A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.Bgl ͉ MS2 ͉ dimerization ͉ DIS ͉ fluorescent protein

Bovine serum albumin (BSA) conjugated gold (Au) nanoparticles (NPs) were synthesized to explore their applications as drug delivery vehicles in systemic circulation. They showed little hemolysis and cytotoxic responses essentially required for such applications. This study shows some of the important physiochemical aspects needed for an appropriate synthesis of BSA-conjugated NPs where unfolded BSA is an essential reaction component. Unfolding of BSA was carried out under different experimental conditions in the presence of different ionic/ zwitterionic surfactants and monitored simultaneously by UV−visible studies. Cationic surfactants induced unfolding at relatively lower temperatures than anionic and zwitterionic surfactants due to stronger electrostatic interactions with BSA. TEM analysis revealed the presence of NPs with almost similar shapes and sizes for different samples, and all NPs were stabilized by a coating of unfolded BSA. Isoelectric point of unfolded BSA coating on NP surface was close to 4.7 in all cases, which was similar to that of unconjugated BSA. BSA free and cationic surfactant coated Au NPs were used as controls. They showed high hemolytic activity and very low cell viability under identical conditions. Thus, BSA coated NPs were considered to be the best vehicles for drug release and other possible biomedical applications.

Glioblastoma is the most common malignant brain tumor of adults and one of the most lethal cancers. The secreted growth factor pleiotrophin (PTN) promotes glioblastoma migration and proliferation, initiating its oncogenic activities through two cell surface receptors, the protein tyrosine phosphatase receptor (PTPRZ1) and the anaplastic lymphoma kinase (ALK), respectively. Here, we report on the presence and purification of two naturally occurring forms of PTN (18 and 15 kDa) that differentially promote glioblastoma migration and proliferation. Using a panel of glioblastoma cell lines, including low passage patient-derived cultures, we demonstrate that PTN15 promotes glioblastoma proliferation in an ALKdependent fashion, whereas immobilized PTN18 promotes haptotactic migration of glioblastoma cells in a PTPRZ1-dependent fashion. Mass spectrometric analysis indicated that PTN15 differs from PTN18 by processing of 12 C-terminal amino acids. To demonstrate clinical relevance, we show that PTN15, PTN18, and PTPRZ1 are significantly overexpressed in glioblastoma relative to normal brain at both mRNA and protein levels using microarray, Western blot, and tissue microarray analyses on human tumors. These results indicate that the PTN18-PTPRZ1 and the PTN15-ALK signaling pathways represent potentially important therapeutic targets for glioblastoma invasion and growth.Glioblastoma is the most frequent and lethal primary brain tumor of adults, with a median survival time of 12 months from the time of diagnosis despite aggressive surgery, radiation, and chemotherapy. Although refractory to standard treatments, glioblastomas are well suited for targeted molecular therapies (1). The characterization of molecules or pathways critical to glioblastoma pathogenesis may thus lead to the discovery of novel targets for therapy.The secreted growth factor PTN 1 (also called HARP, HB-GAM, HBNF, and OSF-1) may represent such a potential target.PTN is a heparin-binding, developmentally regulated protein that is expressed in the embryonic and postnatal nervous system (2-5) and has been shown to be overexpressed in a number of cancers, including glioblastomas (6 -11). A variety of biological activities have been attributed to PTN including mitogenesis, angiogenesis, neurite outgrowth, and cell migration (12). PTN promotes glioblastoma growth via the receptor tyrosine kinase ALK in a process that can be mediated via the phosphatidylinositol 3-kinase and MAPK signaling pathways (13,14). PTN also engages a second receptor, the receptor tyrosine phosphatase PTPRZ1 (also termed RPTP␤ and PTP) (15,16), and functional data suggest that this interaction may promote glioblastoma migration (17). Like PTN, both ALK and PTPRZ1 are predominantly expressed in the developing central nervous system (18 -21) and have also been shown to be overexpressed in glioblastomas (11,13,17).To date, the molecular mechanisms underlying PTN-mediated invasion and proliferation through these receptors have yet to be fully elucidated. Because the two receptor pathways ha...

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