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Toward a functional analysis of salivary proteins from the African malaria vector Anopheles gambiae: the characterization of gSG6.
No full textIl lavoro della dottoranda riportato in questa tesi è in parte incluso in una review sulla rivista Parassitologia (Lombardo F et al., 2006) ed è alla base di due pubblicazioni rispettivamente su PLoS ONE (Poinsignon et al, 2008) ed Insect Biochem Mol Biol (2009)
Display of Plasmodium adhesive domains on the surface of lambda phage.
No full textWith the aim to better understand the molecular interactions underlying mosquito salivary gland invasion by plasmodium sporozoites, we are displaying on the surface of lambda phages putative adhesive domains from few selected plasmodium proteins which may be involved in the invasion of mosquito salivary glands (domains from TRAP, CS, MAEBL, CRMPs). The lambda system is particularly suitable for our purpose since it makes possible to display a variety of relatively large protein domains (up to 400 aminoacids) as fusions to the C-terminus of the abundant viral capsid protein gpD. Thus, a phage library displaying a collection of plasmodium adhesive domains could be employed for the development of affinity selection experiments targeted to the identification of salivary gland receptors. As a first step toward this direction, adhesive domains from P. falciparum and P. berghei surface proteins putatively involved in recognition/invasion of mosquito salivary glands have been fused to the C-terminus of the lambda phage capsid protein gpD. In order to create the mini-library each domain was amplified by PCR, cloned in a plasmid vector, sequenced and transferred into the lambda vector λD4 (kindly provided by Dr. S. Nasi, University La Sapienza, Rome). After packaging, the resulting libraries were screened for the isolation of recombinant lambda clones. Pilot western blot analysis indicates that, as expected, all the lambda clones identified and isolated to date correctly express the fusion proteins. Screening and verification of correct display of all the selected domains is in progress. The collection of lambda phages will be used for the development of binding assays and used for the affinity selection of salivary gland ligands
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Toward the understanding of function and complexity of the Anopheles gambiae salivary glands
No full textThe mosquito salivary glands are an interesting target of molecular entomology studies both for their involvement in the transmission of pathogens and for the pharmacological activities carried by the salivary secretions (anti-hemostatics, immunomodulators, etc.). Using a cloning strategy based on the selective trapping of cDNAs encoding signal peptide-containing proteins we identified, in the last few years, twenty-two novel genes whose expression is either tissue-specific or highly enriched in the Anopheles gambiae salivary glands. Among these are the platelet inhibitor apyrase (AgApy), a group of D7-related genes (D7r) and several polypeptides whose functions still need to be clarified. Thereafter we mainly focused our attention on (i) the identification of regulatory sequences capable to drive tissue-specific expression in the salivary glands and on (ii) the production of recombinant proteins to be employed for functional studies. Salivary gland promoter analysis. Promoters of the An. gambiae female salivary gland-specific genes AgApy, D7r2 and D7r4 were analyzed in the fruitfly D. melanogaster and in the asian malaria mosquito An. stephensi using the E. coli LacZ as reporter gene. The AgApy promoter could drive transcription of LacZ in adult transgenic An. stephensi. However, β-galactosidase activity could be detected only in one line carrying multiple copies of the transgene and the expression pattern only in part overlapped the expression profile of AgApy in An. gambiae. We also analyzed the ~1 kb intergenic regions located immediately upstream of the D7r2 and D7r4 genes first in D. melanogaster, where they worked very nicely, and then in An. stephensi. D7r4-driven LacZ expression could be detected by RT-PCR in adult males and females of all An. stephensi transgenic lines; moreover, in two lines (Bα and Eδ) transcription appeared to be restricted to salivary glands. However, histochemical stainings failed to reveal β-galactosidase activity on dissected glands indicating low levels of expression and/or translation of the transgene. These results suggest that salivary gland expression in mosquitoes may require some tissue-specific enhancer or other essential elements and that its regulation is apparently more complex than in the fruitfly. The frequent cluster organization adopted by several salivary gland genes, as revealed by the recently completed An. gambiae genome sequence, may be connected to a more complex regulation. Finally, sequence comparison of regulatory regions located immediately upstream of several salivary gland genes shows the presense of consensus binding sites for factors of the fkh/HNF-3 family suggesting that they may play a role in salivary gland-specific gene expression in mosquitoes. Functional analysis. A remarkable outcome of studies on mosquito salivary glands is the inability to assign functions to several salivary proteins. As a first step toward functional and structural studies we started the expression of recombinant salivary proteins in the yeast Pichia pastoris. gSG6 and gSG7 were recently obtained and purified in small amounts. The first may be a serineproteinase inhibitor and is distantly related to a family of anticoagulants from the parasitic nematode Ancylostoma caninum, the second shows a weak similarity to phospholipase A2 from snake venom. Expression of additional salivary proteins is underway. The recombinant proteins will be assayed for their ability to affect the hemostatic and/or inflammatory/immune response of the host and evaluated as possible markers of exposure to mosquito bites. Receptor studies. In the attempt to get insights into the molecular interactions underlying mosquito salivary gland invasion by plasmodium sporozoites we are planning to display on the surface of lambda phages putative adhesive domains of a few selected plasmodium proteins which may be involved in the invasion (TRAP, CS, MAEBL, CRMPs). This mini phage display library may be useful for the development of binding assays to whole salivary glands and for additional analyses targeted to the identification of salivary gland receptors
At the interface between parasite and host: the salivary glands of the African malaria vector Anopheles gambiae.
No full textMultiple complex interactions take place between the three organisms involved in malaria transmission: Plasmodium, Anopheles and the human host. The mosquito salivary glands are placed at the parasite-host interface and they represent the last barrier that sporozoites must overcome to infect the vertebrate. Moreover, the salivary glands of disease vectors produce and secrete a large variety of pharmacologically active compounds which can have profound effects on pathogen transmission and on the physiological responses of the human host both to the parasite and to the mosquito bite. Recent studies significantly expanded the Anopheles gambiae salivary transcriptome/proteome and they are expected to represent a starting point toward a functional analysis of mosquito salivary secretions. We believe that these investigations will contribute to a better understanding of the complex parasite-vector-host relationships and, perhaps, will be helpful in devising novel strategies to fight malaria
Display of Plasmodium adhesive domains on lambda phages.
No full textEfficient transmission of the malaria parasite by the mosquito vector requires successful invasion of the salivary glands, a process involving specific molecular interactions. Although different studies in the last decade highlighted the possible role of a few sporozoite surface proteins the key players of the invasion process have remained elusive so far. With the objective to improve our understanding of sporozoites-salivary gland interaction, we are using an approach that involves the display on the lambda surface of putative adhesive domains from a few selected Plasmodium proteins possibly implicated in salivary gland recognition/invasion. This collection of lambda phages can be employed for the development of affinity selection experiments targeted to the identification of salivary gland receptors. The lambda system allows the display of relatively large domains (up to 400 aminoacids) and, therefore, it is particularly suitable for our purpose. As a first step toward this direction, adhesive domains from selected P. falciparum and P. berghei surface proteins (such as TRAP, CS, MAEBL, etc) have been fused to the C-terminus of the abundant capsid protein gpD. The different domains were PCR-amplified, cloned in a plasmid vector, sequenced and finally ligated into the lambda vector λD4 (kindly provided by Dr. S. Nasi, CNR, Rome). After packaging, different recombinant lambda clones were randomly isolated from this phage collection and analyzed by western blot to verify the expression of the fusion proteins. All lambda clones identified and isolated so far seem to express recombinant proteins of the expected size. Verification of correct display for all selected domains, as well as binding assays to mosquito salivary glands, is in progress
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Display of Plasmodium adhesive domains on lambda phages.
No full textEfficient transmission of the malaria parasite by the mosquito vector requires successful invasion of the salivary glands, a process involving specific molecular interactions. Although different studies in the last decade highlighted the possible role of a few sporozoite surface proteins the key players of the invasion process have remained elusive so far. With the objective to improve our understanding of sporozoites-salivary gland interaction, we are using an approach that involves the display on the lambda surface of putative adhesive domains from a few selected Plasmodium proteins possibly implicated in salivary gland recognition/invasion. This collection of lambda phages can be employed for the development of affinity selection experiments targeted to the identification of salivary gland receptors. The lambda system allows the display of relatively large domains (up to 400 aminoacids) and, therefore, it is particularly suitable for our purpose. As a first step toward this direction, adhesive domains from selected P. falciparum and P. berghei surface proteins (such as TRAP, CS, MAEBL, etc) have been fused to the C-terminus of the abundant capsid protein gpD. The different domains were PCR-amplified, cloned in a plasmid vector, sequenced and finally ligated into the lambda vector λD4 (kindly provided by Dr. S. Nasi, CNR, Rome). After packaging, different recombinant lambda clones were randomly isolated from this phage collection and analyzed by western blot to verify the expression of the fusion proteins. All lambda clones identified and isolated so far seem to express recombinant proteins of the expected size. Verification of correct display for all selected domains, as well as binding assays to mosquito salivary glands, is in progress
Anopheles gambiae salivary gland promoter analysis.
No full textThe application of the powerful tools of genetics and molecular biology to studies on mosquito vectors and malaria parasites has contributed to a significant advancement in the basic research in mosquito and parasite biology, and in the understanding of the complex interactions between Anopheles and Plasmodium. Moreover, the development of genetic manipulation techniques for An. gambiae and P. falciparum, and the availability of the complete sequences of human, mosquito and parasite genomes, raised great expectations for the development of novel malaria control strategies. In this context the availability of salivary gland-specific promoters, which may allow the expression of a given foreign gene in the salivary glands of transgenic mosquitoes, may prove a useful tool both for studies on vector-parasite interactions and, potentially, for the development of novel strategies for vector control. Recently, we described the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4 in transgenic An. stephensi (Lombardo et al. 2005). Specifically, an 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-lateral rather than in the distal-lateral lobes of female glands. We proposed some explanations to elucidate those data and to plan future work. First of all, the AgApy promoter employed in the An. stephensi transformation could be lacking in some of the sequence infomation needed for the correct and strong expression in the female salivary glands; moreover, the transcriptional machinery of An. stephensi could not perfectly recognize all the transcriptional information eventually enclosed in the An. gambiae promoter region. Taking advantage of the recent improvements in transformation techniques that made possible the genetic manipulation of An. gambiae mosquitoes, we established transgenic An. gambiae lines carrying a larger portion of the AgApy promoter (~ 2,4 kb) driving the LacZ reporter gene. A transformation vector based on the piggyBac transposon and marked with the dsRed gene under control of the 3xP3 promoter was used for the microinjection experiments. A number of different transgenic An. gambiae G1 founders were obtained: the progeny was analyzed by Southern blot revealing the presence of single as well as multiple copies of the transgene. Three independent lines containing one (E9), three (D6) and four copies (D4) of the transgene were selected and established for further analysis. RT-PCR expression analysis with LacZ-specific primers showed different expression profiles in the three transgenic lines suggesting that position effect and copy number affect reporter gene expression. In fact, LacZ mRNA was ubiquitously detectable in transgenic mosquitoes from the D4 line whereas individuals from the D6 line exhibited an expression profile restricted to female salivary glands and adult males. Finally, transgene expression in E9 mosquitoes appeared strong in larval and pupal stages, absent in female carcasses and rather weak in salivary glands and males. Hystochemical assays on whole glands and western blot analysis on salivary protein extracts failed so far to reveal β-galactosidase protein in transgenic mosquitoes of the different lines. Further analysis are therefore in progress to completely characterize the transactivation properties of this AgApy regulatory region in the An. gambiae transgenic lines
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