Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Sequence Analysis of Active Mariner Elements in Natural Populations of Drosophila Simulans

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.     

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.     

Differential expression of kunitz and bowman-birk soybean proteinase inhibitors in plant and callus tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.     

A new powdery mildew resistance allele at the Pm4 wheat locus transferred from einkorn (Triticum monococcum)

Powdery mildew is one of the most destructive foliar diseases of wheat. A set of differential Blumeria graminis f.sp. tritici (Bgt) isolates was used to test the powdery mildew response of a Triticum monococcum-derived resistant hexaploid line, Tm27d2. Segregation analysis of 95 F_2:3 lines from a Chinese Spring/Tm27d2 cross revealed that the resistance of Tm27d2 is controlled by a single dominant gene. Using monosomic analysis and a molecular mapping approach, the resistance gene was localized to the terminal end of chromosome 2AL. The linkage map of chromosome 2AL consisted of nine simple sequence repeat markers and one sequence-tagged site (STS) marker (ResPm4) indicative for the Pm4 locus. According to the differential reactions of 19 wheat cultivars/lines with known powdery mildew resistance genes to 13 Bgt isolates, Tm27d2 carried a new resistance specificity. The complete association of the resistance allele with STS marker ResPm4 indicated that it represented a new allele at the Pm4 locus. This new allele was designated Pm4d. The two flanking markers Xgwm526 and Xbarc122 closely linked to Pm4d at genetic distances of 3.4 and 1.0 cM, respectively, are present in chromosome bin 2AL1-0.85-1.00.     

Genetic Dissection of Segregation Distortion II. Mechanism of Suppression of Distortion by Certain Inversions

In(2L+2R)Cy and In(2LR)Pm² are inversion-bearing chromosomes, the former carrying a paracentric inversion in each arm and the latter carrying a long pericentric. Both chromosomes produce normal segregation ratios when present in heterozygous males with certain segregation distorter chromosomes. The apparent suppression of distortion by these chromosomes was long attributed to a failure of synapsis, but this hypothesis has fallen out of favor recently because a large number of chromosome aberrations, particularly translocations and inversions, suppress distortion even though their breakpoints fall into no recognizable pattern. Although failure of synapsis does not appear to be the mechanism of suppression of distortion, what is responsible for the suppression remains unknown. In this paper it is shown that In(2L+2R)Cy and In(2LR)Pm² suppress segregation distortion because they carry Rsp, a component of the segregation distorter system that renders a chromosome insensitive to distortion. Both chromosomes induce "suicide" of chromosomes carrying Sd Rsp⁺.     

Evaluation of a vermicompost and leachates on <i>Solidago x hybrida</i> and organic carbon mineralization under aerobic incubations

In the floriculture region of Tenancingo in the State of Mexico, the application of stabilized organic matter, such as vermicompost and leachates, contributes to improve the quality of the soil and plant nutrition. However, it is important to know the chemical composition of a vermicompost and the mineralization process. This is because the amount and speed of nutrient release which will be available to the crop will depend on that knowledge. The objective of the study was to evaluate the effect of the application of a vermicompost and leachates on various quantitative variables of Solidago x hybrid, and the mineralization of organic carbon under aerobic incubations. Vermicompost (74 and 36 g/kg soil), leachates (5 and 10 L/kg soil), 0.33 g/kg soil of chemical fertilizer Ca (NO₃)₂, and not treated soil (control) were applied under greenhouse conditions to evaluate their effects on plant growth variables. Mixtures of 100 g of soil with vermicompost and leachates were made in the laboratory which were incubated during 9 weeks to obtain the potentially mineralizable organic carbon (Corg PM) and the rate of mineralization (k) after adjusting an exponential model. In the greenhouse experiment there were no statistical differences after applying vermicompost and leachates on the quantitative variables (number of stems per plant, diameter of the panicle, fresh weight, plant length and stem diameter) with respect to the control (p>0.05). The effect among the applied doses was evident only for variables such as fresh weight, panicle length and stem diameter with respect to the control. In incubated soils, k values ranged between 0.209-0.325 C mg/kg soil/week. Only with the application of leachates in high doses two pools of organic matter were shown: one soluble labile (102.9 mg/kg soil) and the other hydrolysable (819 mg/kg soil). The soluble, labile fraction favored nutrient availability immediately after its application to the soil. However, a single pool of hydrolysable organic C (987-1074 mg/kg soil) was found when vermicompost was applied. It was associated with a release of organic matter during crop development, and to a possible stimulation of microbial activity. High values of electrical conductivity in vermicompost and leachates (8.2-11.7 mS/m) suggest a moderate application of both products.     

Genetic dissection of hybrid incompatibilities between Drosophila simulans and D. mauritiana. I. Differential accumulation of hybrid male sterility effects on the X and autosomes

The genetic basis of hybrid incompatibility in crosses between Drosophila mauritiana and D. simulans was investigated to gain insight into the evolutionary mechanisms of speciation. In this study, segments of the D. mauritiana third chromosome were introgressed into a D. simulans genetic background and tested as homozygotes for viability, male fertility, and female fertility. The entire third chromosome was covered with partially overlapping segments. Many segments were male sterile, while none were female sterile or lethal, confirming previous reports of the rapid evolution of hybrid male sterility (HMS). A statistical model was developed to quantify the HMS accumulation. In comparison with previous work on the X chromosome, we estimate that the X has approximately 2.5 times the density of HMS factors as the autosomes. We also estimate that the whole genome contains approximately 15 HMS "equivalents"-i.e., 15 times the minimum number of incompatibility factors necessary to cause complete sterility. Although some caveats for the quantitative estimate of a 2.5-fold density difference are described, this study supports the notion that the X chromosome plays a special role in the evolution of reproductive isolation. Possible mechanisms of a "large X" effect include selective fixation of new mutations that are recessive or partially recessive and the evolution of sex-ratio distortion systems.     

Characterization of a receptor for heat shock protein 70 on macrophages and monocytes

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8+ T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range (Kd < 100 nM). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.