Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction.

Poliovirus RNA polymerase requires a host factor to initiate RNA synthesis in vitro. The host factor was previously purified to near homogeneity from HeLa cells but was not assigned an enzymatic activity. This report describes the purification of a terminal uridylyltransferase that can act as host factor. By all criteria examined it is identical to the factor purified previously. It has the same molecular weight (68,000), chromatographic properties, and cellular localization. We present evidence that terminal uridylyltransferase can add uridine residues to the 3' poly(A) end of virion RNA and that these anneal back to the poly(A) and form a hairpin primer for polymerase.     

Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Double-stranded RNAs approximately 21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.     

Synthesis of immunoglobulin mu chain gene products precedes synthesis of light chains during B-lymphocyte development.

Immunoglobulin (Ig) gene expression has been followed during the later stages of development of the murine fetal liver. Biosynthetic labeling and immunoprecipitation were used to isolate Ig-related polypeptides from fetal and neonatal livers. By examination of the specific immune precipitates, the earliest detectable Ig was shown to consist only of mu heavy chain. At about the time of birth, when light chain synthesis became evident, separation of surface Ig-positive cells from surface Ig-negative cells by using anti-Ig-coated dishes showed that cells lacking surface Ig (pre-B lymphocytes) synthesized only mu chains. Thus, commencement of light chain synthesis was closely coordinated with the appearance of surface Ig. Ig RNA species were examined by electrophoretic fractionation and hybridization with cloned Ig DNA sequences. The sizes and amounts of Ig mRNA were found to correlate with the pattern of mu and light chain protein biosynthesis. mu chain RNA species appeared earlier in gestation than light chain RNA did, and only after birth did light chain sequences reach levels equivalent to those of mu chain. Cell populations enriched in pre-B lymphocytes also contained an excess of mu over light chain mRNA.     

The Yin and Yang of microRNAs: leukemia and immunity

Yin and Yang are two complementary forces that together describe the nature of real-world elements. Yin is the dark side; Yang is the light side. We describe microRNAs having both Yin and Yang characteristics because they can contribute to normal function (Yang) but also to autoimmunity, myeloproliferation, and cancer (Yin). We have been working on a number of microRNAs that have these dual characteristics and here we focus on two, miR-125b and miR-146a. We have concentrated on these two RNAs because we have very extensive knowledge of them, much of it from our laboratory, and also because they provide a strong contrast: the effects of overexpression of miR-125b are rapid, suggesting that it acts directly, whereas the effects of miR-146a are slow to develop, suggesting that they arise from chronic alterations in cellular behavior.     

Inhibition of translation by poliovirus: inactivation of a specific initiation factor.

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an acitivity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation.     

Binding of Bruton''s tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction.

Bruton's tyrosine kinase (Btk) is a recently described B-cell-specific tyrosine kinase. Mutations in this gene lead to human X chromosome-linked agammaglobulinemia and murine X-linked immunodeficiency. Although genetic evidence strongly suggests that Btk plays a crucial role in B-lymphocyte differentiation and activation, its precise mechanism of action remains unknown, primarily because the proteins that it interacts with have not yet been identified. Here, we show that Btk interacts with Src homology 3 domains of Fyn, Lyn, and Hck, protein-tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-aa motifs in Btk. An analogous site with the same specificity is also present in Itk, the T-cell-specific homologue of Btk. Our data extend the range of interactions mediated by Src homology 3 domains and provide an indication of a link between Btk and established signaling pathways in B lymphocytes.     

MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output

The production of blood cells depends on a rare hematopoietic stem-cell (HSC) population, but the molecular mechanisms underlying HSC biology remain incompletely understood. Here, we identify a subset of microRNAs (miRNAs) that is enriched in HSCs compared with other bone-marrow cells. An in vivo gain-of-function screen found that three of these miRNAs conferred a competitive advantage to engrafting hematopoietic cells, whereas other HSC miRNAs attenuated production of blood cells. Overexpression of the most advantageous miRNA, miR-125b, caused a dose-dependent myeloproliferative disorder that progressed to a lethal myeloid leukemia in mice and also enhanced hematopoietic engraftment in human immune system mice. Our study identifies an evolutionarily conserved subset of miRNAs that is expressed in HSCs and functions to modulate hematopoietic output.     

The hospital clinical preceptor: essential preparation for success

Hospitals have a responsibility to provide preceptors with the knowledge and skills required to provide bedside instruction to and evaluation of orientees. Formal preceptor preparation programs that provide practical information for immediate application are necessary for successful transition of orientees into patient care environments. Essential content includes the importance of socialization, skill building techniques, critical thinking facilitation, and assignment management. Preceptor preparation courses need to be based on adult learning principles and incorporate interactive and creative teaching strategies.