Nga Y. Nguyen

Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.

The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with β-tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified β-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)-3-demethylthiocolchicine ([14C]3CTC). This β-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and β-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Å of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Å distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal β-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.

Synthetic peptides as an antigenic base in an ELISA for laboratory diagnosis of schistosomiasis mansoni

A pool of five synthetic peptides was used as an antigenic base in an ELISA (ELISA-Pp) for laboratory diagnosis of Schistosoma mansoni. Serum samples were obtained from individuals with acute (n=23) and chronic (n=30) schistosomiasis, with other parasitoses (n=39) or without parasitic infections (n=100). ELISA-Pp was compared with other immunoenzymatic methods for detection of IgM (IgM-ELISA) or IgG (IgG-ELISA) as well as an immunofluorescence test for detection of IgM antibodies (IgM-IFT). The sensitivity and specificity of ELISA-Pp was 86.8% and 94.2% when tested on the schistosomiasis group and the non-schistosomiasis group, respectively. Comparison of ELISA-Pp with other serological methods resulted in kappa concordance indices varying from 0.59 to 0.75. Evaluation of anti-peptide IgG antibodies showed higher levels in patients with acute compared with chronic schistosomiasis (P=0.001). ELISA-Pp showed satisfactory sensitivity and high specificity and may constitute a potentially useful method for laboratory diagnosis of schistosomiasis mansoni.

DNA-binding activities in Streptococcus gordonii: identification of a receptor-nickase and a histonelike protein.

Abstract. Extraction of Streptococcus gordonii cells with the mild chaotropic agent, LiCl, drastically decreased DNA transforming ability, had little effect on viability, and released both DNA nicking and binding activities. Both activities were Mg2+ and Ca2+ independent and were not competence specific. Southwestern blot analysis of the extract identified putative surface proteins of 56 kDa and 68 kDa in strain Challis and Wicky, respectively. Extracts also contained a 10-kDa DNA-binding protein, designated HSgo, that belongs to the eubacterial histonelike class of proteins.

The Translational Hop Junction and the 5′ Transcriptional Start Site for the Prevotella loescheii Adhesin Encoded by plaA

Abstract. The Prevotella loescheii adhesin gene, plaA, contains a coding gap between a small open reading frame (ORF-1) and a large open reading frame (ORF-2). Translation of the plaA mRNA requires bypassing this 29-nt coding gap on the plaA transcript. We have determined the N-terminal peptide sequence of the SO34 adhesin beyond the gap sequence. This sequence shows that the peptide junction between ORF-1 and ORF-2 is continuous in the adhesin and supports the conclusion that synthesis of the SO34 adhesin occurs by a ribosomal hop mechanism. To elucidate upstream signals, we used the 5′ RACE (rapid amplification of cDNA ends) technique to map the start point of the plaA mRNA. DNA sequencing of plasmids with the 5′ RACE products placed the 5′ end of plaA mRNA 270 nt upstream from the plaA start codon. A region corresponding to a Bacteroides fragilis promoter consensus sequence precedes this start site.

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne.

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10(2) colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.

Role of the N-Terminal Amino Acid of Bacillus anthracis Lethal Factor in Lethal Toxin Cytotoxicity and Its Effect on the Lethal Toxin Neutralization Assay

ABSTRACT The cytotoxic activity of lethal factor (LF), a critical reagent used in the cell-based lethal toxin neutralization assay to assess anthrax vaccines, was shown to depend on the identity of its N-terminal amino acid, which plays a role in the targeting of LF to the proteasome for degradation. These results demonstrate that care must be taken to ensure that LF preparations used in standardized cell-based assays are not altered at their N-terminal ends.

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site.

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the β-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.

Application of synthetic peptides in development of a serologic method for laboratory diagnosis of schistosomiasis mansoni

The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003) on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019). These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni.

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.

Amino acid composition of proteins extracted from endemic goiter glands

Proteins were isolated and characterized in thyroid tissue of six patients from Brazil with endemic goiter. Biochemical studies of these thyroidal proteins included gel filtration, electrophoresis, and amino acid analysis. In addition to thyroglobulin, two of the most abundant proteins found in all goiters studied had molecular weights of 68 and 14 kDa. One protein was identified as albumin based on its immunoreactivity with antibodies to serum albumin. The other protein was identified as a hemoglobin subunit using reversephase high-performance liquid chromatography (HPLC). Identification was confirmed by partial N-terminal amino acid sequencing that showed several nonconservative differences in thyroid albumin when compared to human serum albumin (HSA). Biochemical findings were correlated with iodine and hormone contents in serum and thyroglobulin from these patients. Based on these findings, we suggest that hemoglobin and albumin are taken up from the blood by the hyperplastic thyroid tissue. Albumin would be processed by the thyroid follicular cell, becoming iodinated and released into the circulation.