Development of an internally controlled antibody microarray

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.     

Genetic divergence and evidence for sympatric host-races in the highly polyphagous brown tail moth,Euproctis chrysorrhoea (Lepidoptera: Erebidae)

The brown tail moth (BTM) Euproctis chrysorrhoea (Linnaeus 1758) (Lepidoptera: Erebidae) is a forest and ornamental pest in Europe and the United States. Its extreme polyphagy, and documented phenological shift associated with host use suggest the presence of distinct host-races. To test this hypothesis, we sampled BTM infesting different host species in several locations along its distribution, and used DNA sequence data (a total of 1,672 bp from cytochrome c oxidase subunit I, elongation factor 1-alpha, and wingless) to produce haplotype networks and reconstruct the phylogenetic relationships between individuals. Population genetic diversity indices pointed out a higher genetic diversity in Europe, particularly in the samples from southern Spain and southern England. Lower F _ST values were found between geographically closer populations when compared to more distant ones, but analyses of molecular variance and Mantel tests failed to reveal geographically associated genetic differentiation. However, haplotype networks and phylogenetic reconstructions revealed a previously unknown genetic differentiation within the BTM, with one lineage circumscribed to southern Europe. Although BTM haplotypes did not cluster according to their host plant, host-associated haplotypes were observed within certain geographic regions. Hence, our data support the existence of host-races of BTM within southern Spain and southern England, where populations from different hosts occur in sympatry.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils

Background

Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1β production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages.

Methods and Findings

Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients'' neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response.

Conclusions

The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.     

A two thousand year history of a northern Swedish boreal forest stand

Abstract. Successional processes within northern Swedish boreal forest are investigated for the last 2000 years by analysis of pollen, charcoal fragments and insect remains preserved in a deep mor humus layer on a small island in a large lake. Frequent disturbances by fire, blow-down, insect attack and flood have been the chief influence on vegetation dynamics. The gradual climatically induced replacement of Betula pubescens by Picea abies is superimposed on the disturbance dynamics. Picea first became established during the Little Ice Age, which was the longest period without fire in the record. A thin layer of volcanic ash from Iceland confirms the chronology at the onset of the Little Ice Age. The present importance of Pinus sylvestris and Ericaceae on the island is a product of a recent increase in disturbance frequency. The disturbance regime ensures that the communities stay in equilibrium with prevailing climate.     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.     

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus

Thermophilic, faculatatively mixotrophic sulfur-oxidizing bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland. The strain, IT-7254, used thiosulfate and elemental sulfur as electron donors, oxygen and nitrate as electron acceptors, and acetate and other organic compounds as carbon sources. After a few days of growth in the presence of thiosulfate, this strain formed sulfur globules. Comparison of intracellular enzymes and heme proteins of heterotrophically and mixotrophically grown cells showed some differences. The new isolate belonged to Thermus scotoductus because the small subunit (SSU) rRNA gene sequence analysis showed 98.6% sequence similarity and 84% DNA:DNA reassociation to Thermus scotoductus NMX2 A. 1. It is also close to Thermus antranikianii HN3-7, with 98.3% and 79% SSU rRNA sequence similarity and DNA:DNA reassociation, respectively. It was also found that both Thermus NMX2 A.1 and T. antranikianii HN3-7 were able to oxidize thiosulfate but that the T. scotoductus type strain SE-1 was not. This is the first report of Thermus strains that are capable of mixotrophic growth with sulfur oxidation.