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Biomedical subjects

R C Hoeben

Publications and source records attributed to R C Hoeben.

47 records · Page 3Linked to original sources
PubMed1994

Insertion of the human cytomegalovirus enhancer into a myeloproliferative sarcoma virus long terminal repeat creates a high-expression retroviral vector.

A new retroviral vector is characterized in which the enhancer in the long terminal repeat of myeloproliferative sarcoma virus (MPSV) is combined with the strong and universally active immediate-early human cytomegalovirus (hCMV) enhancer. We demonstrate that insertion of the hCMV enhancer increases the amount of vector-specific mRNA in various rodent cell lines and in human diploid fibroblasts, by at least 3-5-fold. The vector may be particularly useful if high expression of two genes is desired.

3T3 Cells↗
PubMed1993

Toward gene therapy for hemophilia A: long-term persistence of factor VIII-secreting fibroblasts after transplantation into immunodeficient mice.

Hemophilia A is caused by the lack of functional blood-clotting factor VIII. We have used retrovirus-mediated gene transfer to generate various cell lines, rodent as well as human, that secrete the human factor VIII protein. To study whether transplantation of genetically modified fibroblasts is a feasible approach for gene therapy of hemophilia A, we implanted the factor VIII-secreting cells into immune-deficient mice. Implantation of factor VIII-secreting primary human skin fibroblasts resulted in long-term persistence of the transplanted cells; cells recovered from the implants up to 2 months post-implantation still had the capacity to secrete factor VIII when regrown in tissue culture. However, we were unable to detect any human factor VIII in plasma samples of the recipient mice. The absence of human factor VIII in the recipients' plasma is shown to be due neither to (epigenetic) inactivation of the retroviral vector in vivo, nor to inability of the stationary cells to secrete factor VIII protein. However, we did note a rapid clearing of the human factor VIII: CAg from plasma upon intravenous injection of plasma-derived human factor VIII in mice (t1/2 < 60 min vs. 10 hr in humans). This phenomenon can fully explain the apparent absence of human factor VIII in the recipients' plasma.

Animals↗
PubMed1992

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.

Aluminum↗
PubMed1992

Toward gene therapy in haemophilia A: retrovirus-mediated transfer of a factor VIII gene into murine haematopoietic progenitor cells.

To study and evaluate the potential of the haematopoietic system as a target for gene therapy in haemophilia A, we have infected murine bone-marrow cells with a recombinant retrovirus encoding blood-coagulation factor VIII and the bacterial enzyme neomycin-phosphotransferase. After transplantation of the infected bone marrow into lethally irradiated mice, the presence of intact vector could be demonstrated in DNA isolated from individual haematopoietic progenitor-cell-derived spleen colonies. About 8% of the spleen colonies were shown to contain the intact vector. Selection for resistance to the neomycin analogue G418 prior to transplantation specifically killed the uninfected bone-marrow cells and, as a result, over 90% of the spleen colonies contained the factor VIII vector. However, expression of factor VIII in vivo, either at the RNA or at the protein level could not be demonstrated. From these data we conclude that: 1) retroviral vectors can be used to transfer factor-VIII cDNA into haematopoietic progenitor cells; 2) the vector sequences are expressed immediately after integration; and 3) transcription of the vector is repressed in the progenitor-cell-derived cells.

Animals↗
PubMed1991

Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position.

The expression of a retroviral vector with the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) promoter after integration into the genome of murine fibroblast cell lines was monitored with the Escherichia coli-derived beta-galactosidase (beta-gal) gene as the reporter. Monoclonal cell lines derived after retroviral infection exhibited a marked heterogeneity in their expression of the reporter gene. We studied two monoclonal cell lines with a single unrearranged copy of the vector provirus integrated into their genome. The first, BB10, expressed the marker enzyme in only 8% of its cell population, whereas in the second, BB16, beta-gal expression could be detected in over 98% of the cells. Treatment of BB10 with the DNA-demethylating agent 5-azacytidine raised the number of beta-gal-positive cells to over 60%. Transfection experiments showed that the Mo-MuLV LTR promoter-enhancer is potentially fully functional in both the BB10 and BB16 cell lines. The inactivated provirus from BB10 cells was cloned and subsequently used to generate retrovirus stocks. The promoter-enhancer activity of its LTR after infection with these BB10-derived viruses showed a variation similar to that of the original virus stocks. Our data showed that (1) inactivation of the Mo-MuLV LTR is a frequent event in murine fibroblast cell lines, (2) inactivation is associated with de novo methylation of cytidine residues, (3) the frequency of inactivation of the provirus must be determined by its chromosomal position, (4) the process of methylation of sequences within the LTR is not necessarily the same as the transcription-repression mechanism that is operating in undifferentiated embryonal carcinoma cells.

Animals↗
PubMed1991

Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

Amino Acid Sequence↗
PubMed1990

Expression of functional factor VIII in primary human skin fibroblasts after retrovirus-mediated gene transfer.

We have developed a retroviral-vector system for the transfer and expression of a cloned blood clotting factor VIII cDNA. Since inclusion of the complete cDNA into existing vectors is precluded by its large size, we deleted most codons for the B-domain, which is also excised during in vivo maturation of factor VIII. When inserted into the retroviral vector M5-neoR (Laker, C., Stocking, C., Bergholtz, V., Hess, N., DeLamarter, J. F., and Ostertag, W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8458-8462), the sequence was shown to be efficiently expressed in murine fibroblast cell lines, as well as in primary human skin fibroblasts. Upon infection of murine fibroblast cell lines, clones containing only a single copy of the integrated vector-provirus secreted up to 125 milliunits of factor VIII antigen/10(6) cells/day. Equivalent amounts were found in a factor VIII activity assay, which signifies that the factor VIII protein secreted by the infected fibroblasts is fully functional. Primary human skin fibroblasts infected with the vector virus secreted up to 30 milliunits/10(6) cells/day.

Animals↗
PubMed1989

Characterization of adenovirus type 5 insertion and deletion mutants encoding altered DNA binding proteins.

We have introduced insertion and deletion mutations in the cloned DNA binding protein (DBP) gene of adenovirus type 5. The mutated DBP genes were subsequently introduced in the viral genome by a combination of in vitro and in vivo methods. The resulting mutant viruses were tested for their viability in human 293 cells and an initial characterization of these viruses was performed. Viable mutants with insertions in the carboxyl-terminal portion of the gene could not be obtained. In contrast, a number of viable mutants were constructed that contained insertions or deletions in the amino-terminal half of DBP. Several of these, which covered the region between amino acid (aa) residues 39 and 81, were phenotypically wild type, implying that this segment is completely dispensable for DBP function. However, mutations altering the region encompassed by aa 2-38 were, at least, partially defective suggesting that this region is important for full activity of the protein.

Adenoviruses, Human↗
PubMed1987

Hydrocortisone stimulates the development of oligodendrocytes in primary glial cultures and affects glucose metabolism and lipid synthesis in these cultures.

Cultures of glial cells were prepared from the brains of one-week-old rat pups. After one day in culture, serum was omitted from the medium and replaced by a combination of growth-stimulating hormones and other factors that enhanced the percentage of oligodendrocytes in the cultures. We investigated the effects of hydrocortisone on the development of oligodendrocytes, on the activities of oligodendrocyte-specific enzymes and on glucose- and lipid-metabolism of the glial cells. Hydrocortisone greatly enhanced the survival of glial cells in culture. The development of galactocerebroside-positive cells and the specific activity of 2',3'-cyclic-nucleotide 3'-phosphodiesterase were stimulated by 50 nM hydrocortisone, whereas these effects were partly reversed at higher concentrations of the hormone. The specific activity of glycerol-3-phosphate dehydrogenase was markedly stimulated by hydrocortisone; 1 microM or higher concentrations of hydrocortisone were required for an optimal effect. The consumption of glucose and the production of lactate were lowered by hydrocortisone whereas the oxidation of [6-14C]glucose to 14CO2 was not affected. Incorporation of [35S]sulfate into sulfolipids was greatly enhanced by hydrocortisone and [14C]incorporation from [1-14C]acetate into cholesterol and fatty acids was also stimulated but to a smaller extent. These results show that hydrocortisone exerts a general trophic function on glial cells in our culture system; enhances the ratio of oligodendrocytes over astrocytes, possibly by directing bipotential progenitor cells to develop into oligodendrocytes; specifically induces glycerol-3-phosphate dehydrogenase in oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures.

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗