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T A Kost

Publications and source records attributed to T A Kost.

At least 37 records · Page 2Linked to original sources
PubMed1994

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one is an active site-directed slow time-dependent inhibitor of human steroid 5 alpha-reductase 1.

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one (finasteride), which has been approved for treatment of benign prostatic hyperplasia, is shown here to be a slow time-dependent inhibitor of human steroid 5 alpha-reductase isozyme 1. This inhibition is characterized by an initial, fast step where the inhibitor binds to the enzyme followed by a slow step that leads to a final enzyme-inhibitor complex (EI*). No recovery of activity from this EI* complex was observed after dialysis for 3 days. The formation of EI* is diminished in the presence of a competitive, reversible inhibitor, indicating that the inhibition is active site-directed. At 37 degrees C and pH 7.0, the rate constant for the second, slow inhibition step, k3, is (1.40 +/- 0.04) x 10(-3) s-1 and the pseudo-bimolecular rate constant, k3/Ki, is (4.0 +/- 0.3) x 10(3) M-1 s-1. This latter rate constant is less than the value of 2.7 x 10(5) M-1 s-1 determined for the inhibition of 5 alpha-reductase 2 by finasteride [Faller, B., Farley, D., & Nick, H. (1993) Biochemistry 32, 5705-5710].(ABSTRACT TRUNCATED AT 250 WORDS)

5-alpha Reductase Inhibitors↗
PubMed1994

Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system.

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinant Drosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by the Drosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.

Animals↗
PubMed1993

High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme.

Alkyl and Aryl Transferases↗
PubMed1992

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.

Centrifugation, Density Gradient↗
PubMed1991

Human immunodeficiency virus infection and syncytium formation in HeLa cells expressing glycophospholipid-anchored CD4.

The CD4 molecule, a glycoprotein expressed primarily on the cell surface of specific T lymphocytes, is thought to function in T-cell antigen recognition and activation. In addition, CD4 serves as a receptor for human immunodeficiency virus type 1 (HIV-1) by a direct interaction with the HIV-1 surface glycoprotein (gp120). To further characterize the HIV-1-cell interaction, a HeLa cell line was established that expressed a chimeric molecule of CD4 and decay-accelerating factor (DAF). In the chimeric CD4-DAF molecule the transmembrane and cytoplasmic domains of CD4 were deleted and replaced with the carboxy-terminal 37 amino acids of DAF. This resulted in the anchoring of the extracellular domain of CD4 to the cell membrane via a glycophospholipid linkage. The glycophospholipid-anchored CD4 had a molecular size of approximately 56 to 62 kDa and was released following treatment of the cells with phosphatidylinositol-specific phospholipase C. HeLa cells expressing the CD4-DAF hybrid could be infected with HIV-1, as evidenced by reverse transcriptase activity, p24 core antigen content, and infectious virus production. In addition, transfection of the HeLa CD4-DAF cells with a plasmid that directs the synthesis of HIV-1 envelope glycoproteins or cocultivation with HeLa cells expressing the virus glycoproteins resulted in syncytium formation. These results indicate that the transmembrane and cytoplasmic domains of the CD4 molecule are dispensable for both HIV infection and syncytium formation.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Regulation of chicken alpha and beta actin genes and their hybrids inserted into myogenic mouse cells.

We have investigated the regulation of intact non-muscle (beta) and muscle-specific (skeletal alpha) chicken actin genes and of hybrids of these two genes (alpha 5'-beta 3' and beta 5'-alpha 3') transferred into the mouse myogenic non-fusing cell line BC3H1. BC3H1 cells express members of the actin multigene family in a differentiation-dependent manner. When proliferating, the cells accumulate large amounts of non-muscle actin mRNA; when the cells are induced to differentiate, the amount of non-muscle actin mRNA decreases and the amount of muscle-specific actin mRNA increases. The transferred beta-actin gene is efficiently expressed in undifferentiated cells and appropriately down-regulated upon differentiation. In contrast, the transferred alpha-actin gene is inefficiently expressed and not consistently up-regulated. Results with the intact and hybrid genes, taken together, are consistent with the hypothesis that both 5' and 3' halves of these genes contain sequences important in regulating the efficiency and/or developmental timing of their expression in BC3H1 cells. By nuclear run-on experiments we found no evidence for gene-specific changes in the rate of transcription of the transferred actin genes during myogenesis. We conclude that the differentiation-dependent changes in expression of the intact beta-actin gene in BC3H1 cells must be regulated at the post-transcriptional level.

Actins↗
PubMed1989

Biological evaluation of glycoproteins mapping to two distinct mRNAs within the BamHI fragment 7 of pseudorabies virus: expression of the coding regions by vaccinia virus.

Several glycoproteins from the unique short region of pseudorabies have been identified and characterized. The genes encoding at least four glycoproteins (gp50, gp63, gl, and gX) are located within the BamHI fragment 7 of pseudorabies. S1 nuclease mapping was used to determine that a 2.4-kb mRNA encompasses the coding region for gp50 and gp63 and probably represents a colinear transcript for these proteins. Using the same technique, a 2.8-kb mRNA was found to encode gl. No other mRNAs were found to be encoded on the opposite strand of DNA in this region. Various recombinant vaccinia vectors were made incorporating the coding regions for these two mRNAs. Pseudorabies recombinant vaccinia infected ST cells expressed glycoproteins that co-migrated with the authentic PRV glycoproteins upon polyacrylamide electrophoresis. Intracranial or intraperitoneal inoculation of mice with the recombinant viruses constructed to contain the mRNA coding regions resulted in various degrees of protection from a lethal challenge of pseudorabies virus.

Animals↗
PubMed1988

Molecular cloning of biologically active proviruses of bovine immunodeficiency-like virus.

A series of independent proviral molecular clones of bovine immunodeficiency-like virus (BIV) obtained from a genomic library of BIV-infected bovine cell DNA were physically and biologically characterized. Heteroduplex mapping shows that two of these BIV clones (106 and 127) contain uninterrupted proviral sequences approximately 9.0 kb in length, flanked by nonhomologous bovine cellular sequences. Microinjection of purified DNA from BIV clone 106 or 127 into susceptible bovine cells produces virus-specific cytopathic effects, including syncytium induction, supernatant reverse transcriptase activity, and infectious virus particle formation, similar to the effects produced by parental virus stock. Using restriction enzyme mapping, it was determined that the two infectious clones share 13 of 14 sites mapped within the provirus; thus, based on this criterion, the two clones are nearly identical, with the exception of a single polymorphic site recognized in the 3' half of the genome. BIV appears to be an exogenous pathogenic virus, because Southern hybridization analyses detected no endogenous sequences related to BIV in DNA from a variety of uninfected bovine cells and tissues. Most of the BIV-related DNA found in cells 96 hr after infection is present as linear unintegrated viral DNA, although the presence of host flanking sequences in our proviral clones indicates that integration takes place. These biologically active clones of BIV will be of use in defining further the mechanisms of BIV pathogenesis and in engineering specific diagnostic reagents to determine the prevalence of BIV in cattle populations.

Animals↗
PubMed1983

The nucleotide sequence of the chick cytoplasmic beta-actin gene.

The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.

Actins↗
PubMed1982

The myeloproliferative sarcoma virus causes transformation or erythroid progenitor cells in vitro.

The myeloproliferative sarcoma virus induces spleen focus formation in vivo and transforms fibroblasts in vitro. We showed in this study that in vitro infection of spleen or bone marrow cells from susceptible mice with the myeloproliferative sarcoma virus leads to the formation of erythroid bursts. Under optimal conditions erythroid bursts formed in the absence of added erythropoietin, but the addition of as little as 0.05 U of erythropoietin per ml to infected cultures resulted in a significant increase in numbers of erythroid bursts and the proportion of hemoglobinized cells. A comparison of the kinetics of burst formation and the size of the induced bursts with those induced with Friend virus suggested that either sarcoma virus such as the myeloproliferative sarcoma virus or the target cells for the two viruses were not the same. Density characterization and heat lability studies indicated that the increased erythroid proliferation in vitro was a virus-induced event, but the possibility that the induced erythroid burst formation is mediated via interaction with a nonerythroid target cell and subsequent release of a soluble factor cannot be ruled out.

Animals↗
PubMed1980

Combined effect of friend polycythemia virus and erythropoietin on erythroid burst formation in vitro.

The infection of mouse bone marrow or spleen cells in vitro with the polycythemia strain of Friend virus (FVP) results in the formation of erythroid bursts after 4-6 days in plasma clot or methylcellulose cultures in the absence of added erythropoietin (Ep). We have now used this cell culture system to study the effect of added Ep on FVP-induced burst formation. The number of bursts that developed following infection of bone marrow cells with equilibrium density gradient purified FVP was increased by the addition of low concentrations of highly purified human urinary Ep (70,000 units/mg protein) to the infected cultures. The addition of as little as 0.001 unit per ml of Ep to infected cultures resulted in a 2-fold increase in burst formation. Increasing the concentration of Ep to 0.05 unit per ml caused a 4-fold increase in the number of bursts that developed. These concentrations of Ep were ineffective in inducing erythroid bursts when added to cultures of uninfected cells. The addition of Ep within 6 hr after FVP infection was necessary to observe increased burst formation. These experiments demonstrate that FVP-burst formation is enhanced by extremely low levels of Ep. Although a precise mechanism for the increase in FVP-induced erythroid bursts with added Ep is not apparent from these experiments, a number of possibilities are discussed.

Animals↗