Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Noninvasive detection of HER2 amplification with plasma DNA digital PCR.

PURPOSE: Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. EXPERIMENTAL DESIGN: The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. RESULTS: In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively. CONCLUSION: Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.

Staurosporine and gossypol are inhibitors of the function of peptide elongation factor 1 alpha from rabbit reticulocytes.

As it has been found, the incubation of [gamma-32P]ATP with elongation factor--1 alpha purified from rabbit reticulocytes resulted in the phosphorylation of several substrate proteins /Tuhácková, Z. (1992) In: Rec. Adv. Cell. Mol. Biol. 4, 79-86, Peeters Press, Leuven/ (1). In the present paper chromatofocusing of the purified eEF-1 alpha demonstrates that the ATP-dependent protein kinase activity is associated with a single protein catalyzing the GTP-dependent binding of aminoacyl-tRNA to ribosomes. Both of these activities are inhibited by staurosporine and gossypol. The inhibition by GDP but not by GTP indicates a possible involvement of conformation changes also in the modulation of the protein kinase activity displayed by eEF-1 alpha.

Purification of various forms of elongation factor 1 from rabbit reticulocytes.

Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (alpha, beta, gamma, and delta). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1 alpha, EF-1 beta delta, EF-1 beta gamma delta) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1 alpha is a single polypeptide of Mr 52,000, and has an isoelectric point of 9.1. EF-1 beta delta and EF-1 beta gamma delta are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1 beta, 5.5 for EF-1 gamma, and 4.8 for EF-1 delta. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1 alpha in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1 beta gamma delta is slightly more active than EF-1 beta delta. The similarity of the amino acid composition of EF-1 gamma and EF-1 delta and the molar ratio of alpha: beta: gamma: delta in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1 delta is probably a breakdown product of EF-1 gamma, and that the native form of EF-1H probably contains only the alpha, beta, and gamma subunits.

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes.

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (Mr greater than 500,000) containing four distinct polypeptides (alpha, beta, gamma, delta). The other form, EF-1 alpha, consists of a single polypeptide which is the same as the alpha subunit of EF-1H. Both EF-1 alpha and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1 alpha is enhanced in polyphenylalanine synthesis by a complementary component, EF-1 beta delta. It is also shown that EF-1 beta delta can facilitate an exchange of EF-1 alpha-bound GDP for GTP. The EF-1 alpha dissociation constants for GDP and GTP were 0.47 and 0.55 microM respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 microM, respectively. Thus, while EF-1 alpha and EF-1H had approximately the same affinities for GDP and GTP, the EF-1 alpha dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1 alpha or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.

[Distribution of elongation factors EF-1 and EF-2 among components of rabbit reticulocyte lysate]. / Raspredelenie faktorov élongatsii EF-1 i EF-2 sredi komponentov lizata retikulotsitov krolika.

The distribution of activity of the elongation factors EF-1 and EF-2 among the components of rabbit reticulocyte lysate separated by sucrose density gradient centrifugation was studied. At low ionic strength (0.01 M KCl) about 30% of the EF-1 activity was found in polyribosomes. At moderate ionic strength (0.1 M KCl) the EF-1 activity was absent in the polyribosomes. An addition of RNA excess to the lysate prior to centrifugation at low ionic strength resulted in elimination of the EF-1 activity from the polyribosomes. This indicates that EF-1 is reversibly bound to the polyribosomes and that EF-1 may be retained on them due to interaction with RNA of polysomes mediated by its RNA-binding site. After dissociation of polyribosomes containing EF-1 in the presence of EDTA and subsequent fractionation of the dissociation products at low ionic strength (0.01 M KCl) the EF-1 activity was revealed in the ribosomal subparticles (predominantly in 60S). At 0.1 M KCl EF-1 mainly sedimented in the zone of distribution of polyribosomal informosomes. The elongation factor EF-2 was not revealed in polyribosomes during lysate centrifugation even at low ionic strength which corresponds to its lower affinity for RNA.