-bis(dimethylamino)-1,3,5-triazine (HMPMM) in which the OH or a dimethylamino group is replaced by a carbohydrate has been explored. Triazinyl beta-glycosides were readily prepared by reaction of sugars with trimethyl-triazinylammonium salts. These were made with one or two methylamino groups on the triazine for reaction with formaldehyde to give the cytotoxic NMeCH2OH group. However, reaction of the triazinyl glycosides with formaldehyde gave complex intractable mixtures. When the carbohydrate portion was changed to the fully protected 2,3,4,6-tetra-O-acetyl glucose a good yield of the 2-[N-(hydroxymethyl)methylamino]-4-(dimethylamino)-1,3,5-triazin-2 -yl tetra-O-acetyl beta-glucoside was obtained. However, de-acetylation using sodium methoxide also removed the N-CH2OH group. We are investigating protection of the base-sensitive N-CH2OH group as trialkylsilyl and benzyl ethers and are looking at de-acetylation methods that are more selective. We have prepared glycosides in which the sugar is joined through the oxygen of the NMeCH2OH group. Coupling of acetobromoglucose with HMPMM catalysed by silver salts was not successful. Although methyl and cyclohexyl derivatives of HMPMM may be produced in high yields by reaction of HMPMM with methyl and cyclohexyl alcohols under acidic catalysis, production of glycosides in this way gave poor yields. MNDO calculations on reactions of HMPMM helped us devise improved reaction conditions for the condensation of 2,3,4,6-tetra-O-acetyl glucose with HMPMM and its derivatives. The best procedure to generate one of the target glycosides is to react 2,3,4,6-tetra-O-acetyl glucose and formaldehyde with 2-methylamino-4,6-bis(dimethylamino)-1,3,5-triazine. The beta-glycoside product was de-acetylated using potassium carbonate in dry methanol.     

An economic synthesis of 1,2,3,4-tetra-O-acetyl-5-thio-D-xylopyranose and its transformation into 4-substituted-phenyl 1,5-dithio-D- xylopyranosides possessing antithrombotic activity

D-Xylose was converted via 1,2-O-isopropylidene-alpha-D-xylofuranose (4) into 3-O-benzoyl-5-S-benzoyl-1,2-O-isopropylidene-alpha-D-xylofuranose which, after methanolysis, acetylation and subsequent acetolysis afforded 1,2,3,4-tetra-O-acetyl-5-thio-alpha-D-xylopyranose (14) in an overall yield of 36%. Reaction of 4 with thionyl chloride gave a mixture of the diastereomeric cyclic sulfites, the structures of which were established by X-ray crystallography. Their oxidation with sodium periodate afforded the corresponding cyclic sulfate 23. Treatment of 23 with potassium thioacetate gave the potassium salt of 5-S-acetyl-1,2-O-isopropylidene-alpha-D-xylofuranose 3-O-sulfonic acid (26) which, after methanolysis, acetylation and subsequent acetolysis afforded 14 in an overall yield of 56%. Treatment of 4 with sulfuryl chloride gave a mixture containing 5-chloro-3-O-chlorosulfonyl-5-deoxy-1,2-O-isopropylidene-alpha-D- xylofuranose, 3,7,9,11-tetraoxa-4-thia-10-dimethyl-tricyclo[6,3,0, 0(2,6)]undecane S-dioxide and 23 in a 2:3:7 ratio. Tetraacetate 14 was converted into the alpha-1-bromide 18 as well as into the alpha-1-O-trichloroacetimidate 17. These three compounds were used as donors for the glycosylation with 4-cyanothiophenol, affording the 4-cyanophenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- (29) and beta-D-xylopyranoside (30) in different ratios, depending on the reaction conditions. When donor 18 was used in the presence of potassium carbonate, besides 29 and 30 two aryl C-glycosylated-thioglycosides, i.e. 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- and beta-D-xylopyranoside (32 and 33) as well as 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl disulfide 34 could be isolated as byproducts. Deacetylation of 30 with sodium methoxide in methanol afforded, besides 4-cyano-phenyl 1,5-dithio-beta-D-xylopyranoside (1), the corresponding 4-[(methoxy)(imino)methyl]phenyl glycoside 2. The 4-cyano group of 1 was converted into the 4-aminothiocarbonyl, the 4-(methyl-thio)(imino)methyl, the 4-amidino and the 4-(imino)(hydrazino)methyl group. All of these glycosides showed a significant antithrombotic activity on rats.     

Synthesis of allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosi de, a core constituent of the lipopolysaccharide from Acinetobacter calcoaceticus NCTC 10305

Reaction of methyl 2,6-anhydro-2,3-dideoxy-D-manno-2-octenoate 1 with 3-chloroperoxybenzoic acid gave the 2,3-anhydro derivative 2, which was converted into the per-O-acetylated anomeric methyl glycosides of D-glycero-D-galacto-2-octulopyranosylonic acid in good yield. Subsequent inversion of the configuration at C-3 and deprotection afforded sodium (methyl beta-D-glycero-D-talo-2-octulopyranosid)onate. Alternatively, 2 was transformed into methyl (alpha-D-glycero-D-talo-2- octulopyranosyl bromide(onate derivatives. Reaction with methanol or allyl 2-acetamido-2-deoxy- 3,4-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-beta-D-g lycopyranoside, promoted by silver triflate, gave good yields of the corresponding orthoester derivatives. Me3Si triflate-catalyzed orthoester rearrangement and removal of the protecting groups afforded sodium O-(methyl alpha-D-glycero- D-talo-2-octulopyranosid)onate and the disacchanide, allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-gl ucopyranoside in high yield.     

Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta

The active protein components of initiation factor M2B (IF-M2B) have been resolved into two homogeneous factors. These proteins, IF-M2Balpha and IF-M2Bbeta, were purified 300- and 500-fold, respectively, with a yield of about 15% of the original starting activity. The low molecular weight (approximately 17,000) of these two proteins is in contrast with the much greater molecular weights that have been found for other initiation factors. IF-M2Balpha is also unique among the initiation factors in that it contains no tryptophan and is capable of self-association. Both proteins are required for model assays which utilize 40 S and 60 S subunits (poly(U)-directed polyphenylalanine synthesis or AUG-directed methionyl-puromycin synthesis). IF-M2Bbeta has been shown to be required for hemoglobin synthesis, however, the presence of high concentrations of IF-M2Balpha in the 100,000 X g lysate supernatant has precluded the demonstration of an IF-M2Balpha requirement in hemoglobin synthesis.     

Effect of CeO_2-ZrO_2 on Pt/C electrocatalysts for alcohols oxidation

The electrocatalytic activity and stability of Pt/C catalyst modified by using CeO_2-ZrO_2 mixed oxides for the alcohols electrochemical oxidation as probes were investigated. The catalyst samples were characterized by X-ray diffraction(XRD) and scanning electron microscopy(SEM). The electrochemical properties were measured by a three electrode system on electrochemical workstation(IVIUM). The results showed that the presence of CeO_2-ZrO_2 might be associated with the presence of Pt, which indicated that possibly there was synergistic effect between CeO_2-ZrO_2 and Pt nanoparticles. The electrocatalytic activity and stability of Pt-MO_x/C(M=Ce, Zr) for methanol and ethanol oxidation was better than that of Pt-CeO_2/C, which was attributed to that CeO_2-ZrO_2 composited oxides enhanced oxygen mobility and promoted oxygen storage capacity(OSC). Furthermore, the best performance was found when the molar ratio of CeO_2 to ZrO_2 was 2:1 for the oxidation of methanol and ethanol. The forward peak current density of Pt-MO_x/C(M=Ce, Zr, Ce:Zr=2:1) towards the methanol electrooxidation was about 3.8 times that of Pt-CeO_2/C. Pt-MO_x/C(M=Ce, Zr) appeared to be a promising and less expensive methanol oxidation anode catalyst.     

Anti-inflammatory agents and allergen-induced beta2-receptor dysfunction in isolated human bronchi

Antigen challenge causes beta2-adrenoceptor dysfunction in sensitized human bronchi (Am. J. Respir. Crit. Care Med. 1997;155:1230-1234). This study investigated whether the dysfunction can be prevented by anti-inflammatory agents. Human bronchial rings (2 to 4 mm) from surgery were passively sensitized to house dust mite and challenged (1) with allergen only, (2) with allergen plus indomethacin (10(-)5 M), (3) with allergen plus nedocromil sodium (10(-)7 M to 10(-)5 M), (4) with allergen plus the H1-receptor antagonist cetirizine (10(-)7 M to 10(-)5 M), and (5) with allergen plus the peptido-leukotriene receptor antagonist iralukast (10(-)7 M to 10(-)5 M). Rings were first contracted with 10(-)6 M carbachol and then relaxed with salbutamol (10(-)9 M to 10(-)4 M). The concentration-relaxation curve to salbutamol was shifted significantly to the right in the rings challenged with allergen only compared with control rings. In the rings challenged with allergen plus nedocromil sodium (10(-)6 M and 10(-)5 M) or iralukast (10(-)6 M and 10(-)5 M) the concentration-relaxation curves to salbutamol were significantly shifted to the left compared with rings challenged in saline alone, suggesting a protective effect against beta2-adrenoceptor dysfunction. Neither allergen plus cetirizine nor allergen plus indomethacin shifted significantly the concentration-relaxation curves to salbutamol compared with rings challenged in saline alone. We conclude that the release of peptido-leukotrienes may play a significant role in causing the allergen-induced beta2-receptor dysfunction in passively sensitized human bronchi.     

Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and -2 degrees C in different concentrations of methanol and sucrose

The hatching performance of common carp (Cyprinus carpio L.) embryos was examined after 12-72-h storage at 4 and -2 degrees C using different concentrations of sucrose (0.1, 0.25, 0.5 and 1.0 M or 3.42, 8.55, 17.10 and 34.2%), methanol (MeOH) (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 M or 1.6, 3.2, 4.8, 6.4, 8.0, 9.6 and 11.2%), or varying concentrations of methanol in 0.5 M (17.10%) sucrose. For sucrose, 0.5 M (17.10%) showed the maximum survival (41+/-1% (12 h) to 11+/-1.5% (72 h)) at 4 degrees C. No survival was observed at -2 degrees C with any concentration of sucrose. At both temperatures employed, hatching was higher with mixed combination of methanol (1.5 M or 4.8%) and 0.5 M (17.10%) sucrose (4 degrees C: 41+/-1.5% (12 h), 38+/-1.2% (72 h); -2 degrees C: 33+/-1.7% (12 h), 28+/-1.2% (72 h)) compared to methanol alone (4 degrees C: 38+/-1.5% (12 h), 35+/-2.5% (72 h); -2 degrees C: 31+/-2.5% (12 h), 25+/-2% (72 h)). The combination of 1.5 M (4.8%) methanol and 0.5 M (17.10%) sucrose produced the best results among all the concentrations tested at both temperatures.     

Structure-activity relationships of 2'-deoxy-2',2'-difluoro-L-erythro-pentofuranosyl nucleosides

Following the recent discoveries that some L-nucleosides are more or equal potent than their D-counterparts, we synthesized 2'-deoxy-2',2'-difluoro-L-erythro-pentofuranosyl nucleosides as potential antiviral agents. The target compounds were synthesized via the key intermediates 7a or 7b from L-gulono gamma-lactone. Compound 2 was oxidatively cleaved and coupled with ethyl bromodifluoroacetate in the presence of activated zinc under Reformatsky conditions to obtain a diasteomeric mixture of 4(R) and 4(S), in a 4:1 ratio. The major 4(R) isomer was cyclized and treated appropriately to obtain the mesylate 8a or 8b, which was condensed with various silyl-protected pyrimidines. Condensation of the alcohol 7a or 7b with 6-chloropurine under Mitsunobu conditions afforded the 6-chlorpurine analogs 53a or 53b and 54a or 54b. Further treatment of the compounds 53a, 54a and 53b, 54b afforded the inosine and adenine derivatives 57-60, respectively. The condensation of 2-amino-6-chloropurine with compound 8a and subsequent treatment with 2-mercaptoethanol/sodium methoxide afforded the guanine analogs 63 and 64. All of the synthesized nucleosides 31-52, 57-60, 63, and 64 were evaluated for antiviral activity and for cellular toxicity. Adenine derivative 57 showed a moderate activity against HIV-1 in PBM cells (3.4 microM). None of the other compounds showed any significant activities against HIV-1, HBV, HSV-1, HSV-2, and toxicity in Vero, CEM, and PBM cell lines up to 100 microM. The X-ray structure of the 5-iodocytosine analog showed a 2'-exo/3'-endo conformation for the carbohydrate moiety, which is different from those of the biologically active compounds (-)-FTC and L-FMAU.     

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203-210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, beta-estradiol, 17 alpha-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10(-4) M). Beta-Estradiol (10(-4) M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following beta-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of beta-estradiol or 17 alpha-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 x 10(-3) M), like Kepone (10(-5) M), suppressed 2B1/2 mRNA induction following phenobarbital (10(-4) M) treatment, while Kepone alcohol (10(-5) M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.     

Heterogeneity of bovine IgG2. VI. Comparative specificity of monoclonal and polyclonal capture antibodies for IgG2a (A1) and IgG2a (A2)

The relative specificity of 26 randomly selected polyclonal and monoclonal anti-bovine IgG2 reagents for the A1 and A2 allotypic variants of IgG2a was evaluated in a direct RIA using the reagents as solid-phase capture antibodies (CAbs). More than 70% of these reagents were significantly allotype-biased and > 80% of those were positively biased to IgG2a (A1). Compared as the ratio of the ng of IgG2a (A1) bound versus ng IgG2a (A2) bound per 50 ng added (Krel), bias for IgG2a (A1) of six of these reagents was greater than two-fold. Compared in terms of their solid-phase equilibrium constants (Keq), differences as great as two-logs among these reagents were observed. Steward-Petty plots suggested that differences in Krel of a select panel of reagents was usually due to differences in Keq, but for two reagents with large differences in Krel, the existence of one population of CAbs recognizing an allotope and another recognizing common IgG2a determinants, was indicated. Eight of ten guinea pigs immunized with IgG2a (A1) responded with highly significant specificity bias for A1 whereas only two of 11 rabbits and two of ten guinea pigs immunized with IgG2a (A2) responded weakly with preference for IgG2a (A2). These results concur with the concept of the immunodominant nature of the A1 allotope, but also suggest that immunization with IgG2a (A2) might be a practical means of avoiding allotype bias in IgG2a reagents. The data indicate that the majority of randomly selected anti-bovine IgG2 reagents are allotype biased to the extent that when used as serological reagents to measure total IgG2 or bovine IgG2 antibody responses, the allotype of the animal tested rather than its total IgG2a concentration or IgG2 antibody titer, can determine the outcome of the serological test.     

Chromatin-bound protease: (3H)diisopropyl fluorophosphate labeling patterns of chromatin

The nuclei and chromatin of rat liver contain three major proteins reacting with diisopropyl fluorophosphate (DFP). The molecular weights of the three proteins determined by gel filtration in the presence of sodium dodecyl sulfate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 70000, 60000, and 25000. The chromatin isolated from whole liver, instead of nuclei, contains an additional DFP-binding protein whose molecular weight is 100000 in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. The small molecular weight DFP-binding protein can be fractionated from chromatin by 0.25 N HC1 and was found to be a protease which is active in the most commonly used solution for chromatin dissociation, that is, 2-3 M NaCl-5 M urea. This enzyme appears to be the major DFP-binding chromatin-bound protease in the chromatin of most rat tissues. The acid-soluble protease is converted from a 25000-dalton form to a 20000-dalton form during 0.25 N HC1 acid extraction from chromatin, which retains proteolytic activity.     

Isolation and characterization of a microsomal arylaminopeptidase from rat kidney

The isolation and characterization of a microsomal arylaminopeptidase from rat kidney is reported. By treatment of a microsomal arylaminopeptidase-phosphatase-complex with trypsin and subsequent gel filtration of the solubilized proteins on Sepharose 6B a electrophoretic homogeneous arylaminopeptidase was obtained (yield, 3%; enrichment, 900 times). The following properties of the purified enzyme were determined: 1. Molecular weight: 182000 (gel filtration on Sepharose 6B) to 192000 (SDS-polyacrylamide gel electrophoresis). 2. Subunit structure: In the presence of 6 M guanidine - HC1 + 1% BETA-mercaptoethanol the enzyme dissociates into subunits (MW 46700, ESTIMATED BY SDS gel electrophoresis method). 3. Isoelectric point: 4,71 (agarose gel electrophoresis method). 4. UV characteristics: E 280nm/E260NM=1.3. 5. Substrate specifity: optimal substrates L-alanyl derivatives (anilide, beta-naphthyl amide, p-nitroanilide, 4-(phenylazo)-phenylamide and hydrazide). Among these compounds the anilide derivative was hydrolyzed most rapidly. Furthermore, di- and tripeptides, especially L-methionyl-L-leucine, were also split. No hydrolysis was observed with hemoglobin (pH 4.5 and 7.5) and amino acid- or peptide-ester substrates. 6. Optimal pH: 7.5 +/- 0,1; optimal temperature: 45 to 50 degrees C. 7. The enzyme has no transamidation activity with L-alanyl amide both as aminoacyl donator and -acceptor. 8. Influence of effectors: Heavy metal ions (Ni2+, Cd2+, Cu2+, Zn2+), chelating agents (EDTA, o-phenanthroline) and puromycin inhibit the enzyme significantly. SH-group reagents are without any influence. 9. L-alanyl-L-alanyl-4 (phenylazo)-phenylamide, a dipeptide aryl aminopeptidase substrate, is hydrolyzed by the purified enzyme preparation according to a consecutive or step by step mechanism.     

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex

Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.     

Purification of the inlB gene product of Listeria monocytogenes and demonstration of its biological activity

Entry of Listeria monocytogenes into nonphagocytic cells requires the inlAB gene products. InlA and InlB are bacterial cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into tissue culture cell lines in greater detail.     

Mode of reconstitution of chicken erythrocyte and reticulocyte chromatin

The mode of reconstitution of chicken erythrocyte and reticulocyte chromatin has been investigated. Chromatin was dissociated in 2 M NaCl, 5 M urea, and 0.01 M potassium phosphate (pH 7.2) and was dialyzed against various NaCl concentrations in 5 M urea and 0.01 M potassium phosphate (pH 7.2). Histone reassociation to DNA occurs with the binding of histone H5 at 0.5 M NaCl in 5 M urea, followed by histone H1 at 0.4 M NaCl in 5 M urea. All the classes of histones are reassociated with DNA at 0.2 M NaCl in 5 M urea and binding of all classes of histones is complete in 0.1 M NaCl and 5 M urea. Nonhistone proteins reassociate with DNA before and at the same time that histones reassociate with DNA. Binding of nonhistone proteins to DNA appears to be complete in 5 M urea and 0.01 M potassium phosphate (pH 7.2). There is also found in both erythrocyte and reticulocyte chromatin a nonhistone protein present in relatively high concentrations, which remains associated with DNA in 2 M NaCl and 5 M urea. This tightly bound protein appears as one major band when chromatographed on sodium dodecyl sulfate-polyacrylamide gels, with a molecular weight of 95 000. This protein is soluble in phenol and sodium dodecyl sulfate but is insoluble in 5 M urea or 4 M guanidine hydrochloride. A fraction of reticulocyte nonhistone proteins was found to bind to DNA-cellulose in 5 M urea. The majority of these proteins elute at 0.15 M NaCl in 5 M urea but a significant fraction elutes at NaCl concentrations at which the bulk of the histones do not bind to DNA. The proteins that bind to free DNA have low molecular weights and do not show species speciificity. Approximatley 50% of the reticulocyte nonhistone protein does not bind to a DNA-cellulose column in 5 M urea and may require histones for complete reassociation.     

Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim

The purpose of this study was to determine whether the two monoclonal anti-cardiac troponin T (cTnT) antibodies (MAbs) used in the second generation cTnT assay by Boehringer Mannheim (BM, capture Ab, M11.7; detection Ab, M7) would detect cTnT isoforms expressed in human skeletal muscle in response to chronic renal disease (CRD). cTnT expression was examined in skeletal muscle biopsies obtained from 45 CRD patients, as well as nondiseased human heart (n = 3) and skeletal muscle (n = 3). cTnT proteins were resolved by modified 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the following anti-cTnT MAbs: M11.7; M7; JS-2, Lakeland Biomedical; and 13-11, Duke University. All four antibodies detected the cTnT isoforms (Ta, Te) expressed in human myocardium. In 20 of 45 skeletal muscle biopsies, MAb M11.7 recognized its epitope in one to three proteins, molecular mass 34-36 kDa, designated Te, Td, and Tc; the strongest signal was that of Te. The same proteins were recognized by MAbs JS-2 and 13-11. The BM M7 antibody did not detect the cTnT isoforms in the molecular mass range of 34-36 kDa. However, MAb M7 did detect a cTnT isoform, molecular mass 39 kDa, in 2 of 45 biopsies. This isoform had an electrophoretic mobility similar to the predominant heart cTnT isoform, Ta. We conclude that cTnT isoforms are expressed in the skeletal muscle of CRD patients. However, given the epitopes recognized by the BM MAbs M7 and M11.7 and the variable presence of these cTnT isoforms in skeletal muscle, the second generation BM cTnT assay will not detect these isoforms if they are released from skeletal muscle into the circulation.     

Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.     

Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E

The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). Isogenic uspA1, uspA2, and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, the uspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 by M. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and the uspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and the uspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis.     

Rapid and sensitive high-performance liquid chromatographic analysis of halogenopyrimidines in plasma

Recent studies have stressed the need for individual adjustment of 5-fluorouracil (5-FU) dosage. Most of the techniques previously reported are not well adapted to routine application. We describe a sensitive, selective and simple HPLC technique under isocratic conditions for the quantitation of 5-FU and other halogenopyrimidines. The proportion of reagents and internal standard were optimised to allow the use of minitubes, particularly adapted to large series of plasma assays. High extraction yield, 75% for 5-FU and 90% for 5-bromouracil and 5-chlorouracil, was obtained using 1.2 ml isopropanol-ethyl acetate (15:85, v/v) following precipitation of plasma proteins with 300 mg ammonium sulfate. The mobile phase was 0.01 M phosphate buffer (pH 3.0). Uracil and 5-fluorouracil were fully resolved with Spherisorb ODS2 column. The limits of quantitation and detection in human plasma were 6 ng ml(-1) and 3 ng ml(-1), respectively, for all compounds studied. The total analysis time required for each run was 25 min. Final results could be given within 90 min of blood sampling. At least 50 plasma samples could be analysed per day. This method has been successfully used for monitoring 5-FU-based treatments.     

Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins

Elastin fibres can be decomposed into their constituting proteins using several processes; in particular by saponification of some bonds with KOH in aqueous tertiobutyl alcohol, elastin solubilized proteins - ESP- of 10 to 200 KDa were produced with a good yield (70-80%). It is demonstrated that some of these proteins were capable of tightly re-associating with the native elastin fibres and remained bound on the fibres, in spite of several drastic washes using 1 M Guanidinium, HCl at 37 degrees C for 1 h. At pH 4-5, approximately 30-40 microg ESP were retained per mg elastin. The same association is also shown to occur, under similar conditions, with Poly-ethylene-terephthalate, Poly-hexamethylene diamine-adipic acid but not with polyurethanes. The optimal conditions of the coupling were described as depending on ESP concentration, time, pH, ionic strength and Ca++. It was also shown that opposite pH conditions, i.e. pH 14, 0.5 M NaOH, could allow the retained proteins to desorb from polyesters. Hence, it will be possible to determine the sort of proteins which could be involved. This property of ESP allows us (1) to better understand the exceptional capacity of tissue repair certainly due to adhesive properties of the artificial connective matrices containing elastin or ESP, developed in our laboratory, (2) to provide a new approach for elucidating elastin microstructure and function, (3) especially to provide a new mode for coating certain fibres, yielding materials with bioactive and biofunctional qualities.