Human polysaccharide-specific B cells are responsive to pokeweed mitogen and IL-6

The responsiveness of polysaccharide-specific B cells to PWM was examined in vitro. Spleen cells from six patients immunized with Haemophilus influenzae type b-diphtheria toxoid, pneumococcal and meningococcal vaccines were T cell-depleted and separated by Percoll density gradient centrifugation. In each B cell fraction, spontaneous antibody production was demonstrated to capsular polysaccharides as well as diphtheria toxoid. The peak of spontaneous antibody production was demonstrated to be five to seven days after immunization. When T cells and PWM were added, the total Ig secretion increased in all B cell fractions. PWM also enhanced IgG antibody directed to each of three polysaccharide Ag measured. This enhancement was most noticeable for nonresting B cells. The PWM effect was not confined to IgG, as IgM and IgA to Neisseria meningitidis type C were measured and also enhanced. The kinetics of the PWM response demonstrated the most IgG antibody to polysaccharide Ag from spleens immunized five to seven days before splenectomy. When the patients were immunized either 2 days or 4 mo before splenectomy, no spontaneous IgG antibody to polysaccharides was detected although PWM induced small amounts of antibody. Finally, anti-IL-6 antibody blocked PWM-induced total and polysaccharide-specific antibody production. We conclude that human polysaccharide-specific B cells are responsive to PWM and IL-6. We suggest that polysaccharide B cells are not truly "T cell-independent" and may respond to T cell lymphokines and thus are similar to protein-specific B cells.     

Improved recovery of active recombinant laccase from maize seed

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.     

Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.     

Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of allergic airway disease

Stromal cell-derived factor-1alpha/beta (SDF-1alpha/beta) is phylogenetically a primitive chemokine widely expressed in a variety of tissues and cell types. This expression is detectable in the absence of stimuli provided by bacterial or viral infections and allergic or autoimmune disorders. Based on these and other findings, SDF-1alpha has not been considered an inflammatory chemokine, but, rather, has been believed to be involved in certain homeostatic processes, such as leukocyte recirculation. SDF-1alpha is a potent chemoattractant for lymphocytes and monocytes that mediates its activity via the chemokine receptor CXCR4. Study of the role of SDF-1alpha/CXCR4 in vivo during inflammation has been limited by the fact that transgenic mice that have been made deficient in either molecule die early in life due to developmental defects. The present study was aimed at evaluating the functional relevance of the SDF-1alpha/CXCR4 axis during an inflammatory process. Neutralizing Abs to CXCR4 reduced lung eosinophilia (bronchoalveolar lavage fluid and interstitium) by half, indicating that CXCR4-mediated signals contribute to lung inflammation in a mouse model of allergic airway disease (AAD). This reduction in inflammation was accompanied by a significant decrease in airway hyper-responsiveness. SDF-1alpha neutralization resulted in similar reduction in both lung allergic inflammation and airway hyper-responsiveness. Retroviral delivery of a CXCR4 cDNA to leukocytes resulted in greater inflammation when transduced mice were subjected to a mouse model of AAD. These results highlight that, although considered a noninflammatory axis, the involvement of CXCR4 and SDF-1alpha is critical during AAD, and this receptor and its ligand are potentially relevant in other inflammatory processes.     

Assessing the mating 'health' of commercial honey bee queens

Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices.     

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.     

False-positive human immunodeficiency virus enzyme immunoassay results in pregnant women

Objective

Examine whether false-positive HIV enzyme immunoassay (EIA) test results occur more frequently among pregnant women than among women who are not pregnant and men (others).

Design

To obtain a large number of pregnant women and others tested for HIV, we identified specimens tested at a national laboratory using Genetic Systems HIV-1/HIV-2 Plus O EIA from July 2007 to June 2008.

Methods

Specimens with EIA repeatedly reactive and Western blot-negative or indeterminate results were considered EIA false-positive. We compared the false-positive rate among uninfected pregnant women and others, adjusting for HIV prevalence. Among all reactive EIAs, we evaluated the proportion of false-positives, positive predictive value (PPV), and Western blot bands among indeterminates, by pregnancy status.

Results

HIV prevalence was 0.06% among 921,438 pregnant women and 1.34% among 1,103,961 others. The false-positive rate was lower for pregnant women than others (0.14% vs. 0.21%, odds ratio 0.65 [95% confidence interval 0.61, 0.70]). Pregnant women with reactive EIAs were more likely than others (p<0.01) to have Western blot-negative (52.9% vs. 9.8%) and indeterminate results (17.0% vs. 3.7%) and lower PPV (30% vs. 87%). The p24 band was detected more often among pregnant women (p<0.01).

Conclusions

False-positive HIV EIA results were rare and occurred less frequently among pregnant women than others. Pregnant women with reactive EIAs were more likely to have negative and indeterminate Western blot results due to lower HIV prevalence and higher p24 reactivity, respectively. Indeterminate results may complicate clinical management during pregnancy. Alternative methods are needed to rule out infection in persons with reactive EIAs from low prevalence populations.     

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.     

Discovery of GS-9451: an acid inhibitor of the hepatitis C virus NS3/4A protease

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.