Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.     

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.     

Design and synthesis of 4-methoxyphenylacetic acid esters as 15-lipoxygenase inhibitors and SAR comparative studies of them

A group of 4-methoxyphenylacetic acid esters were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compounds 7d–e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 3.8 and 1.9 μM, respectively). All compounds were docked in SLO active site and showed that carbonyl group of compounds is oriented toward the Fe^III–OH moiety in the active site of enzyme and fixed by hydrogen bonding with hydroxyl group. It is assumed that lipophilic interaction of ligand–enzyme would be in charge of inhibiting the enzyme activity. The selectivity of the synthetic esters in inhibiting of 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.During the 1980s to 1990s, the worldwide increase in human Salmonella enterica serovar Enteritidis infections was associated with the consumption of contaminated eggs and egg products (13, 26, 28). In the United States, grade A shell eggs were identified as a major source contributing to Salmonella infections (19, 26, 29), and the percentage of S. Enteritidis among all Salmonella isolated from outbreaks increased from 5% to 26% from 1976 to 1996 (4). Although outbreak-associated cases due to S. Enteritidis decreased from 974 during 1998 to 2000 to 692 cases in 2004 to 2006, the 28 outbreaks in 2006 still remained above the Healthy People 2010 target of 22 (6). Despite efforts directed at reducing egg-related outbreaks, S. Enteritidis infections are still among those with the highest incidence of the seven most-reported serotypes of Salmonella (5).The large proportion of S. Enteritidis serotypes involved in food-borne outbreaks is partly attributed to the adherence elicited by surface fimbriae. Fimbriae are nonflagellar filamentous surface appendages which consist of helically arranged repeating subunit proteins called fimbrins (24). Four serologically distinct fimbriae of S. Enteritidis have been characterized according to their size (kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels: SEF21, SEF18, SEF17, and SEF14 (9-11, 21). Fimbriae can mediate the aggregation of bacteria and their attachment to inert surfaces (2) and to the surfaces of eukaryotic cells, especially to carbohydrate receptors (8, 11, 36).SEF14 fimbriae are detected in all S. Enteritidis strains and are not widely distributed among the Enterobacteriaceae (10). These fimbriae consist of a repeating major subunit protein of 14.3 kDa (SefA) encoded by the gene sefA (9, 33). The results of studies by Peralta et al. (25) and Thiagarajan et al. (31) indicate that SEF14 fimbriae may have a role in pathogenesis by mediating attachment to eukaryotic cells. We focused on SEF14 fimbriae because of their limited distribution and their role as a main immunological target in the serological response to infection by S. Enteritidis in chickens (12). The objectives of this study were to clone and investigate the functional properties of the SEF14 fimbrin, SefA, as part of a fusion protein in Escherichia coli and to determine whether it could mediate adherence to tissue culture cells in vitro.Bacterial strains, tissue culture cells, and plasmids used in this study are described in Table ​Table1.1. Bacterial cultures were grown in LB medium (Fisher Scientific, Raleigh, NC) supplemented with 0.2% glucose and shaken at 220 revolutions min⁻¹ or in colonization factor broth (30) at 37°C. Chloramphenicol (Cm; 10 μg ml⁻¹) and ampicillin (Amp; 50 μg ml⁻¹) were added as needed (Sigma-Aldrich, St. Louis, MO). Avian ovary granulosa cells were grown in 5% CO₂ atmosphere at 37°C in M199 medium supplemented with 26 mM NaHCO₃, 0.1% bovine serum albumin, 100 U ml⁻¹ penicillin G, and 100 μg ml⁻¹ streptomycin sulfate (Gibco-BRL, Gaithersburg, MD). HEp-2 cells were obtained from ATCC (Rockville, MD) and grown in 5% CO₂ atmosphere at 37°C in minimum essential medium (Sigma-Aldrich), pH 7.2, supplemented with 26 mM NaHCO₃, 7% fetal bovine serum, 100 U ml⁻¹ penicillin G, and 100 μg ml⁻¹ streptomycin sulfate (Gibco-BRL and Sigma-Aldrich).

TABLE 1.

Bacterial strains, tissue culture cells, and plasmids

Theoretical investigations on the hydrolysis pathway of tin verdoheme complexes: elucidation of tin’s ring opening inhibition role

In order to obtain a better molecular understanding of inhibitory role of tin metal in the verdoheme ring opening process, hydrolysis of three possibly six, five, and four coordinate verdoheme complexes of tin(IV) and (II) have been studied using DFT method. The results of calculations indicate that, in excellent accord with experimental reports, hydrolysis of different possibly coordinated tin(IV) and (II) verdohemes does not lead to the opening of the macrocycle. Contrary to iron and zinc verdohemes, in five and four coordinate verdoheme complexes of tin(IV) and (II), formation of open ring helical complexes of tin are unfavorable both thermodynamically and kinetically. In these pathways, coordination of hydroxide nucleophile to tin metal due to the highly charged, exclusive oxophilicity nature of the Sn center, and high affinity of Sn to increase coordination state are proposed responsible as inhibiting roles of tin via the ring opening. While, in saturated six coordinate tin(IV) and (II) verdoheme complexes the ring opening of tin verdohemes is possible thermodynamically, but it is not predicted to occur from a kinetics point of view. In the six coordinate pathway, tin plays no coordination role and direct addition of hydroxide nucleophile to the positive oxo-carbon centers and formation of closed ring hydroxy compounds is proposed for preventing the verdoheme ring opening. These key points and findings have been corroborated by the results obtained from atomic charge analysis, geometrical parameters, and molecular orbital calculations. In addition, the results of inhibiting ring opening reaction of tin verdoheme complexes could support the great interest of tin porphyrin analogues as pharmacologic means of chemoprevention of neonatal jaundice by the competitive inhibitory action of tin porphyrins on heme oxygenase.     

Plant quality effects on intraguild predation between Orius laevigatus and Aphidoletes aphidimyza

To understand the influence of plant quality on intraguild predation and consequently on the suppression of a shared prey population as well as on plant yield, the interactions between Aphis gossypii Glover (Hemiptera: Aphididae) (shared prey), Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae) (intermediate predator), and Orius laevigatus Fieber (Hemiptera: Anthocoridae) (top predator) were investigated in 25‐day experiments on cucumber, Cucumis sativus L. (Cucurbitaceae) at various N fertilization levels (90, 150, and 190 p.p.m.) in microcosm set‐ups under greenhouse conditions. The final aphid population size was significantly affected by an interactive effect of N fertilization and predator application. Regardless of the N fertilization levels, O. laevigatus alone was more effective in aphid suppression than A. aphidimyza alone. In addition, the risk for aphids of being predated upon by both predators together was significantly reduced in the low and medium‐N fertilization levels, whereas it was additive in the high‐N fertilization treatment. The A. aphidimyza population was suppressed by O. laevigatus in both the 90 and 150 p.p.m. N treatments. However, there was no intraguild predation of O. laevigatus on A. aphidimyza at the 190 p.p.m. N level. Total plant yield depended on predator treatments and N fertilization levels, with the highest yield produced at the 150 p.p.m. N fertilization level in treatments with either O. laevigatus alone or with both predators together. Our results demonstrate that the weak asymmetric intraguild predation among A. aphidimyza and O. laevigatus does not influence the ability of both predators together to diminish bottom‐up effects on aphid populations and the yield losses associated with aphid infestations.     

Identification and genetic characterization of metallo-beta-lactamase-producing strains of Pseudomonas aeruginosa in Tehran, Iran

Metallo-beta-lactamases (MBLs) are being reported with increasing frequency worldwide. The aim of this study was to investigate the prevalence of blalMP-1, blaVIM-1,2 and blaSPM-1 genes encoding metallo-beta-lactamases (MBLs) among a collection of Pseudomonas aeruginosa strains isolated from patients at different hospitals in Tehran and to trace the disseminated clones at these hospitals by pulsed field gel electrophoresis (PFGE). Susceptibility of 610 P aeruginosa to 14 different antibiotics was determined using disc diffusion method. Isolates showing resistance to imipenem and ceftazidime were subjected to micro broth dilution assay to determine their MIC values. The blaIMP-1, blaVIM-1, blaVIM-2, and blaSPM-1, genes were amplified by PCR. Isolates containing blaVIM-1 were analyzed by PFGE. Sixty-eight isolates were resistant to imipenem (MIC > or = 4 microg/ml) of which 16 isolates carried blaVIM-1 gene using PCR assay. No other MBL genes were detected in this study. Three different unrelated patterns were found for isolates containing blaVIM-1 gene by PFGE of which pattern A was predominant. All isolates were susceptible to colistin and polymixin B. blaVIM-1 was the main gene encoding MBL among the isolates of P aeruginosa in our study. Clonal spread of isolates containing blaVIM-1 had occurred at Tehran hospitals. However, heterogeneous clones also were involved in the outbreaks.