Double‐stranded RNA (dsRNA) unwinding/modifying activity, which is present in a wide range of eukaryotic cells, has been previously shown to convert up to 50% of adenosine residues to inosines within intermolecular dsRNA. In the present study, we report that this activity also modifies, though slightly less efficiently, intramolecular double‐stranded regions of synthetic RNAs. Our results widen the range of the possible biological substrates for the activity since many stem and loop type RNA secondary structures (intramolecular dsRNA), present in eukaryotic as well as viral transcripts, can potentially serve as substrates. In addition, we have found that the dsRNA unwinding/modifying activity requires a double‐stranded region of at least 15–20 base pairs (bp) for substrate recognition. Furthermore, modification efficiency was found to be critically dependent on the length of the double‐stranded region; as the size decreased below 100 bp, it dropped precipitously. Our results suggest that efficient modification may occur only with relatively long (greater than 100 bp) dsRNA, perhaps because multiple copies of the enzyme must be bound.

Morphological diversity of dendrites contributes to specialized functions of individual neurons. In the present study, we examined the molecular basis that generates distinct morphological classes of Drosophila dendritic arborization (da) neurons. da neurons are classified into classes I to IV in order of increasing territory size and/or branching complexity. We found that Abrupt (Ab), a BTB-zinc finger protein, is expressed selectively in class I cells. Misexpression of ab in neurons of other classes directed them to take the appearance of cells with smaller and/or less elaborated arbors. Loss of ab functions in class I neurons resulted in malformation of their typical comb-like arbor patterns and generation of supernumerary branch terminals. Together with the results of monitoring dendritic dynamics of ab-misexpressing cells or ab mutant ones, all of the data suggested that Ab endows characteristics of dendritic morphogenesis of the class I neurons.

The Drosophila CNS midline cells constitute a specialized set of interneurons, motorneurons, and glia. The utility of the CNS midline cells as a neurogenomic system to study CNS development derives from the ability to easily identify CNS midline-expressed genes. For this study, we used a variety of sources to identify 281 putative midline-expressed genes, including enhancer trap lines, microarray data, published accounts, and the Berkeley Drosophila Genome Project (BDGP) gene expression data. For each gene, we analyzed expression at all stages of embryonic CNS development and categorized expression patterns with regard to specific midline cell types. Of the 281 candidates, we identified 224 midline-expressed genes, which include transcription factors, signaling proteins, and transposable elements. We find that 58 genes are expressed in mesectodermal precursor cells, 138 in midline primordium cells, and 143 in mature midline cells--50 in midline glia, 106 in midline neurons. Additionally, we identified 27 genes expressed in glial and mesodermal cells associated with the midline cells. This work provides the basis for future research that will generate a complete cellular and molecular map of CNS midline development, thus allowing for detailed genetic and molecular studies of neuronal and glial development and function.

The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathione S-transferase (GST) fusion protein in Escherichia coli and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.Genital human papillomavirus (HPV) infection is a common sexually transmitted disease that is the primary cause of cervical cancer, resulting in approximately 400,000 deaths per year worldwide (30). An effective vaccine against HPV infection would potentially prevent the development of most human cervical dysplasias and carcinomas (4). In addition, a vaccine would also reduce the cost (estimated at $6 billion annually in the United States) of screening and treating premalignant cervical disease (16).Since HPV cannot replicate in other animal species, evaluation of potential HPV vaccines requires the use of related animal papillomaviruses. The mucosotropic, oncogenic canine oral papillomavirus (COPV) closely mimics the biology of HPV, and the capsid proteins of COPV are closely related to those of HPV, making COPV a relevant and accepted animal model for testing the efficacy of prophylactic vaccine candidates (2,19,26,27).The L1 capsid protein of papillomaviruses self-assembles into virus-like particles (VLPs) when expressed in insect cells (11,14) or yeast (12,23). These L1 VLPs are morphologically similar to virions, being comprised of 72 pentamers (i.e., capsomeres) of L1 arranged in a Tϭ7 icosahedral lattice, but lacking the L2 capsid protein and the viral genome. Previous studies have shown that immunization with purified VLPs protects against experimental papillomavirus infection in rabbits (5, 8), cows (15), and dogs (27). Conformational epitopes on VLPs appear critical for the induction of neutralizing immunoglobulin G (IgG) and for successful vaccination, since denatured L1 protein fails to generate neutralizing antibodies or protect against experimental infection (10,18,27).Although early attempts to use bacteria for producing papillomavirus L1 protein vaccines were unsuccessful due to poor immunogenicity or inefficient expression (1, 9, 13, 28, 29), recent studies have shown that the HPV type 11 (HPV-11) and HPV-16 L1 proteins can be expressed in Escherichia col...

The limitations of cancer cell lines have led to the development of direct patient derived xenograft (PDX) models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg−/− (NSG) mouse on which a patient’s tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal, and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice – an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice, and provide a more accurate tumor model to guide patient treatment.