Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.     

Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium

Summary We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in anin vitro preparation of bullfrog RPE-choroid. Changes in RPE [K⁺] _i alter the current produced by this pump. Increasing [K⁺] _o in the solution perfusing thebasal membrane increases RPE [K⁺] _i (measured with a K⁺-specific microelectrode), and also depolarizes theapical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0mm [K⁺] _o outside the apical membrane. Removal of Cl^– from the solution perfusing the basal membrane abolishes the K⁺-evoked apical depolarization by preventing the entry of K⁺ (as KCl) into the cell. We conclude that the increase in [K⁺] _i causes the decrease in pump current. This result is consistent with the finding that [K⁺] _i is a competitive inhibitor of the Na⁺–K⁺ pump in red blood cells.It is possible that the light-evoked changes in [K⁺] _o in the distal retina could alter RPE [K⁺] _i , and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.     

Interrelation of mating,flight, and fecundity in navel orangeworm females

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae, Phycitini), is an economically important pest of nut crops in California, USA. Improved management will require better understanding of insect dispersal, particularly relative to when mating occurs. A previous study demonstrated a more robust laboratory flight capacity compared to other orchard moth pests, but it was unclear how mating affects dispersal, and how dispersal affects fecundity. In this study, 1‐ and 2‐day‐old females were allowed to fly overnight on a flight mill either before or after mating, respectively, and were then allowed to oviposit. Data on fecundity were compared between treatments to minimally handled or tethered‐only control females. Females that mated before flight flew longer and covered a greater distance than those flying prior to mating. However, timing of flight relative to mating did not affect fecundity, nor did any measure of flight performance. There was no effect on fecundity when females were forced to fly for designated durations from 3 min to 2 h. Together, our data revealed no obvious trade‐off between flight activity and reproductive output. Distances measured on the flight mills (mean ca. 15 km for mated females) may overestimate net displacement in the field where flight tracks are often meandering. The results suggest that most females mate and oviposit in or near their natal habitat, but that some may disperse potentially long distances to oviposit elsewhere.     

Pink eggs and snails: field oviposition patterns of an invasive snail, Pomacea insularum, indicate a preference for an invasive macrophyte

Oviposition of non-calcareous or thinly shelled eggs represents an important life stage of many insects, amphibians, and several gastropods. A recently identified invasive species of apple snail, Pomacea insularum, exhibits alarming invasive characteristics of high reproductive rates and generalist consumption patterns. This snail takes the opposite approach to egg laying compared to most aquatic insects as adult snails crawl out of the water to place clutches on emergent, or terrestrial, substrates. As fecundity best indicates invasive potential for mollusks, control or management efforts need to understand reproductive behavior in P. insularum to predict, and hopefully impede, its spread throughout the Gulf Coast of the United States. Specific characteristics of wetlands and shallow lakes may facilitate the invasion process of P. insularum by providing females with conditions that permit successful oviposition. In order to investigate this possibility, we studied P. insularum oviposition behavior in an invasive population at two times during the reproductive season in Texas, USA. Based on a subsequent survey (August 2009), plants comprised 78% of the available habitat. Wild taro (Colocasia esculenta) and alligator weed (Alternanthera philoxeroides) represented 48 and 43% of that proportion, respectively. During 2008–2009, no new concrete or metal structures appeared in our sampling reach and consistent amounts of plant stands and woody debris remained dominant. Given this distribution, P. insularum laid disproportionately more clutches on wild taro compared to its availability and less on alligator weed and bulrush (Schoenoplectus californicus) than expected. Owing to limited metal and concrete substrates, we found a higher proportion of clutches on these artificial substrates than expected in both May and August 2008. However, artificial substrates comprised less than 2% of available substrates in the bayou. Our results suggest that wetlands and shallow lakes surrounded by large emergent macrophytes, particularly wild taro, likely provide ideal oviposition sites for P. insularum, promote egg supply, and possibly facilitate invasion into new aquatic ecosystems.     

ATM-dependent CHK2 activation induced by anticancer agent, irofulven

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser(1981), which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.