Packaging cell lines for simian foamy virus type 1 vectors. (33/4345)

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.  (+info)

High-efficiency gene transfer into skeletal muscle mediated by electric pulses. (34/4345)

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.  (+info)

The mycotoxin fumonisin B1 transcriptionally activates the p21 promoter through a cis-acting element containing two Sp1 binding sites. (35/4345)

Fumonisin B1 (FB1) is a food-borne mycotoxin produced by Fusarium moniliforme. Structurally FB1 resembles sphingoid bases, and ingestion of FB1 causes several animal diseases. FB1 will cause hepatic carcinoma in rats and is implicated as a cofactor in esophageal or hepatic carcinoma. Previous studies concluded that FB1 repressed cyclin-dependent kinase 2 (CDK2) activity but induced CDK inhibitors p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) in monkey kidney cells (CV-1). In contrast, CV-1 cells transformed by simian virus 40 are resistant to the antiproliferative or apoptotic effects of FB1. Consequently, FB1 treatment of CV-1 cells leads to cell cycle arrest and apoptosis. In this study, we demonstrate that FB1 transcriptionally activates the p21 promoter. Functional analysis of the p21 promoter by reporter gene assays mapped the FB1-responsive region to -124 to -47. DNase I footprinting analysis revealed two protected motifs that span the FB1-responsive region, -124 to -101 (footprint II) and -89 to -67 (footprint III). Further studies demonstrated that DNA sequences from -124 to -101 were sufficient for FB1 stimulation. DNA sequences from -124 to -101 contain two Sp1 binding sites, and gel shift assays provided evidence that nuclear factors specifically bind to this region. Disruption of the two Sp1 binding sites abrogated the binding of nuclear proteins and prevented activation by FB1. Taken together, these results suggest that Sp1 or Sp1-related proteins mediate FB1-induced activation of the p21 promoter.  (+info)

Semi-automated detection of emesis in the rhesus monkey. (36/4345)

The use of a video recorder and the inclusion of a marker dye in material fed to rhesus monkeys has eliminated the need for the constant presence of an observer to detect an emetic response.  (+info)

Pathogenesis and classification of massive periretinal proliferation. (37/4345)

Massive periretinal proliferation (MPP), a serious complication of retinal detachment, is caused by proliferation and fibrous metaplasia of cells mostly deriving from retinal pigment epithelium and retinal glial cells. Contracting fibrous membranes in the vitreous, and on and also under the retina, cause the intraocular changes of MPP. Early signs such as increased 'tobacco dust', pigmented and unpigmented clumps in the vitreous, and subtle preretinal and even retroretinal membranes are usually overlooked. The late signs such as starfolds, irregular retinal folds, circumferential folds, and funnel-shaped detachments are well known. The pathogenesis of the clinically visible signs is described, and a 4-stage classification of the disease is given.  (+info)

Fetal hemoglobin (HbF) synthesis in baboons, Papio cynocephalus. Analysis of fetal and adult hemoglobin synthesis during fetal development. (38/4345)

Fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis was studied in fetal baboons, Papio cynocephalus, to determine the normal pattern of hemoglobin production during fetal development. Fetuses ranging from 53 to 180 days gestation (term gestation 184 days) were used. Erythroid cells were incubated with 3H-L-leucine, and the rates of globin chain synthesis and the distribution of radioactivity into hemoglobin intermediates and completed hemoglobin molecules were determined. Gamma chain synthesis accounted for approximately 97% of the total nonalpha chain synthesis up to 140 days gestation; beta chain synthesis accounted for the remainder. After 140 days gestation, approximately equal quantities of gamma and beta chain were synthesized in the bone marrow. Prior to 140 days gestation, total alpha chain synthesis was 30% greater than total non-alpha chain synthesis, while there was balanced chain synthesis after 140 days gestation. During the period of excess alpha chain synthesis, fetal erythrocytes contained a large pool of alpha-hemoglobin (alpha chain with heme attached) molecules uncombined with beta or gamma chains. In view of the possibility that alpha chains may have a lower affinity for gamma chains than beta chains, excess alpha chain synthesis may be required to maintain low levels of free gamma chains.  (+info)

Expression of Ricinus communis receptors on epithelial cells in oral carcinomas and oral wounds. (39/4345)

The histological distribution of receptors for Ricinus communis Fraction 1 (RCA1) in oral carcinomas and in oral epithelial cells during wound healing has been studied by use of fluorescein-tagged RCA1. Biopsies from 15 human oral carcinomas and adjacent normal mucosa showed RCA1 receptors at the cell membranes in the basal and spinous layer of the normal epithelium, whereas receptors could not be demonstrated in invading islands of the tumors. In healing oral wounds from eight humans and three monkeys, RCA1 receptors were demonstrated both in normal epithelium adjacent to the wounds and in the epithelial outgrowth from the wound margin. Titrations, however, showed that the epithelial outgrowth reacted more weakly than did the normal adjacent epithelium. These results support previous in vitro studies showing changes in carbohydrate composition of moving normal cells and of malignant cells, a finding that may be of interest in relation to formation of metastases.  (+info)

Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test. (40/4345)

A complement fixation study with human, monkey and rabbit sera, using purified sperm nuclear basic proteins as antigens, led to the following conclusions. (1) Protamines, the sperm-specific basic nuclear proteins, may be immunogenic in mammalians. (2) Antibodies detected in the indirect immunofluorescence test on human swollen sperm heads in sera from infertile and vasectomized men, are directed primarily against human protamines. (3) The results obtained suggested that differences in the immunization site and/or in the configuration of the immunizing protamine, may lead to the formation of antibodies directed against different antigenic determinants. Autoimmunity to protamines, following vasectomy or in infertile men, is accompanied by the formation of antibodies cross-reacting with common antigenic determinants present in protamines of other species. Induction of immunity to protamines by means of immunization with protamines-RNA complexes (in rabbits), or protamine-insulin complexes (in humans), leads to the formation of antibodies reacting more specifically with the immunizing protamine, showing only slight cross-reaction with other protamines. (4) The histone-like fraction present in mature human spermatozoa is composed mainly of histone fraction H2B.  (+info)