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B E Ledford

Publications and source records attributed to B E Ledford.

15 recordsLinked to original sources
PubMed1990

Reversible ADP-ribosylation of the 78 kDa glucose-regulated protein.

Starvation of Mouse hepatoma cells for essential amino acids or glucose results in the mono-ADP-ribosylation of the 78 kDa glucose-regulated protein, GRP78. Here we show that the ADP-ribosylated and non-ADP-ribosylated forms of GRP78 are interconvertible during tryptophan starvation and refeeding. In addition, the ADP-ribosylation of GRP78 was shown to be reversible even during nutritional stress. The overexpressed pool of non-ADP-ribosylated GRP78 synthesized during tunicamycin treatment was available for ADP-ribosylation during subsequent amino acid starvation, especially in the absence of tunicamycin. The reversible ADP-ribosylation of GRP78 could be part of a metabolic control mechanism in operation during nutritional stress.

Adenosine Diphosphate Ribose↗
PubMed1989

ADP-ribosylation of the 78-kDa glucose-regulated protein during nutritional stress.

Starvation of a mouse hepatoma cell line, Hepa, for any essential amino acid results in the mono-ADP-ribosylation of an 80-kDa protein, P80. The ADP-ribose acceptor and its putative precursor were identified in two-dimensional gel patterns and isolated by electroelution. Amino-terminal sequence analysis showed they were the 78-kDa glucose-regulated protein, GRP78. Starvation of Hepa cells for tryptophan or glucose stimulated the relative rate of synthesis, and the ADP-ribosylation of GRP78. Inhibition of N-linked glycosylation by treatment with tunicamycin, 2-deoxy-D-glucose or glucosamine stimulated the synthesis of non-ADP-ribosylated GRP78 up to sixfold with relatively little effect on its ADP-ribosylation. Both forms were identified in mouse liver, lung, heart, kidney, spleen and brain.

ADP Ribose Transferases↗
PubMed1988

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.

Animals↗
PubMed1979

Polyribosome size analysis. Measurement of number-average polyribosome sizes.

The analysis of translational efficiencies of specific mRNAs requires a determination of the polyribosome size. The appropriate value to use in such calculations is the number-average size. A method is described for accurately measuring the number-average size of total and of specific protein synthesizing polyribosomes using isokinetic sucrose density gradients and 125I-labeled antibodies. By this method, we demonstrated that albumin synthesizing polyribosomes from a serum albumin secreting mouse hepatoma cell line exist over a broad range from trimers to 20-mers (mean 6-10). The specificity of antibody interaction with polyribosomes was demonstrated using cells not synthesizing mouse serum albumin, and by demonstrating that 125I-anti ovalbumin does not bind to mouse hepatoma polyribosomes. Treatment of the mouse hepatoma cells with 1 MUM cycloheximide shifted practically all of the monomers into polyribosomes resulting in an increase in the number-average size of the albumin synthesizing polyribosomes. Cycloheximide treatment, however, did not eliminate the size heterogeneity in the albumin synthesizing polyribosomes.

Albumins↗
PubMed1979

Uterine prostaglandin levels following stimulation of the decidual cell reaction: effects of indomethacin and tranylcypromine.

Prostaglandin E (PGE) and F (PGF) levels were measured in mouse uteri at various times after either trauma (hemostat crusing) or oil stimulation of the decidual cell reaction (DCR). The oil induced DCR led to an early increase (within 5 min) in both PGE and PGF levels. Both returned to baseline by 1 h after stimulation. A second peak in PGF levels was observed at 120 min after oil stimulation. This study demonstrates a distinct difference between the pattern of PGE and PGF changes in the uterus following oil stimulation of the DCR. Indomethacin pretreatment completely blocked the oil stimulated DCR as well as all prostaglandin increases following either stimulus. The trauma stimulated DCR was not completely blocked by indomethacin pretreatment. Pretreatment with tranylcypromine, an inhibitor of prostacyclin biosynthesis, did not block the prostaglandin E and F increases, but did block the oil stimulated DCR. These findings suggest that prostacyclin may be an early mediator of the DCR.

Animals↗
PubMed1979

Chick-comb fibroblasts. A new strain of cultured cells sensitive to androgens.

An androgen-responsive cell strain of chick-comb fibroblasts has been established in culture. Cells were obtained from combs of 1--2 day old white Leghorn cockerels and cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 6 g/l glucose. The cells required serum but not androgens for growth. In the presence of androgens, growth rate and incorporation of labeled precursors into protein and RNA were markedly increased in exponentially growing cells, but not in confluent cultures. Androgens exerted these stimulatory effects in a dose-dependent fashion. It is concluded that chick-comb fibroblasts, which respond to androgens in vivo, retain this responsiveness when cultured. They furnish a well-defined system of potential value for studies of the molecular mechanisms of androgen action.

Animals↗
PubMed1978

Changes in RNA polymerase activity in isolated mouse uterine nuclei during the decidual cell reaction.

The artificially stimulated decidual cell reaction has been used as a model to study changes occurring in the uterus at the time of implantation. Activities of RNA polymerases I, II and III were measured in uterine nuclei isolated from ovariectomized non-primed mice, hormonally primed mice, and hormonally primed mice following stimulation of the decidual cell reaction. Activities of all three RNA polymerases increased following hormonal priming of ovariectomized mice. In nuclei from stimulated uterine horns, activities of RNA polymerases I and III increased 9 h after stimulation of the decidual cell reaction and remained elevated through 21 h. RNA polymerase II activity did not change following stimulation of the decidual cell reaction. These changes in RNA polymerase activities occur at the time of increased histone modifications and may result from changes in the template capacity.

Animals↗
PubMed1978

Ayalysis of translational parameters in cultured cells.

A method is described for the measurement of protein synthesis parameters in cultured cells. Ribosome transit times, polyribosome size distribution and relative synthetic rates are measured on individual cultures of cells. This method is applied to an analysis of cycloheximide (1 micrometer) inhibition of protein synthesis in cultured mouse hepatoma cells.

Cells, Cultured↗
PubMed1977

Albumin synthesis in cultured hempatoma cells. Regulation by essential amino acids.

The effects of essential amino acids on albumin synthesis by a mouse hepatoma cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates of [3H]leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of tryptophan producing half-maximal synthesis is 4 micronM. The corresponding concentration for leucine is 100 micronM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15 micronM. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels of these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.

Albumins↗
PubMed1977

The influence of antisera specific for alpha-fetoprotein and mouse serum albumin on the viability and protein synthesis of cultured mouse hepatoma cells.

The cytotoxic potential of heterologous rabbit antibody directed against mouse serum albumin (MSA) and alpha-fetoprotein (AFP) was investigated in vitro with a cell line (Hepa) derived from the mouse hepatoma BW7756. Anti-AFP in the presence of complement could kill Hepa cells at concentrations of anti-MSA that were virtually nontoxic. The specificity of the anti-AFP was defined by demonstrating that Hepa cell toxicity was dependent upon and paralleled the secretion of AFP in synchronized cultures. Furthermore, neither antiserum could be shown to be significantly toxic to mouse neuroblastoma cells (Neuro-2A). Immunoglobulin purified from pools of antisera was also highly effective in producing cytotoxicity even in a complement-free system. This reaction proceeded more slowly, requiring nearly 48 hr to reach maximum effect in comparison to the 12 hr for complement-mediated toxicity. MSA and AFP are secreted during different phases of the cell cycle. In cultures arrested by isoleucine starvation, labeled AFP appears in the medium 10 hr after release of the blockade in association with S phase. The appearance of labeled MSA is delayed until the first mitosis. Cytotoxic effects of anti-AFP parallel the secretion of AFP in synchronous cultures. Both antisera could be inhibitory to the secretion and synthesis of the proteins of their antigenic specificity. MSA synthesis was more susceptible to this inhibition than was AFP synthesis. The significance of this phenomenon and its association with the differential cytotoxicity of the antiserum are discussed.

Antibodies↗