Molecular and histological characterizations of the Om(2D) mutants in Drosophila ananassae.

A series of transposon-induced optic morphology (Om) mutants found in a hypermutable marker stock of Drosophila ananassae provides a useful system for analyzing the molecular mechanism of eye morphogenesis. In the present study, one of the 25 Om loci so far reported, Om(2D), has been subjected to histological and molecular analyses as a first step toward understanding the role of Om genes in eye morphogenesis. Histological abnormalities observed during eye morphogenesis of the mutant, i.e. cell death within the eye-antennal discs of third instar larvae, and loss of the lamina, disorganized ommatidia and atrophied optic lobes in adults, were all comparable to those reported with various eye morphology mutants of D. melanogaster. Approximately 25 kb of genomic DNA including the Om (2D) locus was cloned by tom tagging. Southern blot and cloning analyses of two alleles of the Om (2D) locus revealed that insertions of the tom element occurred at three sites within 359 bp; two tandemly arrayed toms sharing one long terminal repeat at the junction and an internally deleted tom were present 359 bp apart from each other in Om (2D) 63, while a single tom in reverse orientation was present within the 359 bp in Om (2D) 10a. Host DNA sequences at the three insertion sites were TATAT or AATAT, and ATAT was duplicated upon the tom insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.     

A Novel Pollen-Pistil Interaction Conferring High-Temperature Tolerance during Reproduction via CLE45 Signaling

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Post-translational modifications in secreted peptide hormones in plants

More than a dozen secreted peptides are now recognized as important hormones that coordinate and specify cellular functions in plants. Recent evidence has shown that secreted peptide hormones often undergo post-translational modification and proteolytic processing, which are critical for their function. Such 'small post-translationally modified peptide hormones' constitute one of the largest groups of peptide hormones in plants. This short review highlights recent progress in research on post-translationally modified peptide hormones, with particular emphasis on their structural characteristics and modification mechanisms.     

Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Genetic analysis of the Om(2D) locus in Drosophila ananassae.

The Om(2D)63 mutants were mutagenized by gamma-ray irradiation and DEB feeding. A total of nine revertants were recovered and characterized; eight revertants were homozygous-lethal expressing no appreciable abnormality in cuticular pattern and central nervous system, and all failed to complement the lethality with each other. Two of the eight expressed embryonic lethality and were associated with cytologically detectable deletions including the putative Om(2D) locus, while four were associated with rearrangements in a region distal to the insertion sites of the tom elements. No rearrangement was detected in the remaining two by Southern blot analysis. One of the nine revertants was homozygous-viable with wild-type eyes and was associated with a reciprocal translocation with the break points at 48B in 2R (Om(2D) locus) and 96A in 3R. Based on these data, it is concluded that interaction between the region comprised of a single complementation group of the recessive lethal and the inserted tom elements seems to be responsible for the Om(2D) mutant phenotype. In addition, two induced dominant enhancers specific to Om(2D)63 were identified; both mapped on chromosome 2.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis

Peptidomics is a challenging field in which to create a link between genomic information and biological function through biochemical analysis of expressed peptides, including precise identification of post-translational modifications and proteolytic processing. We found that secreted peptides in Arabidopsis plants diffuse into the medium of whole-plant submerged cultures, and can be effectively identified by o- chlorophenol extraction followed by LC-MS analysis. Using this system, we first confirmed that a 12-amino-acid mature CLE44 peptide accumulated at a considerable level in the culture medium of transgenic plants overexpressing CLE44 . Next, using an in silico approach, we identified a novel gene family encoding small secreted peptides that exhibit significant sequence similarity within the C-terminal short conserved domain. We determined that the mature peptide encoded by At1g47485 , a member of this gene family, is a 15-amino-acid peptide containing two hydroxyproline residues derived from the conserved domain. This peptide, which we have named CEP1, is mainly expressed in the lateral root primordia and, when overexpressed or externally applied, significantly arrests root growth. CEP1 is a candidate for a novel peptide plant hormone.