Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

油包水微乳液中抗体酶催化布洛芬酯选择性水解的酶学特性

根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21.     

Inhibition of growth and adhesion ofHelicobacter pylori using egg yolk antibodies

Helicobacter pylori is known as a key pathogen for chronic gastric and duodenal ulcers. Egg yolk antibody, IgY produced from chicken immunized withH. pylori antigen was tested for the inhibition of growth and adhesion ofH. pylori to gastric epithelial cell, AGS. The colony forming ofH. pylori was repressed by 30% using 1 mg/mL of IgY while that ofE. coli was only 7% with the same amount of IgY, which showed the growth inhibition ofH. pylori was mainly due to the specific interaction between IgY andH. pylori. The inhibition ofH. pylori adhesion to AGS was as high as 90% using 0.5 mg/mL of antibody only. More than 80% ofH. pylori attached to AGS could be detached treating with the same amount of IgY for one and a half hr. However, this effect was severely dependant on theH. pylori strains tested. The strain used for immunization of chicken was very sensitive to the antibody treatment but changing the test strain generally showed a variation in adhesion inhibition between 15 and 80%. Further studies are necessary to employ the egg yolk antibodies for the treatment ofH. pylori in vivo.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

Phosphorylcholine Group-immobilized Surface Prepared on Polydimethylsiloxane Membrane by In Situ Reaction for Its Reduced Biofouling

To reduce interactions between biological molecules and the surface of microchip devices including the microchip, which should be conducted to improve sensitivity, reactivity, and the typical phospholipid polar group, the phosphorylcholine group-immobilized surfaces were prepared. The surface modification of polydimethylsiloxane (PDMS) was performed by in situ reaction during curing by cross-linking the PDMS prepolymers. Since it is known that 2-methacryloyloxyethyl phosphorylcholine (MPC) facilitates the preparation of biomedical polymers with excellent biocompatibility and antithrombogenicity, it was used as the reactant for surface modification. The MPC was coated on the glass substrate, and two-liquid-type PDMS prepolymers were then applied. During the curing process of the vinyl groups of poly(dimetylsiloxane-co-methylsiloxane) and poly(dimethylsiloxane-co-methylvinylsiloxane), the methacrylate group in MPC was attached onto the PDMS surface via a hydrosilyl group. Analysis of the surface characteristics by X-ray photoelectron spectroscopy and measurement of the surface contact angle revealed that the introduction of the phosphorylcholine group in the MPC unit on the surface induced hydrophilicity at the surface. Further, protein adsorption on the surface decreased with an increase in the number of phosphorylcholine groups. Based on these results, we concluded that the construction of the phosphorylcholine group-enriched surface on the PDMS substrate could be achieved by immobilization of MPC, and it may facilitate fabrication of biomedical devices, particularly microfluidic devices.     

Evaluation of Biotin-OSu and Biotin-ONp in the Solid Phase Biotinylation of Peptides

Biotinyl-oxysuccinimide and biotin p-nitrophenyl ester were evaluated in the solid phase synthesis of biotinylated peptides. Biotin p-nitrophenyl ester was found to be superior in terms of solubility and reactivity.     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.     

Field evaluation of female sex pheromone components of the cotton bollworm,Heliothis armigera

(Z)-11-hexadecenal and (Z)-9-hexadecenal were ineffective lures forH. armigera males unless combined. Attraction depended upon perception of a 90%–99% combination of (Z)-11-hexadecenal with 1%–10% (Z)-9-hexadecenal. Increasing the level of (Z)-9-hexadecenal in the mixture to 26.2% reduced catches. Adding 2.3% (Z)-7-hexadecenal to the mixture did not enhance or reduce attraction, while adding 8.7% (Z)-11-hexadecenol significantly reduced male catches. The combination of (Z)-11-hexadecenal and (Z)-9-hexadecenal was effective only when released from rubber dispensers but not from polyethylene vials. A load of 2 mg of the mixture on rubber dispensers effectively attracted males for at least 31 days. TheH. zea lure which contained all the pheromonal components of that species was also effective in attractingH. armigera males. TheH. virescens lure attracted significantly fewerH. armigera males than theH. zea lure.

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Résumé Les (Z)-11-hexadecenal et (Z)-9-hexadecenal sont des attractifs sexuels in efficaces pour les mâles deH. armigera. L'attraction dépend de la perception d'un mélange de 90 à 99% de (Z)-11-hexadecenal avec 1 à 10% (Z)-9-hexadecenal, L'augmentation jusqu'à 26,2% de la teneur en (Z)-9-hexadecenal réduit les captures. L'addition de 2,3% de (Z)-7-hexadecenal au mélange ne modifie pas l'attractivité, tandis que celle de 8,7% de (Z)-11-hexadecenol, réduit significativement les captures de mâles. Le mêlange de (Z)-11-hexadecenal et de (Z)-9-hexadecenal n'a été efficace qu'avec des diffuseurs en caoutchouc, par contre il a été sans effet à partir de récipients de polyéthylène. Une charge de 2 mg de mélange dans des diffuseurs en caoutchouc attire effectivement les mâles pendant ou moins 31 jours. L'attractif sexuel deH. zea qui contient tous les constituants de la phéromone de cette espèce attire aussi efficacement les mâles deH. armigera. Celui deH. virescens attire significativement moins de mâles deH. armigera que l'attractif sexuel deH. zea.

     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase     

A kinetic analysis of the electrogenic pump ofChara corallina: IV. Temperature dependence of the pump activity

Summary The sigmoidal current-voltage curve (i _p -V curve) of the electrogenic H⁺-pump of theChara membrane was simulated satisfactorily with a simple reaction kinetic model which assumed consecutive changes in state of H⁺-ATPase. Four rate constants, i.e., forward and backward ones in voltage-dependent and-independent steps could be evaluated from the data. The emf of the pump (E _p ), the voltage at which the pump current changes its sign, varies only slightly with temperature. However, the pump current (i _p ) is highly temperature dependent, and there-fore the conductance (g _p ) of the pump, calculated as the chord conductance from thei _p-V curve, is also highly voltage dependent having a peak at a level somewhat less negative than the resting potential. In contrast tog _p , the conductance (i _p ) of the passive channel does not change appreciably with temperature. Arrhenius plots ofg _p and also of the rate constants showed a clear bend at about 19°C. Great temperature dependence of the kinetic parameters offers useful information on the pumping mechanism of theChara membrane.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase

We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimylistoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an α-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

The definition of a new IA antigenic specificity using the B6.C-H-2 bm12 I-region mutant strain

A new Ia specificity has been defined using theIA-subregion mutant B6.C-H-2 ^bm12. The immunization to produce the antiserum wasbm12 anti-A.BY, as all other immunizations, such asbm12 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of theb, d, p andq H–2 haplotypes were positive, whereasf, k, r ands were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to theIA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW 33 000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost frombm12, is present on the same molecule as other I-A specificities.This work was supported by funds obtained from the N. H. and M. R. C. (Australia) and the National Institutes of Health, Grant No. CA-21224.     

Investigations on the PGM (phosphoglucomutase) polymorphism by isoelectric focusing inDrosophila melanogaster

Pgm allele frequencies of 383 individuals were determined in a sample ofDrosophila melanogaster from three laboratory Sardinian populations, using the techniques of standard electrophoresis, heat denaturation, and isoelectric focusing. The analysis of the progeny obtained from informative crosses showed that the isoelectric focusing patterns segregate in a Mendelian way. ThePgm ^1.00 andPgm ^0.70 electrophoretic alleles displayed different isoelectric points, whereas thePgm ^1.00,tr andPgm ^1.00,ts isoelectrophoretic alleles could not be differentiated when tested by isoelectric focusing. Moreover, thePgm ^0.70,ts allele was split into two classes, with isoelectric points ofpH 6.4 andpH 6.6.     

I/V-Curve studies of the control of a K+ transporter inNitella by temperature

Summary InNitella, current-voltage relationships were measured at different temperatures ranging from 5 to 25°C. Sets of theseI/V curves were subject to curve fitting on the basis of a cyclic reaction scheme (Class I model). Different hypotheses of the mode of action of temperature on theI/V curve were tested, including changes in reaction constants in the transport cycle and deactivation of transport molecules. It was found that models assuming an influence of temperature on pairs of rate constants of the transport cycle gave very bad fits. Good fits were obtained with models implying that temperature influences the number of active transporters. The lazy-state model (the exchange of an inactive state with a stateN ₃ in the transport cycle is influenced by temperature) gave a slightly better fit than the assumption of an unspecific inactivation (independent of the state of the transport molecule). According to the lazy-state analysis, the inactive state is kinetically closer toN _o , the state in which the transport molecule is open to the outside substrate than toN _i , the state in which it is open to the inside substrate. The two inactivation models imply that temperature does not act directly on the properties of the plasmamembrane, but that temperature-sensitive metabolic processes in the cell send signals which control the activation and deactivation of the transporter.     

Effect ofpH on the formation of Compounds II and III of horseradish peroxidase

The formation of Compounds II and III of horseradish peroxidase from Compound I and potassium ferrocyanide and from Compound II and excess hydrogen peroxide, respectively, was studied as a function ofpH at 25°C and a constant ionic strength of 0.11. The yield of Compound II obtained increases progressively with increase inpH; a mixture of Compounds I and II is produced at acidicpH. Pure Compound III is obtained at allpH values, but the highest yield is obtained atpH values between 6.0 and 7.0. The yield of p-670, formed when Compound III is allowed to stand for 60 min, decreases with increase inpH, while the decay of Compound III also decreases with increase inpH. Therefore p-670 is the decay product of Compound III.