Wing vein formation in Drosophila melanogaster: hairless is involved in the cross-talk between Notch and EGF signaling pathways

Wing vein development in Drosophila is controlled by different morphogenetic pathways, including Notch. Hairless (H) antagonizes Notch target gene activation by binding to the Notch signal transducer Suppressor of Hairless [Su(H)]. Accordingly, overexpression of H phenocopies reduction of Notch activity. Deletion of the Su(H)-binding domain in H-C2 results in loss of H activity. However, overexpression of H-C2 induces formation of ectopic veins. In a screen for genetic modifiers of this phenotype, we have identified several genes involved in Notch and epidermal growth factor (EGF) signaling. Most notably veinlet, an activator of EGF signaling, acts downstream of H-C2. H-C2 positively regulates veinlet maybe through inhibition of inter-vein determinants in agreement with a model, whereby Notch and EGF signaling pathways cross-regulate vein pre-patterning.     

Enhancement of the protective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa

The outer membrane protein F gene (oprF) of Pseudomonas aeruginosa was recently shown by us to protect mice from P. aeruginosa chronic pulmonary infection when used as a DNA vaccine administered by three biolistic (gene gun) intradermal inoculations given at 2-week intervals. In the present study, we used two different strategies to improve the protective efficacy of the DNA vaccine. In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus. Both the synthetic peptide conjugate and the chimeric virus contained peptide 10, a previously identified immunoprotective epitope of protein F. The second strategy involved the addition of a second outer membrane protein to the vaccine. DNA encoding a fusion protein comprised of the C-terminal half of protein F fused to OprI was administered by three biolistic intradermal inoculations. Challenge with P. aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide-KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone. Thus, both strategies appear to augment the protection afforded by an oprF-only DNA vaccine.     

Tracing Myelin Protein Zero (P0) <Emphasis Type="Italic">in vivo</Emphasis> by construction of P0-GFP fusion proteins

Background  

Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein.     

Low-mass proteome analysis based on liquid chromatography fractionation,nanoliter protein concentration/digestion,and microspot matrix-assisted laser desorption ionization mass spectrometry

HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult. For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred. However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry. In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC. In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters. About 700 pl of the pre-concentrated fraction is then drawn into a 20-microm I.D. capillary and dried in a small region near the capillary's entrance. This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary. After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits. The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis. The performance of this technique is demonstrated with the use of a standard protein solution. This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E. coli extract.     

Excited state conformational dynamics of semiflexibly bridged electron donor-acceptor systems: a semiempirical CI-study including solvent effects

Semiempirical (AM1) molecular orbital theory with configuration interaction has been used to investigate the harpooning mechanism proposed from experimental studies on semiflexibly bridged electron donor-acceptor systems. Our calculations on the charge-transfer state of N-phenyl-4-(4-cyano-naphth-1-ylmethyl)-piperidine, 1, confirm the proposed harpooning mechanism including an intermediate loosely folded charge-transfer state and reproduce the thermodynamics obtained from our spectroscopic studies closely. The structural details of the extended (ECT), intermediate (ICT) and compact (CCT) charge-transfer states are discussed, as are the transition states connecting them. Solvent effects have been modeled using self-consistent reaction field (SCRF) calculations within the polarized continuum model. The effect of solvent polarity on the stabilities of the three charge-transfer states is discussed.     

Developmental plasticity in the cardiovascular system of fish,with special reference to the zebrafish

During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as is well established for adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages control of blood flow appears to be possible by blood-borne and/or local hormones. This study presents methods based on video-imaging techniques and fluorescence microscopy to visualize cardiac activity, as well as the vascular bed of developing lower vertebrates, and tests the idea that environmental factors, such as hypoxia, may modify cardiac activity, or even the early formation of blood vessels in embryos and larvae. In zebrafish larvae, adaptations of cardiovascular activity to chronic hypoxia become visible shortly after hatching, and the formation of some blood vessels is enhanced under chronic hypoxia. Exposure of early larval stages of zebrafish to a constant water current induces physiological adaptations, resulting in enhanced swimming efficiency and increased tolerance towards hypoxia. Furthermore, application of hormones such as NO can modify cardiac activity as well as peripheral resistance, and they can stimulate blood vessel formation. In consequence, even during early development of fish or amphibian larvae, the performance of cardiac muscle and of skeletal muscle can be modified by environmental influences and peripheral resistance can be adjusted. Even blood vessel formation can be stimulated by hypoxia, for example, or by the presence of specific hormones. Thus, at approximately the time of hatching the physiological performance of vertebrate larvae is already determined by the combined action of environmental influences and of genetic information.     

Oxidative nerve cell death in Alzheimer's disease and stroke: antioxidants as neuroprotective compounds

Many neurodegenerative disorders and syndromes are associated with an excessive generation of reactive oxygen species (ROS) and oxidative stress. The pathways to nerve cell death induced by diverse potential neurotoxins such as peptides, excitatory amino acids, cytokines or synthetic drugs commonly share oxidative downstream processes, which can cause either an acute oxidative destruction or activate secondary events leading to apoptosis. The pathophysiological role of ROS has been intensively studied in in vitro and in vivo models of chronic neurodegenerative diseases such as Alzheimer's disease (AD) and of syndromes associated with rapid nerve cell loss as occuring in stroke. In AD, oxidative neuronal cell dysfunction and cell death caused by protofibrils and aggregates of the AD-associated amyloid beta protein (Abeta) may causally contribute to pathogenesis and progression. ROS and reactive nitrogen species also take part in the complex cascade of events and the detrimental effects occuring during ischemia and reperfusion in stroke. Direct antioxidants such as chain-breaking free radical scavengers can prevent oxidative nerve cell death. Although there is ample experimental evidence demonstrating neuroprotective activities of direct antioxidants in vitro, the clinical evidence for antioxidant compounds to act as protective drugs is relatively scarce. Here, the neuroprotective potential of antioxidant phenolic structures including alpha-tocopherol (vitamin E) and 17beta-estradiol (estrogen) in vitro is summarized. In addition, the antioxidant and cytoprotective activities of lipophilic tyrosine- and tryptophan-containing structures are discussed. Finally, an outlook is given on the neuroprotective potential of aromatic amines and imines, which may comprise novel lead structures for antioxidant drug design.     

Linked unresponsiveness: early cytokine gene expression profiles in cardiac allografts following pretreatment of recipients with bone marrow cells expressing donor MHC alloantigen

Linked unresponsiveness operates to induce specific unresponsiveness to fully mismatched vascularized allografts in recipients pretreated with anti-CD4 antibody and syngeneic bone marrow cells expressing a single donor MHC class I alloantigen. The aim of the study was to evaluate early post transplant cytokine expression in allografts where linked unresponsiveness was required for long term graft survival. CBA (H2(k)) mice were pretreated with CBK (H2(k)+K(b)) bone marrow cells under the cover of anti-CD4 antibody 28 days before transplantation of a CBK or a C57BL/10 (H2(b)) cardiac allograft. In both cases graft survival was prolonged (MST=100 days). Intragraft expression for interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12(p40), IL-18, iNOS, transforming growth factor (TGF)-beta(1) and C-beta was determined on day 1.5, 3, 7 and 14 after transplantation. Whereas rejecting allografts displayed a sharp peak in IFN-gamma, IL-2, IL-4 and IL-10 expression, non-rejecting allografts were characterized by an initial TGF-beta(1) and IFN-gamma production. An increasing IL-4 expression towards day 14 was a unique feature of linked unresponsiveness. All non-rejecting allografts were characterized by an increasing IL-12(p40) production towards day 14. In summary, the early cytokine expression pattern in allografts after bone marrow induced operational tolerance is influenced by the quantity of donor alloantigens expressed on the graft as well as on the bone marrow inoculum.