Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Specific determination of tyrosine-phosphorylated proteins and peptides by differential iodination.

A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.

Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach.

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser10, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser10. Introduction of the phosphate was performed with Et2NP(OtBu)2 and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products.

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Synthesis, purification and biological activity of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin).

Based on the global phosphorylation approach, a selective synthesis of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin), which contains 32 amino acid residues and a disulfide loop is described. The peptide was assembled stepwise on a polyethyleneglycol-polystyrene support using Fmoc-chemistry. The phosphorylation was performed on-resin by phosphitylation with a large excess of di-tert-butyl-N,N-diethylphosphoramidite within 1 hour, followed by oxidation with tert-butylhydroperoxide to the protected phosphopeptide. After cleavage and deprotection the disulfide bridge was introduced without side reactions by iodine titration of the mono-acetamidomethyl protected crude peptide. During the synthetic pathway, the acylation with side chain-unprotected Fmoc-serine and the phosphitylation satisfactorily yielded the expected intermediates. In some phosphorylation experiments a by-product having a reduced mass corresponding to the H-phosphonate was observed. Illustrated with the synthesis of phosphourodilatin, this type of by-product, which could not be separated by HPLC, and the difficult amino acid sequence make the synthesis of a large phosphopeptide a more delicate task than the synthesis of short phosphopeptides, which do not contain oxidation-sensitive amino acids, difficult sequences or additional structural elements such as disulfide loops. The biological activity of phosphourodilatin was compared with non-phosphorylated urodilatin in two assay systems. Both peptides revealed a vasorelaxant effect on aortic smooth muscle strips and induced a cGMP-generation in RFL-6 cells with increasing dose dependency.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.