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Monomeric Red Fluorescent Protein (monomeric + red_fluorescent_protein)
Selected AbstractsFlying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feedingINSECT MOLECULAR BIOLOGY, Issue 3 2010D. S. Yamamoto Abstract ,Flying vaccinator' is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti-SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a ,flying vaccinator'. However, medical safety issues and concerns about informed consent mitigate the use of the ,flying vaccinator' as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva,malaria sporozoite interactions. [source] Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane systemPLANT BIOTECHNOLOGY JOURNAL, Issue 7 2008Sarah L. Irons Summary In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay. [source] Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivoTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2009Patrick W. Howorth Abstract The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4,L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 ± 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4,L5. These neurons were immunopositive for dopamine ,-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception. J. Comp. Neurol. 512:141,157, 2009. © 2008 Wiley-Liss, Inc. [source] Localization of a flavonoid biosynthetic polyphenol oxidase in vacuolesTHE PLANT JOURNAL, Issue 2 2006Eiichiro Ono Summary Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+ -translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower. [source] A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potatoCELLULAR MICROBIOLOGY, Issue 11 2008Anna O. Avrova Summary Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans -membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans. [source] | ||||||||||