Pyrazinoindolone inhibitors of MAPKAP-K2.

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

New serotypes of Pseudomonas cepacia.

O and H serotyping of Pseudomonas cepacia has provided a suitable procedure for epidemiological studies. Our previous reports have described 7 O and 5 H antigens. The study of strains from another geographical origin led us to prepare antisera against those which could not be serotyped and thus to determine 2 new O and 2 new H specificities (O:8 and O:9, H:4 and H:8).

Phenylmercuric acetate biodegradation by environmental strains of Pseudomonas species.

Organomercurial pollution occurring in the Rhine river in 1986 led us to study the possibility of depollution by mercury-resistant environmental aquatic strains. Four species of Pseudomonas were investigated for their ability to biotransform phenylmercuric acetate (PMA). Such biological depollution was demonstrated to be due to an enzymatic activity in whole cells and in cell-free extracts from Pseudomonas fluorescens and other Pseudomonas species. PMA biotransformation was followed by high-performance liquid chromatography. Some of those bacteria growing between 4 and 41 degrees C probably represent a natural means of organomercurial depollution, which acts slowly in interaction with other organisms and non-organic porous surfaces.

Chemical modifications of chicken liver pyruvate carboxylase: evidence for essential cysteine-lysine pairs and a reactive sulfhydryl group.

Inactivations of chicken liver pyruvate carboxylase with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) and o-phthalaldehyde (o-PA) have identified cysteine and lysine residues that are essential for catalytic activity. Protection experiments suggest that the modified residues are located in or near the first and second subsites. At a one- to two-fold molar excess over active site concentration, DACM inactivated approximately 80-90% of the pyruvate carboxylase and ADP/Pi linked oxaloacetate decarboxylase activities by forming a sulfhydryl-DACM adduct with a fluorescence excitation maximum at 385 nm and an emission maximum at 476 nm. o-PA reacted with the enzyme by cross-linking lysine and cysteine residues to form an inactive isoindole-enzyme derivative with a fluorescence excitation maximum at 337 nm and an emission maximum at 415 nm. Incorporation of one equivalent of either DACM or isoindole derivative resulted in an 80-90% decrease in all activities involving chemistry at the first subsite, suggesting that the modification of a sulfhydryl group or a cysteine-lysine ion pair in or near the first subsite inactivates the enzyme. A cysteine-lysine ion pair in the first subsite could function to remove the N-1 proton of biotin to yield enol-biotin, which could be readily carboxylated by the carboxyphosphate intermediate. In the reverse direction, a cysteine-lysine ion pair in or near the second subsite has been proposed to enolize biotin prior to carboxylation by oxaloacetate (P. V. Attwood and W. W. Cleland, 1986, Biochemistry 25, 8197-8205). Enzyme modified with 2 equivalents of isoindole retained only 7% of the oxamate-induced, ADP/Pi-independent oxaloacetate decarboxylase activity, suggesting that there is at least one essential cysteine-lysine ion pair at or near the second subsite.

VO2+(IV) complexes with pyruvate carboxylase: activation of oxaloacetate decarboxylation and EPR properties of enzyme-VO2+ complexes.

Chicken liver pyruvate carboxylase catalyzes a nonclassical ping-pong mechanism in which the carboxylation of biotin at subsite 1 of the active site is coupled to the biotin-dependent carboxylation of pyruvate at subsite 2. The functions of two divalent cation cofactors and at least one monovalent cation cofactor in catalysis are not well understood. The oxyvanadyl cation, VO2+ does not support phosphoryl transfer at the first subsite, and uncouples the decarboxylation of oxaloacetate at subsite 2 from the formation of ATP at subsite 1. Stimulation of this oxaloacetate decarboxylase activity in the presence of substrates and cofactors of the first subsite, including VO2+, VOADP-, Pi, and acetyl CoA, suggests that these cofactors and substrates induce the movement of carboxybiotin from the second subsite to the first subsite, where it is decarboxylated. VO2+ EPR has provided evidence for enzymic and nucleotide divalent cation binding sites within the first subsite. The EPR properties of enzyme bound VO2+ were altered by bicarbonate, suggesting that this substrate ligands directly to VO2+ at the enzymic metal site. Fluorescence quenching experiments suggest that a monovalent cation may interact with bicarbonate at the first subsite as well. The results of this study provide evidence that (i) the extrinsic metal ion cofactors interact with the substrates at the first subsite, and that (ii) divalent cations play a role in coupling catalysis at the two nonoverlapping subsites by inducing the decarboxylation of carboxybiotin at the first subsite.

DNA polymerase beta: structure-fidelity relationship from Pre-steady-state kinetic analyses of all possible correct and incorrect base pairs for wild type and R283A mutant.

The kinetic parameters (kpol, Kd app) for all possible correct and incorrect pairing between the A, T, G, and C bases were determined for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. The base substitution fidelities of these two proteins were then determined for all 12 possible mispairs representing the first complete fidelity analysis of polymerases using pre-steady-state kinetics. The results led to several significant findings: (i) For both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides) and to a small extent by Kd app (increases for the incorporation of incorrect nucleotides). (ii) In general, the fidelity for the Y.X (incorporation of dXTP opposite template dYMP) mismatch is different from that for the X.Y mismatch, reflecting the asymmetry of the active site. (iii) The fidelity of R283A is reduced in all 12 mispairs compared to that of WT. The extent of decrease varies from 200-fold for the A.G mispair to 2.5-fold for the T.C mispair. In general, the differences in fidelity between the mutant and WT are greater for purine.purine mismatches (up to 200-fold) than purine.pyrimidine, pyrimidine. purine, or pyrimidine.pyrimidine mismatches (up to 19-fold). (iv) Overall, the decreases in the fidelity of the R283A mutant are caused mainly by changes in the values of kpol; the kpol values of correct incorporations decrease to a greater extent for the R283A mutant with respect to WT than those of incorrect incorporations. With the exception of G.C, the values of Kd app for the WT and R283A mutant remain constant for correct pairings and vary by less than a factor of 4 for incorrect pairings. (v) For WT pol beta, the Kd app of G.C (8.6 microM) is distinctly smaller than that of other correct base pairs (41-108 microM). For the R283A mutant, the kpol of G.C is higher by a factor of 15-17.

Transfer of anti-HLA-DR antibodies from the mother to the child. Are DR antigens expressed on the placenta?

Materno-foetal transfer of anti-HLA-DR antibodies was studied in 7 women. In 6 cases, the antibody was induced by the current pregnancy and in one case, it was a residual antibody from a previous pregnancy. When the maternal antibody was induced by the current pregnancy, no anti-DR antibody was detected in the child. When the antibody was residual, it was weakly present in the child. A comparison was made between the materno-foetal transfer of anti-HLA-A,B,C antibodies and that of anti-DR antibodies. Anti-HLA-DR antibody placental absorption suggested that the corresponding antigens were expressed on certain placenta cells.

Fetal immunization against the mother's lymphocytes. A report of two cases.

In 2 cases the child's serum at birth contained a lymphocytotoxic antibody whose specificity did not correspond to the maternal serum's specificity. Further investigation suggested that the newborns were immunized against their mother as the antibodies were directed against maternal antigens.

DNA polymerase beta: multiple conformational changes in the mechanism of catalysis.

Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase beta (Pol beta), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate-limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3' end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by the binding of the catalytic Mg2+ ion. The same biphasic kinetics with different rates were also observed with the thio analog dTTPalphaS and incorrect nucleotides dATP, dGTP, and dCTP. The structural nature for the two conformational changes has been discussed in relation to the available structural information of this enzyme. The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.

DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

Molecular characterization of CD40 signaling intermediates.

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.

DNA polymerase beta: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics.

DNA polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol beta. Since the chemical step may not be rate-limiting for wild-type pol beta, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol beta displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.

Clostridium difficile toxin B: characterization and sequence of three peptides.

The cytotoxin, also named toxin B, was isolated from a toxigenic strain of Clostridium difficile, purified to homogeneity and partially characterized. The purification procedure included ultrafiltration followed by anion-exchange chromatography. We noticed that a non-specific nucleic material eluted with the protein during the purification. The presence of these nucleic acids appeared to be important for the toxic activity of the protein. Some characteristics of the cytotoxin were examined, especially the amino acid composition and the sequence of three tryptic fragments.