Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

INTRODUCTION: Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man. METHOD: Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4. RESULTS: We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression. CONCLUSION: Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression

Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes

Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency

An alpha-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major

Submitted by Sandra Infurna ([email protected]) on 2018-02-08T14:28:28Z No. of bitstreams: 1 otacilio_moreira_etal_IOC_2017.pdf: 4048766 bytes, checksum: 739bd60ac93001eb3f167318d867f73c (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-02-08T14:43:56Z (GMT) No. of bitstreams: 1 otacilio_moreira_etal_IOC_2017.pdf: 4048766 bytes, checksum: 739bd60ac93001eb3f167318d867f73c (MD5)Made available in DSpace on 2018-02-08T14:43:57Z (GMT). No. of bitstreams: 1 otacilio_moreira_etal_IOC_2017.pdf: 4048766 bytes, checksum: 739bd60ac93001eb3f167318d867f73c (MD5) Previous issue date: 2017University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Chemistry. Border Biomedical Research Center. El Paso, Texas, USA.Liverpool School of Tropical Medicine. Department of Parasitology. Pembroke Place, Liverpool, United Kingdom.University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Chemistry. Border Biomedical Research Center. El Paso, Texas, USA.Liverpool School of Tropical Medicine. Department of Parasitology. Pembroke Place, Liverpool, United Kingdom.University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Liverpool School of Tropical Medicine. Department of Parasitology. Pembroke Place, Liverpool, United Kingdom / Liverpool School of Tropical Medicine. Department of Vector Biology. Pembroke Place, Liverpool, United Kingdom.University of Texas at El Paso. Department of Chemistry. Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL