Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila

BACKGROUND: The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies. RESULTS: Here we establish a causal link between nutrient availability and insulin-dependent growth. We show that in addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies. CONCLUSIONS: These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms.     

A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.     

Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.

Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.     

A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans

Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues.     

Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori

BACKGROUND: Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (Amx) H. pylori isolates, and is mediated by mutations in penicillin-binding protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional Amx H. pylori isolates has been established. METHODS: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven Amx H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained Amx transformants were determined. RESULTS: Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the Amx H. pylori isolates resulted in an increased MIC (0.5-1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402-404) and third (KTG555-557) conserved penicillin-binding protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. CONCLUSION: In naturally occurring Amx H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests.     

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.     

Altitudinal variation of secondary metabolite profiles in flowering heads of Arnica montana cv. ARBO

The altitudinal variation on the contents of secondary metabolites in flowering heads of Arnica montana was assessed. Plants of A. montana cultivar ARBO were grown in nine experimental plots at altitudes between 590 and 2230m at Mount Patscherkofel near Innsbruck/Austria. The total contents of sesquiterpene lactones and flavonoids were not positively correlated with the altitude of the growing site. However, the proportion of flavonoids with vicinal free hydroxy groups in ring B to flavonoids lacking this feature significantly increased with elevation. Additionally, the level of caffeic acid derivatives also positively correlated with the altitude of the growing site. In particular amounts of 1-methoxyoxaloyl-3,5-dicaffeoylquinic acid significantly increased in higher sites and samples from the summit region contained 85% more of this compound than samples from valley sites. These results are discussed with regards to chemosystematic studies comparing samples collected in different altitudes as well as in the light of a UV-B protective and radical scavenging function of phenolics and their significance for plant life in environments with elevated UV-B radiation.     

New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.     

Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs

Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.