Catfish Oct2 binding affinity and functional preference for octamer motifs, and interaction with OBF-1.

The DNA-binding (POU) domain of the catfish Oct2 transcription factor was shown, by electromobility shift assays and surface plasmon resonance techniques, to have an affinity for the consensus octamer motif (ATGCAAAT) that was slightly higher than its affinity for a variant motif (ATGtAAAT). This observation is consistent with the transcriptional activation potentials of catfish Oct2 alpha and Oct2 beta, which were shown to activate transcription in catfish B and T cell lines to an equivalent extent from both the consensus and variant octamer motifs. When tested in a mouse plasmacytoma cell line, catfish Oct2 alpha and Oct2 beta, as well as mouse Oct2, showed higher transcriptional activation with the variant, as compared to the consensus, octamer motif. Catfish Oct2 was shown to function synergistically with the mammalian co-activator, OBF-1, activating octamer-dependent transcription in catfish T cells. The strong transcriptional activity of OBF-1 in catfish cells was dependent on the presence of octamer motif(s) at the proximal (promoter) rather than the distal (enhancer) position.

ADP-ribosylation of the molecular chaperone GRP78/BiP.

Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway.

Expression of a mouse-channel catfish chimeric IgM molecule in a mouse myeloma cell.

Fusion genes encoding a murine VH domain and the constant region domains of the mu chain from the channel catfish, Ictalurus punctatus, were stably expressed in the lambda light chain producing mouse myeloma cell line J558L. Although the pathways of pre-mRNA processing for expression of membrane (micron and secreted (microsecond) forms of the mu chain differ between mammals and teleosts, mRNAs encoding both catfish micron and microsecond were correctly expressed in the mouse myeloma cells. The mouse-channel catfish chimeric mu chain polypeptide was able to associate covalently with the mouse lambda light chain and assemble, intracellularly, into polymers of covalent structure (microL)2-8 which resembled those seen with native catfish IgM. In contrast to native catfish IgM, the mouse-catfish chimeric IgM showed the property of binding strongly to protein A of Staphylococcus aureus. The mouse-channel catfish chimeric IgM was core-glycosylated, but did not contain terminal sialic acid. Secretion rates for the chimeric IgM were low, and the possibility could not be excluded that extracellular chimeric IgM was released from dead or dying cells. The reason(s) for the intracellular retention of the chimeric IgM (probably in the endoplasmic reticulum) are not known, but those mechanisms involving retention via cysteine residues were excluded.

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.

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.

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.

Translational control of ADP-ribosylation in eucaryotic cells.

Starvation of the mouse hepatoma cell line Hepa for an essential amino acid (Trp, His, Leu, Ile or Phe) stimulated the incorporation of [3H]adenosine as ADP-ribose monomer into an 80,000-Mr protein, P80. Two-dimensional electrophoresis of Hepa proteins showed that P80 was the only protein labeled under starvation conditions. Time course experiments showed that the ADP-ribosylation of P80 was a consequence rather than the cause of reduced translational activity. Cycloheximide treatment and incubation at reduced temperatures also reduced the rate of protein synthesis and stimulated the ADP-ribosylation of P80. Starvation-dependent ADP-ribosylation of P80 was shown to occur in three other cell lines (Chang, Neuro-2a, and chick comb fibroblasts).

Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids.

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid-precipitated from postribosomal supernatants of detergents-solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min-1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.

Purification of transferrin and albumin from mouse ascites fluid.

Mouse ascites fluid, which is readily obtained when cell lines and hybridomas are maintained in host mice, is a convenient source of several plasma proteins. This paper describes procedures for the purification of albumin and transferrin from mouse ascites fluid. Mouse transferrin was prepared from a 50-75% ammonium sulfate fraction of mouse ascites fluid by CM- and DEAE-cellulose chromatography. Mouse albumin was obtained by the same purification route, but required an additional chromatography step on Cibacron Blue F3GA-agarose. Both proteins were shown to be homogeneous by polyacrylamide gel electrophoresis and immunoelectrophoresis. Characterization, which included a determination of amino acid composition, partial N-terminal sequence, molecular weight and extinction coefficient, correlated well with known values reported for human transferrin and albumin. The purified mouse proteins may be useful for biochemical studies, antibody preparation, and as growth factors for hybridomas or other mouse cell lines maintained in culture.

Muscle protein synthesis: regulation of a translational inhibitor.

Insulin and branched-chain amino acids are known to stimulate protein synthesis in skeletal muscle. Extracts prepared from rat diaphragms after incubation in balanced salt solution and glucose alone yielded heat- and acid-stable, TCA-precipitable, nondialyzable factor(s) that inhibit protein synthesis when added to rabbit reticulocyte lysates. Polyribosomal profiles of inhibited lysates were consistent with a defect in peptide-chain initiation. Addition of insulin and amino acids to the diaphragm incubation media partially removed the inhibition seen with the muscle extract and was accompanied by an increase in polysomes and decreased subunits. Similarly, extracts prepared from rat hindlimb muscle 48 h after induction of diabetes were much more inhibitory in rabbit reticulocyte lysates than extracts from control rats. Polyribosomal profiles were consistent with defective peptide-chain initiation. Trypsin treatment before assay abolished the inhibitory activity of muscle extracts from diabetic rats. Because translation-inhibiting peptide(s) appear to be under metabolic and/or hormonal control, their possible role in muscle protein homeostasis warrants further study.

Evidence for the existence of a diverse family of immunoglobulin-related recognition molecules.

All classes of molecule postulated to play a role in recognition or to have some structural homology to immunoglobulins were compared by the S delta Q index of differences in amino acid composition. The results suggest that there is a wide family of immunoglobulin-related molecules that includes vertebrate immunoglobulins, T cell receptors, MHC products, C-reactive proteins, and (some) invertebrate lectins.

Cell cycle patterns of thymidylate synthetase and 5,10-methylenetetrahydrofolate polyglutamates in cultured mouse hepatoma cells.

The regulation of thymidylate synthetase activity was investigated throughout the first cell cycle after release from an isoleucine block in synchronous cultures of mouse hepatoma (Hepa) cells. Activity in cell extracts increased with the onset of S phase and the increased activity was attributed to a parallel increase in enzyme concentration as determined by titration with tritiated fluorodeoxyuridylate. The polyglutamate chain length of reduced folate cofactors, which could also influence thymidylate synthetase activity, was unchanged.

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.

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.