A mis-expression study of factors affecting Drosophila PNS cell identity

Drosophila PNS sense organs arise from single sensory organ precursor (SOP) cells through a series of asymmetric divisions. In a mis-expression screen for factors affecting PNS development, we identified string and dappled as being important for the proper formation of adult external sensory (ES) organs. string is a G2 regulator. dappled has no described function but is implicated in tumorigenesis. The mis-expression effect from string was analysed using timed over expression during adult ES-organ and, for comparison, embryonic Chordotonal (Ch) organ formation. Surprisingly, string mis-expression prior to SOP division gave the greatest effect in both systems. In adult ES-organs, this lead to cell fate transformations producing structural cells, whilst in the embryo organs were lost, hence differences within the lineages exist. Mis-expression of dappled, lead to loss and duplications of entire organs in both systems, potentially affecting SOP specification, in addition to affecting neuronal guidance.     

Parenteral nutrition with lipid or glucose suppresses liver growth and response to GH in adolescent male rats

Our aim was to investigate the effects of modifying the carbohydrate-to-lipid ratio of parenteral nutrition (PN) on body composition and the anabolic actions of insulin-like growth factor I (IGF-I) and growth hormone (GH). Adolescent male Sprague-Dawley rats were randomized to receive 7 days of GH, IGF-I (3.5 mg. kg(-1). day(-1) for both) or placebo while receiving high-carbohydrate PN (CHO-PN), high-lipid PN (L-PN), or an oral diet (chow) (the PN protocols were isonitrogenous and isocaloric). PN impaired muscle growth, which was reversed by GH in the CHO-PN group only (P < 0.03). PN increased carcass lipid (P < 0.02), the effect being greater in the L-PN than in the CHO-PN group (P < 0.001). Visceral lean tissue growth was significantly impaired by PN (P < 0.001). IGF-I reversed this impairment, but GH had no effect. PN impaired the normal increase in hepatic protein and DNA (P < 0.001) and produced liver steatosis (P < 0.001). However, this steatosis was less in L-PN than in CHO-PN (P < 0.001). Serum IGF-I and the acid-labile subunit (ALS) were decreased by PN (P < 0.001) and were not affected by GH during PN treatment. However, GH significantly increased serum ALS concentrations in the chow-fed rats (P = 0.032). In conclusion, modifying the CHO-to-L ratio of PN had no significant effect on IGF-I action, but CHO-PN increased the peripheral effect of GH. L-PN increased carcass lipid significantly and decreased hepatic steatosis. Nevertheless, PN caused significant liver steatosis and profound impairment of hepatic cell growth, which was associated with relative hepatic GH resistance.     

Population structure of North Atlantic and North Pacific sei whales (<Emphasis Type="Italic">Balaenoptera borealis</Emphasis>) inferred from mitochondrial control region DNA sequences and microsatellite genotypes

Currently, three stocks of sei whales (Balaenoptera borealis) are defined in the North Atlantic; the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks, which are mainly based upon historical catch and sighting data. We analyzed mitochondrial control region DNA (mtDNA) sequences and genotypes from 7 to 11 microsatellite loci in 87 samples from three sites in the North Atlantic; Iceland, the Gulf of Maine and the Azores, and compared against the North Pacific using 489 previously published samples. No statistically significant deviations from homogeneity were detected among the North Atlantic samples at mtDNA or microsatellite loci. The genealogy estimated from the mtDNA sequences revealed a clear division of the haplotypes into a North Atlantic and a North Pacific clade, with the exception of one haplotype detected in a single sample from the Azores, which was included in the North Pacific clade. Significant genetic divergence between the North Atlantic and North Pacific Oceans was detected (mtDNA Φ_ST?=?0.72, microsatellite Weir and Cockerham’s ? = 0.20; p?<?0.001). The coalescent-based estimate of the population divergence time between the North Atlantic and North Pacific populations from the sequence variation among the mtDNA sequences was at 163,000 years ago. However, the inference was limited by an absence of samples from the Southern Hemisphere and uncertainty regarding mutation rates and generation times. The estimates of inter-oceanic migration rates were low (Nm at 0.007 into the North Pacific and at 0.248 in the opposite direction). Although estimates of genetic divergence among the current North Atlantic stocks were low and consistent with the extensive range of movement observed in satellite tagged sei whales, the high uncertainty of the genetic divergence estimates precludes rejection of multiple stocks in the North Atlantic.     

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2⁺ donors, but no responses were detectable in 9 HLA-A2^– healthy donors or 4 HLA-A2^– melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.     

Motion repulsion arises from stimulus statistics when analyzed with a clustering algorithm

Motion repulsion is the perceived enlargement of the angle between the directions of motion of two transparently moving patterns. An explanation of this illusion has long been sought for in the neural circuitry of the brain. We show that motion repulsion already arises from the statistical properties of the motion transparency problem when analyzed with a clustering algorithm.     

Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling

A novel antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expression of genes altered upon exposure to the Curvularia haloperoxidase system was analyzed by using DNA microarrays. Only a limited number of genes were involved in the response to the Curvularia haloperoxidase system. Among the induced genes were the ibpA and ibpB genes encoding small heat shock proteins, a gene cluster of six genes (b0301-b0306) of unknown function, and finally, cpxP, a member of the Cpx pathway. Knockout mutants were constructed with deletions in b0301-b0306, cpxP, and cpxARP, respectively. Only the mutant lacking cpxARP was significantly more sensitive to the enzyme system than was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of E. coli--namely, the Cpx pathway, which is important for managing the stress response from the Curvularia haloperoxidase system.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.