No abstract

A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection approximately 10-100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity approximately 5-50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi-gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi-gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.

HcLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely supprcsscd in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with intcrphase cells treated in the same way. Cell-free protein synthesis and RNA polymcrase activity were also greatly depressed in extracts of metaphasc cells. The dcoxyribonuclcoprotein (DNP) of condenscd chromosomes from mitotic cells was less cfficicnt as a template for Escherichia coli RNA polymcrase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of mctaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell cxtracts. These rcsults suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is deprcsscd at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in mctaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.

The efforts of 69 elementary school teachers to instruct children in cognitive processing activities were observed. Although the teaching of such activities was relatively infrequent, it varied by grade (occurring more often in grades 2-3 than in higher or lower grades) and by the content of instruction. Teachers of grade 4 and above more often provided rationales for the use of cognitive strategies than did teachers of younger children. In a second study, children of three achievement levels were selected from classrooms in which teachers varied in their use of suggestions regarding cognitive processes. Subsequent to training in the use of a memory strategy, children's performance on a maintenance trial was evaluated: Among average and low achievers, those whose teachers were relatively high in strategy suggestions showed better maintenance and more deliberate use of the trained strategy than did children whose teachers rarely made strategy suggestions. The role of school experience in the development of children's memory skills is discussed.

The pathogenicity of infection produced following intracerebral (i.c.) inoculation of wild-type vesicular stomatitis virus (VSV) or temperature-sensitive (ts) mutants of VSV was compared. ts mutants used were ts 31 (VSV complementation group II) and ts 41 (VSV complementation group IV). The i.c. injection of wild-type VSV in weanling Swiss mice produced a rapidly fatal encephalitis with death of mice in 2 to 3 days. Histopathologically, such mice exhibited minimal changes of encephalitis on light microscopy. In contrast to the highly virulent, rapidly fatal central nervous system (CNS) infection seen after i.c. inoculation of wild-type VSV, infection with ts 31 VSV produced a more slowly progressive CNS infection characterized by hind limb paralysis and death 6 to 9 days after infection. Histopathologically, CNS infection with ts 31 is associated with previously unreported extensive spongiform changes in the gray matter of the spinal cord. The inoculation of ts 41 i.c., on the other hand, did not result in either clinical illness or histopathological changes in the spinal cords or brains of infected mice. The absence of clinical and histopathological lesions following i.c. infection ofts 41 VSV suggests that the capacity to alter the pathogenesis of VSV CNS infection may be a function of only certain ts mutants of VSV.