Gene expression signatures from three genetically separable resistance gene signaling pathways for downy mildew resistance

Resistance gene-dependent disease resistance to pathogenic microorganisms is mediated by genetically separable regulatory pathways. Using the GeneChip Arabidopsis genome array, we compared the expression profiles of approximately 8,000 Arabidopsis genes following activation of three RPP genes directed against the pathogenic oomycete Peronospora parasitica. Judicious choice of P. parasitica isolates and loss of resistance plant mutants allowed us to compare the responses controlled by three genetically distinct resistance gene-mediated signaling pathways. We found that all three pathways can converge, leading to up-regulation of common sets of target genes. At least two temporal patterns of gene activation are triggered by two of the pathways examined. Many genes defined by their early and transient increases in expression encode proteins that execute defense biochemistry, while genes exhibiting a sustained or delayed expression increase predominantly encode putative signaling proteins. Previously defined and novel sequence motifs were found to be enriched in the promoters of genes coregulated by the local defense-signaling network. These putative promoter elements may operate downstream from signal convergence points.     

LXR-dependent gene expression is important for macrophage survival and the innate immune response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.     

Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers

Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.     

Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.)

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5-ACGTGGC-3 and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.     

4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors

Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24.     

New fatty acid oxidation inhibitors with increased potency lacking adverse metabolic and electrophysiological properties

New inhibitors of palmitoylCoA oxidation were synthesized based on a structurally novel lead, CVT-3501 (1). Investigation of structure-activity relationships was conducted with respect to potency of inhibition of cardiac mitochondrial palmitoylCoA oxidation and metabolic stability. Potent and metabolically stable analogues 33, 42, and 43 were evaluated in vitro for cytochrome P450 inhibition and potentially adverse electrophysiological effects. Compound 33 was also found to have favorable pharmacokinetic properties in rat.     

Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus

Annual outbreaks of influenza A infection are an ongoing public health threat and novel influenza strains can periodically emerge to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed at least 40 million people worldwide and pandemics in 1957 and 1968 caused hundreds of thousands of deaths. The influenza A virus is capable of enormous genetic variation, both by continuous, gradual mutation and by reassortment of genome segments between viruses. Both the 1957 and 1968 pandemic strains are thought to have originated as reassortants in which one or both human-adapted viral surface proteins were replaced by proteins from avian influenza strains. Analyses of the genes of the 1918 pandemic virus, however, indicate that this strain might have had a different origin. The haemagglutinin and nucleoprotein genome segments in particular are unlikely to have come directly from an avian source that is similar to those that are currently being sequenced. Determining whether a pandemic influenza virus can emerge by different mechanisms will affect the scope and focus of surveillance and prevention efforts.     

Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations

Pluronic F-68 (PF-68) is routinely used as a shear-protection additive in mammalian cell cultures. However, most previous studies of its shear protection mechanisms have typically been qualitative in nature and have not covered a wide range of PF-68 and cell concentrations. In this study, interactions between air bubbles along with the associated cell damage were investigated using the novel adenovirus-producing cell line PER.C6, a human embryonic retinoblast transfected with the adenovirus type 5 E1 gene. A wide range of PF-68 and cell concentrations (approximately 3 orders of magnitude) were used in these studies. At low PF-68 concentrations (0.001 g/L), cells had a very high affinity for bubbles, indicated by a more than 10-fold increase in cell concentration in the foam layer liquid versus the bulk liquid. At high PF-68 concentrations ( approximately 3 g/L), however, the cell concentration in the foam layer liquid was only approximately 40% of that in the bulk cell suspension. The number of cells associated with each bubble decreased from approximately 1000 cells at 0.001 g/L PF-68 to approximately 120 cells at 3 g/L PF-68. Despite the lower cell affinity for bubbles at a high PF-68 concentration, at high cell concentrations (10(7) cells/mL and 1 g/L PF-68) significant cell entrapment occurred in the foam layer, on the order of 1000 cells/bubble. For the cells carried by the bubbles, quantitative cell damage data revealed that the probability of cell death from bubble rupture was independent of bulk cell concentration but was affected by PF-68 concentration. These quantitative studies further indicated that even at a low PF-68 concentration of 0.03 g/L, approximately 30% of the attached cells were killed during the bubble rupture process. At the same time, at low PF-68 concentration (<0.1 g/L), significant cell death occurred prior to bubble rupture. On average, a bubble disrupted more cells in the bulk liquid and/or foam layer than during rupture. For both mechanisms, the number of cells damaged by each bubble increased with decreasing PF-68 concentration and increasing bulk cell concentration.     

Evolution of pigment cell regression in the cavefish Astyanax: a late step in melanogenesis

Pigmentation and eyes are often lost in cave-adapted animals. Although the mechanisms of eye degeneration are beginning to be understood, little is known about the evolutionary and developmental processes involved in pigment cell regression. In teleost embryos, a population of neural crest cells migrates into the body wall and differentiates into melanophores, xanthophores, and iridophores. All three pigment cell types are present in the eyed surface-dwelling form (surface fish) of the teleost Astyanax mexicanus. However, melanophores are absent or substantially reduced in number in various derived populations of the conspecific blind cave-dwelling form (cavefish). We show here that tyrosinase-positive melanoblasts are present in cavefish. DiI labeling revealed a population of trunk neural crest cells in cavefish embryos that migrate to locations normally occupied by differentiated melanophores. We also discovered a cell population in cavefish embryos and adults resembling melanoblasts in several features, including the ability to synthesize melanin when supplied with the tyrosinase substrate l-dopa. DiI-tyrosinase double-labeling and neural keel explant experiments showed that the tyrosinase-positive cells are derived from the neural crest. The number of melanoblasts varies in different adult cavefish populations relative to the extent of melanophore reduction. Although cavefish melanoblasts can synthesize melanin from exogenous l-dopa, they are unable to convert exogenous l-tyrosine to l-dopa and melanin. We conclude that pigment cell regression in cavefish is mediated by an evolutionary change late in melanogenesis that may involve an impediment in the ability to convert l-tyrosine to l-dopa and melanin.