The ability to entrain to long photoperiods differs between 3 Drosophila melanogaster wild-type strains and is modified by twilight simulation

The ability to adapt to different environmental conditions including seasonal changes is a key feature of the circadian clock. Here, we compared the ability of 3 Drosophila melanogaster wild-type strains to adapt rhythmic activity to long photoperiods simulated in the laboratory. Fruit flies are predominantly crepuscular with activity bouts in the morning (M) and evening (E). The M peak follows dawn and the E peak follows dusk when the photoperiod is extended. We show that this ability is restricted to a certain extension of the phase angle between M and E peaks, such that the E peak does not delay beyond a certain phase under long days. We demonstrate that this ability is significantly improved by simulated twilight and that it depends additionally on the genetic background and the ambient temperature. At 20 °C, the laboratory strain CantonS had the most flexible phase angle between M and E peaks, a Northern wild-type strain had an intermediate one, and a Southern wild-type strain had the lowest flexibility. Furthermore, we found that the 3 strains differed in clock light sensitivity, with the CantonS and the Northern strains more light sensitive than the Southern strain. These results are generally in accord with the recently discovered polymorphisms in the timeless gene (tim) that affect clock light sensitivity.     

Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.     

The Macrostomum lignano EST database as a molecular resource for studying platyhelminth development and phylogeny

We report the development of an Expressed Sequence Tag (EST) resource for the flatworm Macrostomum lignano. This taxon is of interest due to its basal placement within the flatworms. As such, it provides a useful comparative model for understanding the development of neural and sensory organization. It was anticipated on the basis of previous studies [e.g., Sánchez-Alvarado et al., Development, 129:5659–5665, (2002)] that a wide range of developmental markers would be expressed in later-stage macrostomids, and this proved to be the case, permitting recovery of a range of gene sequences important in development. To this end, an adult Macrostomum cDNA library was generated and 7,680 Macrostomum ESTs were sequenced from the 5′ end. In addition, 1,536 of these aforementioned sequences were sequenced from the 3′ end. Of the roughly 5,416 non-redundant sequences identified, 68% are similar to previously reported genes of known function. In addition, nearly 100 specific clones were obtained with potential neural and sensory function. From these data, an annotated searchable database of the Macrostomum EST collection has been made available on the web. A major objective was to obtain genes that would allow reconstruction of embryogenesis, and in particular neurogenesis, in a basal platyhelminth. The sequences recovered will serve as probes with which the origin and morphogenesis of lineages and tissues can be followed. To this end, we demonstrate a protocol for combined immunohistochemistry and in situ hybridization labeling in juvenile Macrostomum, employing homologs of lin11/lim1 and six3/optix. Expression of these genes is shown in the context of the neuropile/muscle system.     

Is Bagging Effective in the Classification of Small-Sample Genomic and Proteomic Data?

There has been considerable interest recently in the application of bagging in the classification of both gene-expression data and protein-abundance mass spectrometry data. The approach is often justified by the improvement it produces on the performance of unstable, overfitting classification rules under small-sample situations. However, the question of real practical interest is whether the ensemble scheme will improve performance of those classifiers sufficiently to beat the performance of single stable, nonoverfitting classifiers, in the case of small-sample genomic and proteomic data sets. To investigate that question, we conducted a detailed empirical study, using publicly-available data sets from published genomic and proteomic studies. We observed that, under t-test and RELIEF filter-based feature selection, bagging generally does a good job of improving the performance of unstable, overfitting classifiers, such as CART decision trees and neural networks, but that improvement was not sufficient to beat the performance of single stable, nonoverfitting classifiers, such as diagonal and plain linear discriminant analysis, or 3-nearest neighbors. Furthermore, as expected, the ensemble method did not improve the performance of these classifiers significantly. Representative experimental results are presented and discussed in this work.     

T Cell-Dependence of Lassa Fever Pathogenesis

Lassa virus (LASV), the causative agent of Lassa fever (LF), is endemic in West Africa, accounting for substantial morbidity and mortality. In spite of ongoing research efforts, LF pathogenesis and mechanisms of LASV immune control remain poorly understood. While normal laboratory mice are resistant to LASV, we report that mice expressing humanized instead of murine MHC class I (MHC-I) failed to control LASV infection and develop severe LF. Infection of MHC-I knockout mice confirmed a key role for MHC-I-restricted T cell responses in controlling LASV. Intriguingly we found that T cell depletion in LASV-infected HHD mice prevented disease, irrespective of high-level viremia. Widespread activation of monocyte/macrophage lineage cells, manifest through inducible NO synthase expression, and elevated IL-12p40 serum levels indicated a systemic inflammatory condition. The absence of extensive monocyte/macrophage activation in T cell-depleted mice suggested that T cell responses contribute to deleterious innate inflammatory reactions and LF pathogenesis. Our observations in mice indicate a dual role for T cells, not only protecting from LASV, but also enhancing LF pathogenesis. The possibility of T cell-driven enhancement and immunopathogenesis should be given consideration in future LF vaccine development.     

Synthesis of functional poly(1,4-ketone)s bearing bioactive moieties by Pd-catalyzed insertion polymerization

The synthesis of novel alternating polyketones bearing pendent bioactive moieties is presented. These materials were prepared by palladium catalyzed coordination polymerization of carbon monoxide and alpha-olefins substituted with protected tyrosine or with dipeptide sequences such as tyrosine-glycine, tyrosine-alanine, and tyrosine-valine. Copolymerization experiments of CO with monomers containing vitamin E or testosterone were also successfully performed under mild reaction conditions. The dicationic Pd (II) bis(phoshine) complex [Pd(dppp)(NCCH(3))(2)](BF(4))(2) was used as catalyst precursor giving rise to macromolecules with well-defined structures.     

The Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation

The Saccharomyces cerevisiae mutant cwh8 was previously found to have an anomalous cell wall. Here we show that the cwh8 mutant has an N -glycosylation defect. We found that cwh8 cells were resistant to vanadate and sensitive to hygromycin B, and produced glycoforms of invertase and carboxypeptidase Y with a reduced number of N -chains. We have cloned the CWH8 gene. We found that it was nonessential and encoded a putative transmembrane protein of 239 amino acids. Comparison of the in vitro oligosaccharyl transferase activities of membrane preparations from wild type or cwh8 Delta cells revealed no differences in enzyme kinetic properties indicating that the oligosaccharyl transferase complex of mutant cells was not affected. cwh8 Delta cells also produced normal dolichols and dolichol-linked oligosaccharide intermediates including the full-length form Glc3Man9GlcNAc2. The level of dolichol-linked oligosaccharides in cwh8 Delta cells was, however, reduced to about 20% of the wild type. We propose that inefficient N -glycosylation of secretory proteins in cwh8 Delta cells is caused by an insufficient supply of dolichol-linked oligosaccharide substrate.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.     

The significance of muscle cells for the origin of mesoderm in bilateria

Muscle tissue may have played a central role in the early evolutionof mesoderm. The first function of myocytes could have beento control swimming and gliding motion in ciliated vermiformorganisms, as it still is in such present-day basal Bilateriaas the Nemertodermatida. The only mesodermal cells between epidermisand gastrodermis in Nemertodermatida are myocytes, and conceivablythe myocyte was, in fact, the original mesodermal cell type.In Nemertodermatida as well as the Acoela, myocytes are subepithelialfiber-type muscle cells and appear to originate from the gastrodermalepithelium by emigration of single cells. Other mesodermal cellsin the acoels are the peripheral parenchyma (connective tissue)and tunica cells of the gonads, and these also arise from thegastrodermis. Musculature in many of the coelomate protostomesand deuterostomes, on the other hand, is in the form of epitheliomuscular(myoepithelial) cells, and this cell type may also have beenan early form of the mesodermal myocyte. The mesodermal bandsin the small annelid Polygordius and in juvenile enteropneustshave cells intermediate between mesenchymal and epithelial intheir histological organization as they develop into myoepithelia.If acoelomates were derived from coelomates by progenesis, thenthe fiber-type muscles of acoelomates could be products of foreshorteneddifferentiation of such tissue. The precise serial patterningof circular muscle cells along the anterior-posterior axis duringembryonic development in the acoel Convoluta pulchra providesa model for early steps in the gradual evolution of segmentationfrom iterated organ systems.