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P Hsieh

Publications and source records attributed to P Hsieh.

At least 55 records · Page 3Linked to original sources
PubMed1986

Folylpolyglutamate synthetase inhibition and cytotoxic effects of methotrexate analogs containing 2,omega-diaminoalkanoic acids.

The properties of a series of methotrexate analogs containing 2,omega-diaminoalkanoic acids have been investigated. The compounds were potent inhibitors of dihydrofolate reductase but, unlike methotrexate, they were also inhibitors of mammalian folylpolyglutamate synthetases. The potency of synthetase and reductase inhibition increased with increasing length of the 2,omega-diaminoalkanoate moiety. The most cytotoxic compound and the most potent inhibitor of both dihydrofolate reductase (I50 = 2.5 to 4 nM) and folylpolyglutamate synthetase (Ki ca. 4 microM) contained 2,5-diaminopentanoic acid (ornithine). These compounds were 70- to 100-fold less cytotoxic than methotrexate to human leukemia cell lines; however, they retained their potency against sublines resistant to methotrexate via defective transport. Their dual loci of enzyme inhibition and their efficacy against methotrexate transport-defective cell lines indicate that these compounds may be an important new class of antifol.

Aldehyde Oxidase↗
PubMed1986

Partial purification and characterization of a recombinase from human cells.

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.

Adenosine Triphosphate↗
PubMed1986

Effects of corneal extracts on rabbit corneal stromal cells in culture.

Rabbit corneal tissues were treated sequentially with phosphate buffered saline (PBS) and 4 M guanidine hydrochloride (GuHCl), dialyzed, and lyophilized. The interaction of the individual PBS and the GuHCl extracts with cultured rabbit corneal stromal cells was assessed. The PBS extract stimulated stromal cell growth. These cells had a thinner spindle-shaped appearance, a greater tendency toward multilayer formation, and a approximately 40-60% higher final density than the controls. The cells subjected to the GuHCl extract exhibited no such changes. When the PBS extract was heated to 80 degrees C, the stimulatory activity was replaced by an inhibitory activity, indicating that the PBS extract contained both the stimulatory and the inhibitory factors. Using a high performance liquid chromatograph system, such factors could be separated. The effects of corneal extracts on connective tissue synthesis were examined after labeling confluent stromal cultures with either (35S)sulfate or (3H)proline for 20 hr. The PBS and the GuHCl extracts significantly promoted the incorporation of (35S)sulfate into glycosaminoglycans. Neither extract altered the types of glycosaminoglycans synthesized or the collagen synthesis of stromal keratocytes in culture.

Animals↗
PubMed1985

Role of methotrexate polyglutamates in methotrexate- and sequential methotrexate-5-fluorouracil-mediated cell kill.

The synthesis of methotrexate (MTX) polyglutamates has been investigated in a human leukemia cell line, CCRF-CEM. An increase in either the extracellular MTX concentration (0.1-10 microM) or exposure time (4-24 h) allowed a greater accumulation of polyglutamate derivatives. The species which predominated (di-, tri-, or tetraglutamate) was also time and concentration dependent. Derivatives up to the pentaglutamate were readily detectable, with hexaglutamate present under some conditions. Polyglutamate derivatives were preferentially retained when the cells were transferred to drug-free medium. MTX itself rapidly exited the cells under these conditions. In the absence of extracellular MTX, the performed intracellular MTX polyglutamates continued to be elongated. Accumulation of MTX polyglutamates, particularly triglutamate and longer species, positively correlated with cytotoxicity to CCRF-CEM cells as measured by a clonogenic assay. Investigation of the methotrexate-fluorouracil interaction revealed no effect of 5-fluorouracil on MTX polyglutamate accumulation under various conditions where widely different degrees of cytotoxicity were found. The synergy observed when MTX precedes fluorouracil treatment is therefore not an effect on MTX polyglutamylation. MTX polyglutamates may, however, play an indirect role in this synergy.

Cell Survival↗
PubMed1985

Effects of fibroblastic and endothelial extracellular matrices on corneal endothelial cells.

Extracellular matrices (ECM) isolated from chick embryo fibroblast and human and rabbit corneal stromal cells induce polarization and elongation of corneal endothelial cells in culture. ECM isolated from rabbit corneal or bovine aortic endothelial cells neither polarize nor elongate corneal endothelial cells in culture. By indirect immunofluorescence, fibronectin is seen as arrays of long fibers in fibroblastic ECM, whereas in endothelial ECM, fibronectin is found in discreet foci as short fibers. The morphology corneal endothelial cells in culture is associated with the structure of the ECM laid down; short fibers in clusters associated with a typical polygonal shape, long polarized fibers inducing a fibroblastic-like appearance. ECM isolated from both fibroblastic or endothelial sources promote cell growth. Since coating the plastic with plasma fibronectin or denatured collagen does not promote endothelial cell growth, the authors assume the polymerized fibronectin network or a component bound to this network enhances the growth of corneal endothelial cells cultured in the presence of serum.

Animals↗
PubMed1984

Enzymatic synthesis of polyglutamate derivatives of 7-hydroxymethotrexate.

7-Hydroxymethotrexate, an important metabolite of methotrexate, is a substrate for folylpolyglutamate synthetase (FPGS) isolated from rat liver and several human leukemia cell lines. The substrate activity it displays over a wide range of concentrations (0-200 microM) is nearly equivalent to that of methotrexate. The 7-hydroxy derivative of dichloromethotrexate is also a substrate for FPGS. The pattern of polyglutamate products synthesized by rat liver FPGS was nearly identical with both 7-hydroxymethotrexate and methotrexate. In addition, conversion of MTX polyglutamates to the corresponding 7-hydroxy compounds was demonstrated using partially purified rabbit liver aldehyde oxidase. The rate of conversion was concentration dependent, and the relative rate decreased as the MTX polyglutamate chain length increased. We propose that 7-hydroxymethotrexate polyglutamates may be formed by initial hydroxylation of methotrexate and subsequent polyglutamate formation or by direct hydroxylation of methotrexate polyglutamates. It was further shown that the relative substrate activity of folate analogs for folylpolyglutamate synthetase is dependent on the source of the enzyme.

Aldehyde Oxidase↗
PubMed1984

Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells.

We have examined the synthesis and processing of asparagine-linked oligosaccharides from Aedes albopictus C6/36 mosquito cells. These cells synthesized a glucose-containing lipid-linked oligosaccharide with properties identical to that of Glc3Man9GlcNAc2-PP-dolichol. Results of brief pulse label experiments with [3H]mannose were consistent with the transfer of Glc3Man9GlcNAc2 to protein followed by the rapid removal of glucose residues. Pulse-chase experiments established that further processing of oligosaccharides in C6/36 cells resulted in the removal of up to six alpha-linked mannose residues yielding Man3GlcNAc2 whose structure is identical to that of the trimannosyl "core" of N-linked oligosaccharides of vertebrate cells and yeast. Complex-type oligosaccharides were not observed in C6/36 cells. When Sindbis virus was grown in mosquito cells, Man3GlcNAc2 glycans were preferentially located at the two glycosylation sites which were previously shown to have complex glycans in virus grown in vertebrate cells. These Man3GlcNAc2 structures are the most extensively processed oligosaccharides in A. albopictus, and as such, are analogous to the complex glycans of vertebrate cells. We suggest that determinants of oligosaccharide processing which reside in the polypeptide are universally recognized despite evolutionary divergence of the oligosaccharide-processing pathway between insects and vertebrates.

Aedes↗
PubMed1983

Host-dependent variation of asparagine-linked oligosaccharides at individual glycosylation sites of Sindbis virus glycoproteins.

We examined the Asn-linked oligosaccharides at individual glycosylation sites of the two envelope glycoproteins of Sindbis virus, E1 and E2. The analysis was done by separating tryptic glycopeptides by reverse phase high performance liquid chromatography and analyzing the oligosaccharides from isolated glycopeptides by gel filtration chromatography. Both E1 and E2 have two glycosylation sites each in virus grown in chick embryo fibroblasts, baby hamster kidney cells, and Chinese hamster ovary cells. Our results indicate that previously observed host-dependent variation in the oligosaccharides of Sindbis virus glycoproteins are probably attributable to differences in oligosaccharide composition at individual sites of E1 and E2 and not to differences in the frequency of glycosylation of these sites. One of the two glycosylation sites in E1 has exclusively complex type oligosaccharides regardless of the host cell type. However, the second glycosylation site in E1 has high mannose oligosaccharides in virus grown in chick embryo fibroblasts, complex oligosaccharides in virus from baby hamster kidney cells, and both complex and high mannose oligosaccharides in virus from Chinese hamster ovary cells. E2 has one glycosylation site which has primarily high mannose oligosaccharides and one site which has primarily complex type oligosaccharides. Certain sites which have predominantly complex type glycans also have a small number of two specific classes of oligosaccharides which are cleaved by endo-beta-N-acetylglucosaminidase H and may be high mannose type. Our findings are discussed with regard to regulation of oligosaccharide processing in animal cells.

Animals↗
PubMed1983

In vitro methotrexate polyglutamate synthesis by rat liver folylpolyglutamate synthetase and inhibition by bromosulfophthalein.

We have investigated the properties of the rat liver folylpolyglutamate synthetase using methotrexate (MTX; 4-NH2-10-CH3-PteGlu) as a substrate. Many characteristics of the synthetase (e.g., the apparent Km values for L-glutamate and ATP, and the optimal concentrations of KC1 and 2-mercaptoethanol) are virtually identical whether MTX or tetrahydrofolate is the "folate" substrate. There are, however, several significant differences between the reactions catalyzed with these two substrates. The length of products synthesized from tetrahydrofolate are inversely related to the initial monoglutamate concentration. Low tetrahydrofolate concentrations allow synthesis of longer (n greater than or equal to 3) polyglutamates, up to pentaglutamate length, while high concentrations lead to predominantly diglutamate synthesis. However, 4-NH2-10-CH3-PteGlu2 predominates regardless of the initial MTX concentration, under otherwise identical conditions. Also, tetrahydrofolate can be readily converted to pentaglutamate lengths, the same as predominates in rat liver in vivo. In contrast, MTX forms species containing only up to a total of three glutamates, i.e., 4-NH2-10-CH3-PteGlu3. Finally, the ultimate product of synthesis from tetrahydrofolate, H4PteGlu5, is a fairly good inhibitor of synthetase activity with either MTX or tetrahydrofolate as the substrate. The ultimate product of MTX synthesis, 4-NH2-10-CH3-PteGlu3, however, is a poor inhibitor of activity with either substrate. We have also investigated the inhibitory effect of bromsulfophthalein (BSP) on the rat liver synthetase. Gewirtz et al., showed that this organic anion inhibited the uptake of MTX and 5-CH3-tetrahydrofolate into hepatocytes, and presented indirect evidence that BSP effected polyglutamate biosynthesis. We have demonstrated that BSP is a potent (Ki = 1 microM) inhibitor of the rat liver synthetase. Inhibition is noncompetitive with rspect to L-glutamate, ATP, and MTX. Pre-incubation and time course experiments demonstrated that inhibition is not stoichiometric and is not caused by slow inactivation of the synthetase. Since BSP is transported into mammalian cells, it is the first inhibitor of polyglutamate biosynthesis which has potential for use in in vivo studies.

Animals↗
PubMed1983

Behavior of cells seeded in isolated fibronectin matrices.

Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.

Animals↗
PubMed1982

Cell types associated with fibronectin in long-term mouse bone marrow cultures.

The formation of fibronectin matrix was studied in long-term mouse bone marrow cultures. Stromal and hematopoietic cells were observed in situ under phase contrast optics and quantified according to their staining characteristics on smear preparations. Surface fibronectin was demonstrated by indirect immunofluorescence. While only stromal and no hematopoietic cells participated, various stromal cell types differed in their expression of cell surface fibronectin: Reticulum cells contributed the major portion of fibronectin matrix. Elongated, meshwork-forming histiocytes expressed some surface fibronectin, while the flattened, macrophagic histiocytes remained fibronectin negative. These findings were recapitulated during regeneration of scrape wounds in the adherent layers. Isolated fibronectin matrix did not support hematopoietic cell adherence or maintenance, although it had marked effects on stromal cells.

Adipose Tissue↗
PubMed1980

Enzymatic synthesis of folylpolyglutamates. Characterization of the reaction and its products.

Rat liver folylpolyglutamate synthetase was partially purified and its reaction and products were characterized. The preparation contained no conjugase activity. Gel filtration analysis revealed a molecular weight of 69,000. The synthetase was optimally active at pH 8.4 (37 degrees C), required mercaptoethanol and a monovalent cation, and was highly specific for L-glutamate. Only purine nucleoside triphosphates served as the energy source for the reaction; ATP and dATP gave the best activity. All naturally occurring folates (including 5-methyl-tetrahydrofolic acid) as well as a number of folate analogs (including methotrexate) served as substrates. The unnatural diastereoisomer of at least one folate, 5,6,7,8-tetrahydrofolic acid, was also a substrate. Modifications of the terminal, acceptor glutamate led to loss of substrate activity, as well as loss of binding. High pressure liquid chromatography analysis, conjugase digestion, double radiolabel studies, and amino acid analysis of acid-hydrolyzed product confirmed that folylpoly-gamma-glutamates were synthesized. High concentrations of (dl)-5,6,7,8-tetrahydrofolic acid favored accumulation of short chain (predominantly diglutamate) products while low concentrations favored accumulation of longer chains (predominantly tetraglutamate). This inverse relationship between concentration and chain length may be of regulatory significance. Synthesis of pentaglutamate forms, the predominant chain length of rat liver folates in vivo, was detected at low (dl)-5,6,7,8-tetrahydrofolic acid, but hexaglutamate was not detected. Synthetic (l)-5,6,7,8-tetrahydropteroylpentaglutamate was a poor substrate for the synthetase but it inhibited formation of polyglutamates from monoglutamates. These observations indicate that the predominant chain length of folates in rat liver may be determined solely by the substrate specificity of the rat liver synthetase. Inhibition by the specificity of the rat liver synthetase. Inhibition by the pentaglutamate derivative offers a means by which folylpolyglutamates could regulate their own synthesis.

Adenosine Triphosphate↗
PubMed1980

Effect of [3H]methotrexate impurities on apparent transport of methotrexate by a sensitive and resistant L1210 cell line.

Transport of methotrexate by L1210 sensitive and resistant cell lines was studied using [3H]methotrexate and methotrexate. The intracellular and cellular efflux of drug was analyzed by radioligand binding assay and high performance liquid chromatography. It was shown that the initial, rapid uptake of [3H]methotrexate was not methotrexate but rather [3H]p-aminobenzoylglutamate, even when the [3H]methotrexate was greater than 97% homogeneous. In the mutant cell line, the impurity could account for all of the apparent methotrexate uptake at 1 to 10 micro M extracellular drug. Similarly, the rapid efflux of [3H]methotrexate was also shown to be all, or in part, 3H-impurities, in both the mutant and sensitive cell lines. These results do not conflict with the currently accepted model of a carrier-mediated process for reduced folate and antifolate transport but do suggest that the quantitative interpretation of the early time points of transport experiments be more critically evaluated, especially when mutant cell lines are being analyzed.

4-Aminobenzoic Acid↗
PubMed1980

Antisera inhibiting mammalian cell spreading and possible cell surface antigens involved.

Antiserum against a rat neuronal tumor cell line (B103) has been prepared in rabbit by intravenous injection of live cells. This immune serum (anti-B103) precipitates a few cell surface proteins recognizable by two-dimensional gel electrophoresis as common radioiodinatable spots in 15 different rat neural cell lines and in mouse and rat fibroblast cell lines. The apparent molecular weight of one major common protein (II4) is estimated by SDS gel electrophoresis to be somewhere between 80,000 and 90,000 and another protein (I3) to be 120,000. These two proteins are consistently recognized in various cell lines by this antiserum. Furthermore, at a 1:20 dilution, this serum causes monolayer cells to round up usually within 0.5 h and detach from the plate within 3 h. It also inhibits spreading of freshly plated cells. These effects of the antiserum are reversible. Upon absorption of the antiserum with cells (e.g., absorbed with a glial cell line, B27), the serum no longer causes the rounding up of the monolayer cells, it does not inhibit cell spreading, and it does not immune-precipitate the two common proteins from the cell surface of various cell lines. Antisera against several other rat cell lines also precipitate the same common proteins (II4 and I3) from the cell surface and prevent cell spreading. These data suggest that the antibody acts first at the cell surface and then inhibits cell spreading or rounding up of spread cells. The consistent pattern of the immunoprecipitated cell surface proteins on the two-dimensional gel electrophoresis makes these two common surface proteins (II4 or I3 or both) possible candidates for target proteins to which the antibody binds. Thus, they may play a critical role in cell spreading.

Animals↗
PubMed1980

Studies of fibronectin matrices in living cells with fluoresceinated gelatin.

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC-gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC-gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC-gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.

Animals↗
PubMed1979

Possible role of fibronectin in malignancy.

Frozen sections of tumors induced by injecting virally transformed cells into animals were stained for fibronectin by immunofluorescence. Many tumor cell lines do not express fibronectin in tumors in situ even though some of them express fibronectin in culture. Cell shape and hormones appear to influence the expression of fibronectin in culture; however, it is unclear how fibronectin expression is regulated in vivo.

Animals↗