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Biomedical subjects

D W Salter

Publications and source records attributed to D W Salter.

At least 19 recordsLinked to original sources
PubMed1993

A new defective retroviral vector system based on the Bryan strain of Rous sarcoma virus.

We have constructed a helper cell line and vector system based on the Bryan high titer (BH) strain of Rous sarcoma virus (RSV). BH-RSV is a defective virus which lacks an env gene; however, if env is supplied in trans, it replicates to a very high titer. Like BH-RSV, the vector contains gag and pol genes and lacks an env gene. The helper cell line supplies env in trans and permits the production of infectious virions. To construct the helper cell line the subgroup A env gene from the Schmidt-Ruppin-A (SRA) RSV was stably transfected into Qt6 cells, a chemically transformed quail fibroblast line. To minimize homology between the vector and helper cell line, transcription of the env gene is driven by a MuLV LTR, and 3' processing is controlled by the simian virus 40 (SV40) polyadenylation signal. This combination of vector and helper cells can be used to produce high-titer viral stocks in which recombinant replication-competent virus have not been detected even when the stocks were used to inoculate chickens. This system should be useful for developing transgenic chickens, studying cell lineage, and introducing genes into cultured cells.

Animals↗
PubMed1992

Appropriate in vivo expression of a muscle-specific promoter by using avian retroviral vectors for gene transfer [corrected].

The promoter regions of the chicken skeletal muscle alpha-actin (alpha sk-actin) and the cytoplasmic beta-actin genes were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Replication-competent retroviral vectors were used to introduce these two actin/CAT cassettes into the chicken genome. Chickens infected with retroviruses containing the alpha sk-actin promoter expressed high levels of CAT activity in striated muscle (skeletal muscle and heart); much lower levels of CAT activity were produced in the other nonmuscle tissues. In contrast, chickens infected with retroviruses containing the beta-actin promoter linked to the CAT gene expressed low levels of CAT activity in many different tissue types and with no discernible tissue specificity. Data are presented to demonstrate that the high levels of CAT activity that were detected in the skeletal muscle of chickens infected with the retrovirus containing the alpha sk-actin promoter/CAT cassette were not due to preferential infectivity, integration, or replication of the retrovirus vector in the striated muscles of these animals.

Actins↗
PubMed1992

A transgene, alv6, that expresses the envelope of subgroup A avian leukosis virus reduces the rate of congenital transmission of a field strain of avian leukosis virus.

A major mode of transmission of avian leukosis virus (ALV) is from a dam that is viremic with and immunologically tolerant to ALV, through the egg to the progeny. The authors have produced a line of chickens transgenic for a defective ALV provirus that expresses envelope glycoprotein, but not infectious virus, and is very resistant to infection with Subgroup A ALV. In the present experiment the authors sought to prevent or reduce congenital transmission by mating viremic-tolerant hens to males carrying the inserted provirus, thus introducing a gene for resistance into the progeny. Mature viremic females were mated with males hemizygous for the transgene to produce over 80 progeny each with and without the transgene. The chicks were hatched and maintained for 36 wk and observed for viremia, antibody, and the incidence of bursal lymphomas. Over 90% of the transgene-negative controls remained viremic through 36 wk of age and 51% developed bursal lymphomas. In contrast, 27% of the transgene-positive birds remained viremic and 18% died with bursal lymphomas. Thus, expression of Subgroup A envelope protein in the developing embryo reduced but did not eliminate congenital infection.

Analysis of Variance↗
PubMed1990

Expression of retroviral genes in transgenic chickens.

The development of transgenic technology in poultry has lagged behind that in mammals because of the unique reproductive system of birds. Therefore, we chose to use wild-type and recombinant replication-competent avian leukosis viruses to determine whether these retroviruses could be artificially introduced into the germ line by injecting them near the blastoderm of fertile eggs just before incubation. We generated 23 proviral inserts that were stably inherited through two generations. Twenty-one inserts coded for complete avian leukosis virus. Two interesting inserts failed to produce complete virus. One coded for envelope glycoprotein only and the other coded for the group-specific antigen and envelope glycoprotein. Cell culture and animal studies showed that one of these inserts was very resistant to infection and oncogenesis by subgroup A field strains of avian leukosis virus. Therefore, this represents a model system for introducing genes from the pathogen into the host to induce host resistance to the pathogen. Future studies should be aimed at developing more efficient systems for introducing replication-defective retroviral vectors or cloned DNA into the germ line of poultry so that the regulation of gene expression can be studied and transgenic technology can be applied to the improvement of this highly reproductive group of farm animals.

Animals↗
PubMed1987

Transgenic chickens: insertion of retroviral genes into the chicken germ line.

We infected early chicken embryos by injection of wild-type and recombinant avian leukosis viruses into the yolk of unincubated, fertile eggs. The viremic males (designated generation 0 (G-0] were tested for transmission of proviral DNA to their G-1 progeny. Nine of 37 G-0 viremic males were mosiac and proviral DNA was transmitted to their progeny at frequencies varying from 1 to 11%. All of the G-1 progeny examined by restriction enzyme analysis for clonality of proviral junction fragments had one to three simple but different fragments. The proviral DNA was transmitted from G-1 to the G-2 progeny in a Mendelian fashion thus proving that retroviral genes have been inserted into the chicken germ line. One of the viruses is a candidate vector for insertion of foreign genes into the chicken germ line.

Animals↗
PubMed1986

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.

Animals↗
PubMed1986

Gene insertion into the chicken germ line by retroviruses.

We injected chick syncytial strain of reticuloendotheliosis virus (CS-REV) and wild type and recombinant avian leukosis virus (ALV) near the blastoderm of unincubated fertilized embryos and CS-REV intra-abdominally at day of hatch, and we progeny tested the surviving ALV viremic males and REV viremic males and females for transmitted viral genetic material. A number of positive progeny were identified and their deoxyribonucleic acid (DNA) analyzed for restriction enzyme fragments that hybridized with viral genetic material. Most of the progeny had simple restriction enzyme patterns unlike the viremic parents or congenitally infected progeny. This is suggestive evidence that retroviral genetic information has been inserted into the germ line of chickens.

Animals↗
PubMed1986

Design of retroviral vectors for the insertion of foreign deoxyribonucleic acid sequences into the avian germ line.

Because the available avian leukosis viral (ALV) vectors are moderately oncogenic in vivo, they are not suitable for insertion into the germ line. A significant reduction in the oncogenicity of the ALV vectors can be achieved by substituting the noncoding long terminal repeats (LTR) regions of the ALV virus with the LTR of the nononcogenic endogenous RAV-O virus. There is good evidence that the resulting RAV-O LTR vectors can be inserted into the germ line of domestic chickens and have the potential for inserting cloned sequences that can be used for poultry improvement.

Animals↗
PubMed1986

Cryopreservation of chicken semen of inbred or specialized strains.

Pooled semen from several inbred special chicken strains was diluted with solutions containing glycerol or dimethylacetamide as a cryoprotectant. One-half milliliter samples in capped glass vials were frozen at 3 C/min to -35 C in a programmable freezer and stored in a nitrogen vapor tank. One vial of thawed semen was used to inseminate 4 hens by intravaginal, intrauterine, or intramagnal procedures. The intramagnal technique required minor surgery but always produced chicks in seven lines in contrast to the nonsurgical methods. Frozen semen of one strain stored for 29 weeks produced 12 to 14 chicks per vial when inseminated into 4 hens. This method, therefore, will reliably rescue gene pools from semen after long-term storage.

Animals↗
PubMed1984

Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells.

Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate.

3-O-Methylglucose↗
PubMed1984

Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus.

Transport rates of a number of nutrients and ions have been surveyed in chicken embryo fibroblasts that were density inhibited, growing exponentially, or transformed by Rous sarcoma virus. All the transport systems examined displayed changes associated with changes in growth rate. The rate of ouabain-sensitive potassium transport declined in density-inhibited cells, and increased rapidly in response to serum stimulation. This transport system was regulated both by changes in the activity of the transporters and by the number of transporters in the cell membrane. The rate of transport of the amino acid analog alpha-aminoisobutyric acid declined when cells became density inhibited, but also showed alterations in regulation that were associated with malignant transformation. The rate of glucose transport displayed both growth state-related and transformation-specific changes. The increased rate of glucose transport seen in transformed cells is due to an increase in the number of glucose transporters in the cell membrane. Increased glucose transport is necessary for subsequent changes in glycolysis, and temporally precedes some of the changes in activity of glycolytic enzymes.

Amino Acids↗
PubMed1982

Proteins antigenically related to the human erythrocyte glucose transporter in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

Antibody raised against the purified human erythrocyte glucose transporter specifically precipitated four proteins from normal and Rous sarcoma virus-transformed chicken embryo cells: a major protein of Mr 41,000 and minor proteins of Mr 68,000, 73,000, and 82,000. The Mr 41,000 and 82,000 proteins were found only in a membrane fraction, not in the soluble fraction, and displayed a heterogeneous mobility on NaDodSO4/polyacrylamide gel electrophoresis, suggesting glycosylation. The Mr 41,000 and 82,000 proteins were increased in amount after malignant transformation in direct proportion to the increase in hexose transport rate, and the increase was dependent on the expression of the src gene product, as revealed with a temperature-conditional src mutant. We suggest that the Mr 41,000 and 82,000 proteins are the glucose transporter of chicken embryo fibroblasts, or a component of the glucose transporter. These experiments provide direct evidence that malignant transformation increases the rate of glucose transport by increasing the number of transporters in the membrane.

Animals↗
PubMed1978

Quercetin inhibits hexose transport in a human diploid fibroblast.

The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK = 7.65) is the inhibitory form of the molecule (K1 = 10micron). Quercetin, unlike phloretin, is rapidly degraded in 0.1 N NaOH; the degradation products are weakly inhibitory to hexose transport.

Biological Transport, Active↗