Bacillus Anthracis Endospores Regulate Ornithine Decarboxylase and Inducible Nitric Oxide Synthase Through ERK1/2 and p38 Mitogen-Activated Protein Kinases

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (^?NO) from inducible nitric oxide synthase (NOS2) by competing for l-arginine, producing l-ornithine at the expense of ^?NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of l-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores’ germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.     

Ecological distributions of <Emphasis Type="Italic">Chaoborus</Emphasis> species in small,shallow lakes from the Canadian Boreal Shield ecozone

Small, shallow lakes and ponds are often the dominant landscape features in many regions, but are comparably less studied than larger lakes. Shallow lakes are more likely to lack fish populations; however, it is often difficult to ascertain whether these sites were naturally fishless or lost their fish populations due to anthropogenic or natural stressors. We examined the distributional abundances of four Chaoborus species by identifying and enumerating their larval mandibles in the surface sediments of 146 randomly selected lakes from northwestern Ontario, Canada, to determine the key environmental gradients influencing chaoborid distributions. Chaoborus mandibles were encountered at 110 lakes and, in 65% of those lakes, total counts were ≥10. Direct gradient analyses were then used to show that lakewater total aluminum concentrations (negatively correlated with pH), lakewater sodium concentrations, lake surface area, and maximum water depth were significant predictors of the distributional abundances of Chaoborus. Generalized linear models indicated that Chaoborus species varied in their responses to significant environmental factors. C. (Sayomyia) was not significantly associated with any environmental variable and the abundances of larger chaoborids may be an important biotic factor affecting this taxon. Chaoborus americanus, an indicator of fishless lakes, was significantly correlated with all five key variables and demonstrated a clear threshold of occurrence in relatively small lakes (i.e., <10 ha in surface area). Furthermore, based on the occurrence and abundance of C. americanus, we estimated that 20% of the lakes we surveyed are currently fishless. These lakes significantly differ in several geomorphic and water-chemistry measures compared to the other study lakes.     

A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

Deciphering species complexes: Puccinia andropogonis and Puccinia coronata, examples of differing modes of speciation

Species of macrocyclic, heteroecious grass rusts often have been defined with wide host ranges and variation in spore morphology. Consequently, some are species complexes and contain genetically distinct forms. Molecular analyses, together with morphological and biological methods, provide powerful means to dissect these complexes. Puccinia coronata is a complex species that has a broad telial host range including more than 45 genera of grasses and a narrow aecial host range. Phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from 15 aecial and telial collections grouped P. coronata into six distinct clades supporting separation of this complex into four distinct species. Puccinia andropogonis, a common rust of tall prairie grasses in North America, is also a complex species. However, in contrast to P. coronata, P. andropogonis has a narrow telial host range and a broad aecial host range. DNA sequence analysis grouped 15 collections of P. andropogonis into six distinct clades representing at least four distinct species. Speciation of P. coronata appears to have occurred primarily by radiation onto new telial hosts, whereas in P. andropogonis speciation appears to have occurred primarily by radiation onto new aecial hosts.     

Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody

CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family.     

Inbreeding levels of two Ustilago maydis populations

Little is known about the population mating behavior of the smut fungus Ustilago maydis DC (Corda). To determine the amount of inbreeding that occurs in local U. maydis populations, two cornfields were sampled, one in North America (NA) at Le Sueur, Minnesota, and one in South America (SA) at Tarariras, Uruguay. These fields were chosen because of their geographic isolation and host management differences. Inbreeding coefficients (F(is)) were calculated using data derived from amplified fragment length polymorphism (AFLP) markers. Mean F(is) values estimated for both the NA (-0.08), and the SA (-0.02) populations statistically are not different from zero. The results of this study demonstrate that the U. maydis population structure in both cornfields results predominately from out-crossing and suggests that teliospores infrequently act as single infection units. The genetic differentiation between populations was high (F(st) = 0.25).     

Exogenous Carbohydrate Utilisation by Explants of Brassica napus Cultured in vitro

In 5; non-coding regions of genes for phytochrome A from horseradish (ArPHYAs) were fused with the 35S promoter of cauliflower mosaic virus in the antisense direction (CaMV35SantiArPHYAs) and introduced into horseradish hairy roots. Phytochrome levels of proximal areas in many hairy roots that had been transformed with CaMV35SantiArPHYAs decreased to levels of about one-half to one-quarter those of control hairy roots. The extent of the light-induced formation of adventitious shoots from hairy roots with less than half of the control level of phytochrome was lower than in the controls and not different between the three ArPHYAs. In contrast, the efficiency of phytochrome on the extent of shoot formation differed in hairy roots transformed with CaMV35SantiArPHYAs when phytochrome levels were more than 0.02 (;(;A) g^–1). The efficiency of ArPHYA3 to initiate shoot formation was greatest and that of ArPHYA2 was smallest. Furthermore, previous reports on hairy roots overexpressing ArPHYAs showed a similar efficiency of phytochrome on shoot formation. These results indicate that the ArphyA1 and/or ArphyA3 play major roles in the light-induced formation of adventitious shoots and that ArphyA2 has a minor role.     

Characterization of a series of far-red-absorbing thiobarbituric acid oxonol derivatives as fluorescent probes for biological applications

Chromophores that absorb in the far-red region of the spectrum are increasingly being utilized for applications in the biosciences. We have synthesized and evaluated a novel series of fluorescent oxonols based on thiobarbituric acids containing aryl and heteroaryl substituents. The novel chromophores possess narrow absorption spectra ( approximately 40-nm bandwidths), reasonable Stokes shifts ( approximately 25 nm), and quantum yields of up to 0.67 in organic solvents and 0.3 in aqueous solvents, with absorption wavelength maxima at 620-640 nm. The spectral properties of the compounds are sensitive to base and exhibit a loss of far-red absorbance that is concentration and time dependent. Derivatives have been synthesized that can be used for the labeling of macromolecules such as proteins and DNA. The probes show environment sensitivity and the oligonucleotide conjugates sense the formation of duplex DNA. These novel far-red fluorophores have potential applications in diagnostic and research applications.