Validation of quantitative ELISAS for measuring anti-Yersinia pestis F1 and V antibody concentrations in nonhuman primate sera.

This study systematically validated two quantitative enzyme-linked immunosorbent assays (ELISAs) for determining Yersinia pestis anti-F1 or anti-V IgG concentration in cynomolgus macaque sera. The results demonstrated that these ELISAs are reliable, reproducible, and suitable for their intended use to measure both anti-F1 and anti-V IgG in monkey sera following vaccination with a heterologous recombinant fusion F1-V protein (rF1-V). Statistical analysis demonstrated assay precision, accuracy, specificity, linearity/dilutional linearity, and robustness for both assays. The quantitative ranges of standard curves were defined as 40-700 ng/mLfor both anti-F1 and anti-V IgG. Either serological assay could be used to determine potency of F1/V antigen-based vaccines in surrogate clinical studies or to define correlates of protective immunity against plague under the Food and Drug Administration's (FDA) two-animal rule.

Primary prevascular and retropsoas hernias: incidence of rare abdominal wall hernias.

OBJECTIVE: To describe the incidence and treatment of prevascular and retropsoas hernias in a large-volume general surgery practice. BACKGROUND: Femoral hernias are considered uncommon with an incidence between 2 and 8 % of groin hernias. There are no large studies describing the subtypes of femoral hernias or retropsoas hernias, and therefore no reported incidence or standardized treatment recommendations for these hernias exist. METHODS: This study is a retrospective review of all patients undergoing total extraperitoneal (TEP) laparoscopic herniorrhaphy between August 1993 and December 2011. A single surgeon performed all the repairs. Demographics and patient outcomes were reported. RESULTS: 2,436 patients underwent 3,242 TEP repairs. The subtypes were: indirect 1,523 (46.9 %), direct 1,473 (45.4 %), femoral 156 (4.8 %), obturator 35 (1.1 %), prevascular 25 (0.77 %), Spigelian 20 (0.61 %), retropsoas 3 (0.09 %). Prevascular hernias accounted for 16 % of femoral hernias. Patients with prevascular hernias had a mean age of 70.3 years and were all male. 13 of the 25 patients (52 %) with prevascular hernias had other associated defects and four (16 %) of the patients had prevascular hernias as a recurrence from a prior hernia operation. There were three patients with retropsoas hernias that only would not have been seen from an anterior open approach. There are no intraoperative complications or known recurrences from this study group. CONCLUSIONS: Prevascular and retropsoas hernias are uncommon, but have a higher incidence than previously believed. Prevascular hernias tend to be associated with older age and other defects. The diagnosis and management of these hernias are readily achieved using the laparoscopic TEP approach.

High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.

The synthetic C5a gene was initially found to be expressed poorly in Escherichia coli. We undertook studies to determine the reasons for poor expression and to increase expression. The work was focused on the role of the mRNA structure in C5a expression and stability of its product in E. coli. We present data on the effects of varying the sequence at the 5' end of mRNA as well as different ribosome-binding sites on expression. Evaluation of the stability of C5a showed rapid degradation of C5a in wild-type E. coli (half-life 3-5 min). Screening of several protease-deficient strains of E. coli showed that C5a was much more stable in an htpR strain carrying a mutation in the sigma subunit of RNA polymerase that is specific for heat shock promoters. The mutation is associated with a proteolytic deficiency. The half-life of C5a was increased to 20 min. By manipulating the expression vector, the regulatory region for the C5a gene, the host strain, growth conditions and methods for recovering the protein, C5a levels were increased 300-fold over previously reported amounts to about 3% of total cellular protein.

Purification, characterization and crystallization of ERA, an essential GTPase from Escherichia coli.

ERA is an essential GTPase widely conserved in bacteria. Homologues of ERA are also present in higher eukaryotic cells. ERA is involved in bacterial cell cycle control at a point preceding cell division. In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein. Here, we report the purification and crystallization procedures and results. The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity. ERA is one of the very few multi-domain GTPases crystallized to date.

A technique for placement of a bioabsorbable prosthesis with fibrin glue fixation for reinforcement of the crural closure during hiatal hernia repair.

INTRODUCTION: Level 1 data suggest that mesh reinforcement of the crural closure for hiatal hernia repair decreases the recurrence of hernia. The fear of erosion of the prosthetic into the esophagus has kept the use of mesh for hiatal hernia repair from becoming routine. A recent study found several cases of esophageal stenosis/erosion from the use of a biologic mesh. For these reasons, we evaluated a new resorptive prosthetic and new method of fixation of the prosthetic for crural reinforcement during hiatal hernia repair. METHODS: From February 2009 until December 2010, 70 patients underwent hiatal hernia repair using a synthetic bioabsorbable prosthetic made of polglycolide and teimethylene carbonate (Gore BioA Tissue Reinforcement™, Flagstaff, AZ). There were 48 patients with paraesophageal hiatal hernias and 22 with large sliding hiatal hernias. In this study, a square piece of mesh just the size to cover the crural closure only was utilized. Fibrin glue (Tisseel™) was applied over the suture closure of the crura, the mesh was then placed over the glue and held in place for several seconds, and then more fibrin glue was applied on top of the mesh. RESULTS: The new bioabsorbable polymer mesh was readily placed through a 10-mm trocar, had good handling characteristics laparoscopically, and no pre-operative preparation was required of the prosthetic. The material and the fibrin glue created a very substantial reinforcement of the crural closure, and the average time to place and fix the mesh was approximately 5 min. There were no short-term complications from the mesh, and no patient has had any significant post-operative sequelae. CONCLUSION: Crural closure reinforcement during hiatal hernia repair can be done readily with this new bioabsorbable polymer-based mesh. Fibrin glue fixation of this new prosthetic can be done quickly and it creates a strong, fixed barrier that may decrease the chance of erosion. Further studies will need to be done to evaluate long-term efficacy and complications associated with its use.

Quantitative anti-F1 and anti-V IgG ELISAs as serological correlates of protection against plague in female Swiss Webster mice.

A recombinant fusion protein composed of Yersinia pestis fraction 1 capsule (F1) and virulence-associated V antigen (V) (F1-V) has been developed as the next-generation vaccine against plague. In this study, female Swiss Webster mice received a single intramuscular vaccination with one of eight doses of the F1-V vaccine and exposed 4 weeks later to either Y. pestis CO92 or C12 organisms by the subcutaneous or aerosol routes of infection. Quantitative anti-F1 and anti-V immunoglobulin G (IgG) ELISAs were used to examine the relationship between survival outcome and antibody titers to F1 and V. Results suggested that each 1log(10) increase in week 4 quantitative anti-F1 and anti-V IgG ELISA titers were associated with a 1.7-fold (p=0.0051) and 2.5-fold (p=0.0054) increase in odds of survival, respectively, against either bubonic or pneumonic plague and may serve as serological correlates of protection.

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Control of ftsZ expression, cell division, and glutamine metabolism in Luria-Bertani medium by the alarmone ppGpp in Escherichia coli.

Inactivation of transcription factor sigma54, encoded by rpoN (glnF), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84. Mutational defects in three other genes involved in general nitrogen control (glnD, glnG, and glnL) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress ftsZ84, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.

Specific binding of VirG to the vir box requires a C-terminal domain and exhibits a minimum concentration threshold.

The positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA-binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989). This paper is an extension of that research and presents evidence on the in vivo DNA-binding properties of VirG using a transcription interference assay. VirG protein bound specifically to a 'vir box' response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box. A biphasic response in specific DNA-binding was observed upon increasing virG expression, suggesting that specific binding was co-operatively affected by protein concentration. Certain TrpE'-'VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg-118 of the VirG polypeptide. These data further localize a DNA-binding domain within VirG, and support a modified model for the regulation of virulence genes in which transphosphorylation by the coregulator VirA functions to stabilize specific DNA-binding by low concentrations of VirG, resulting in gene activation. Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987).

virG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNA-binding protein.

Virulence genes of the Agrobacterium tumefaciens Ti plasmid are positively regulated by the products of virA and virG. To study the DNA-binding properties of the VirG protein, a translational fusion between virG and the trpE gene of Escherichia coli was constructed, and antiserum was raised against the encoded fusion protein. Using this antiserum, a protein of Mr congruent to 29,000, a size similar to that calculated from the virG nucleotide sequence, was detected in an E. coli strain harbouring a virG expression vector. Both the virG protein and the fusion protein were found, by filter-binding and gel retardation analyses, to bind DNA nonspecifically. These data support an existing model for the two-component regulatory systems of bacteria.

[Demonstration of 3 functional domains responsible for a kinase activity in VirA, a transmembrane sensory protein encoded by the Ti plasmid of Agrobacterium tumefaciens]. / Mise en évidence de trois domaines fonctionnels responsables d'une activité kinase chez VirA, une protéine sensorielle transmembranaire codée par le plasmide Ti d'Agrobacterium tumefaciens.

High-level expression of chimeric virA genes were obtained by replacing the first codons of virA with the first codons of trpE. The fusion proteins encoded by these constructs were partially purified and one of them exhibited autokinase activity. Therefore, protein phosphorylation may be an important feature of VirA function. This allowed us to define the existence of three functional domains inside VirA.

lac Up-promoter mutants with increased homology to the consensus promoter sequence.

Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.

Characterization of mutations affecting the Escherichia coli essential GTPase era that suppress two temperature-sensitive dnaG alleles.

Two suppressor mutations of the temperature-sensitive DNA primase mutant dnaG2903 have been characterized. The gene responsible for suppression, era, encodes an essential GTPase of Escherichia coli. One mutation, rnc-15, is an insertion of an IS1 element within the leader region of the rnc operon and causes a polar defect on the downstream genes of the operon. A previously described polar mutation, rnc-40, was also able to suppress dnaG2903. The other mutation, era-1, causes a single amino acid substitution (P17R) in the G1 region of the GTP-binding domain of Era. Analysis of the GTPase activity of the Era-1 mutant protein showed a four- to five-fold decrease in the ability to convert GTP to GDP. Thus, lowered expression of wild-type Era caused by the polar mutations and reduced GTPase activity caused by the era-1 mutation suppresses dnaG2903 as well as a second dnaG allele, parB. Phenotypic analysis of the era-1 mutant at 25 degrees C showed that 10% of the cells contain four segregated nucleoids, indicative of a delay in cell division. Possible mechanisms of suppression of dnaG and roles for Era are discussed.

Analysis of the rnc locus of Coxiella burnetii.

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of its ability to suppress mucoidy in Escherichia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by beta-galactosidase expression in lon- cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rnc- E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E. coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35, 27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens.

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.

Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58.

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two-component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure-function model for the gene product are discussed.

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli.

Era is a low-molecular-weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell cycle at the predivisional two-cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature-sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit.

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.

Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant.

Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.