Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal

Journal ArticlePURPOSE: This study had three objectives: (1) to characterize the structures of the chicken GCAP1 and GCAP2 genes; (2) to determine if GCAP1, GCAP2, and GC1 genes are expressed in chicken pineal gland; (3) if GC1 is expressed in chicken pineal, to determine if the GC1 null mutation carried by the retinal degeneration (rd) chicken is associated with degenerative changes within the pineal glands of these animals. METHODS: GCAP1 and GCAP2 gene structures were determined by analyses of chicken cosmid and cDNA clones. The putative transcription start points for these genes were determined using 5'-RACE. GCAP1, GCAP2 and GC1 transcripts were analyzed using Northern blot and RT-PCR. Routine light microscopy was used to examine pineal morphology. RESULTS: Chicken GCAP1 and GCAP2 genes are arranged in a tail-to-tail array. Each protein is encoded by 4 exons that are interrupted by 3 introns of variable length, the positions of which are identical within each gene. The putative transcription start points for GCAP1 and GCAP2 are 314 and 243 bases upstream of the translation start codons of these genes, respectively. As in retina, GCAP1, GCAP2 and GC1 genes are expressed in the chicken pineal. Although the GC1 null mutation is present in both the retina and pineal of the rd chicken, only the retina appears to undergo degeneration. CONCLUSIONS: The identical arrangement of chicken, human, and mouse GCAP1/2 genes suggests that these genes originated from an ancient gene duplication/inversion event that occurred during evolution prior to vertebrate diversification. The expression of GC1, GCAP1, and GCAP2 in chicken pineal is consistent with the hypothesis that chicken pineal contains a functional phototransduction cascade. The absence of cellular degeneration in the rd pineal gland suggests that GC1 is not critical for pineal cell survival

Emerging Roles of Primary Cilia in Glioma

Primary cilia are microtubule-based organelles that are typically present on cells during the G0 or G1-S/G2 phases of the cell cycle. Recent studies of glioblastoma (GBM) biopsies, a brain tumor that is notorious for its aggressive growth and resistance to treatment, show that many cells in the tumor lack cilia. At this point, it remains unclear whether primary cilia promote or suppress glioma tumorigenesis. In this review, we will discuss the different roles that have been proposed for primary cilia in glioma and how cilia may contribute to the resistance of these tumors to current therapies

Expression of GCAP 1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina

AbstractWe cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rd/rd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAPI and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAPI levels are reduced by more than 90%. The specific downregulation of GCAPI in rd/rd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction

Lentiviral Expression of Retinal Guanylate Cyclase-1 (RetGC1) Restores Vision in an Avian Model of Childhood Blindness

BACKGROUND: Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase–1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%–20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. METHODS AND FINDINGS: A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. CONCLUSIONS: Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness

Bicistronic Lentiviruses Containing a Viral 2A Cleavage Sequence Reliably Co-Express Two Proteins and Restore Vision to an Animal Model of LCA1

The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase -1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis – 1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2)

Lung carcinoma with hypertrophic osteoarthropathy in a teenager

Hypertrophic osteoarthropathy (HOA) characterised by arthralgia, clubbing and periosteal proliferation of long bones, is rarely encountered in children and adolescents. Whereas in adults over 80% of cases are associated with malignancy, in children the majority of cases are due to non-neoplastic causes such as cystic fibrosis, bilary atresia and congenital heart disease. Up to 5% of adults with lung cancer demonstrate signs of HOA. However, lung cancer is extremely uncommon in children and young people. Here we report a case of lung adenocarcinoma in an 18 year old male associated with HOA present both at diagnosis and at subsequent disease progression

A Preclinical Assessment of Neural Stem Cells as Delivery Vehicles for Anti-Amyloid Therapeutics

Transplantation of neural stems cells (NSCs) could be a useful means to deliver biologic therapeutics for late-stage Alzheimer's disease (AD). In this study, we conducted a small preclinical investigation of whether NSCs could be modified to express metalloproteinase 9 (MMP9), a secreted protease reported to degrade aggregated Aβ peptides that are the major constituents of the senile plaques. Our findings illuminated three issues with using NSCs as delivery vehicles for this particular application. First, transplanted NSCs generally failed to migrate to amyloid plaques, instead tending to colonize white matter tracts. Second, the final destination of these cells was highly influenced by how they were delivered. We found that our injection methods led to cells largely distributing to white matter tracts, which are anisotropic conduits for fluids that facilitate rapid distribution within the CNS. Third, with regard to MMP9 as a therapeutic to remove senile plaques, we observed high concentrations of endogenous metalloproteinases around amyloid plaques in the mouse models used for these preclinical tests with no evidence that the NSC-delivered enzymes elevated these activities or had any impact. Interestingly, MMP9-expressing NSCs formed substantially larger grafts. Overall, we observed long-term survival of NSCs in the brains of mice with high amyloid burden. Therefore, we conclude that such cells may have potential in therapeutic applications in AD but improved targeting of these cells to disease-specific lesions may be required to enhance efficacy