A convenient procedure for purification of thymidylate synthase from L1210 cells

Several laboratories have described procedures for purification of thymidylate synthase (TMP synthase) that utilize folates or folate analogs covalently attached to a matrix. The principle of separation is the formation of a ternary complex between dUMP, TMP synthase, and the bound ligand and the subsequent elution of the enzyme with buffers that do not contain dUMP. We have successfully used 10-formylfolic acid as the bound ligand for the purification of TMP synthase. As compared to other ligands that have been used, 10-formylfolic acid has the advantages that it can be easily synthesized, it is stable, and the enzyme is eluted as a sharp peak. Application of this procedure to L1210 leukemia cells gave 1765-fold purification of TMP synthase with a recovery of 39%. The native enzyme had a molecular weight of 78,000, which is about the same as that reported.     

Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.     

Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.     

Changes in the insulin and glucagon receptors in the regenerating liver following intoxication with carbon tetrachloride.

The response of rat liver plasma membrane adenylate cyclase was studied from one to 14 days after a single dose of carbon tetrachloride (CCl₄). The response to glucagon was diminished to a greater extent than that of fluoride and was due to a deficiency in hormone binding. In contrast, insulin binding increased 300% over control; the change was due to increased number of binding sites. The “affinities” of receptors for either hormone were not altered. The tissue levels of adenosine 3′:5′ -monophosphate increased following CCl₄ poisoning reaching a peak precisely when the adenylate cyclase response to glucagon was at its lowest level. These studies present evidence that receptors for pancreatic hormones change differently when liver is damaged and during its regeneration following CCl₄ intoxication. The change in pattern is remarkably similar to changes reported previously in fetal liver development or following partial hepatectomy of adult rat.     

The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells.

The ability of cultured midgestation mouse trophoblast cells to synthesize progesterone from pregnenolone has been monitored by radioimmunoassay or chromatography and crystallization. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of Δ⁵,3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Since there is little or no further metabolism of the progesterone formed, the ability of trophoblast cells to convert pregnenolone to progesterone in vitro is an accurate reflection of the activity of 3β-HSD in these cells.Midgestation cultures of giant trophoblast cells have a substantially higher 3β-HSD specific activity than the smaller ectoplacental cone cells. Neither giant trophoblast nor ectoplacental cone cell cultures show an increased 3β-HSD specific activity in response to a variety of hormones, including gonadotrophins. In fact, regardless of the gestation age at which the trophoblast cultures are initiated, 3β-HSD activity inevitably follows the same temporal pattern observed in vivo. Taken together, these facts suggest that the levels of 3β-HSD in trophoblast cells are intrinsically controlled and that, unlike the ovary, progesterone production by trophoblast cells in vivo is not influenced by gonadotrophic hormone levels.     

Phosphatidyl glycerol phosphate phosphatase in BHK-21 cells. A comparison with rat liver mitochondria.

Unlike mitochondria from rat liver, BHK-21 mitochondria are not autonomous for polyglycerolphosphatide biosynthesis. The phosphatase which converts phosphatidyl glycerol phosphate to phosphatidyl glycerol is located in the post-microsomal supernatant. It is somewhat heat stable, non-dialyzable, sulfhydryl-containing and its activity is similar to the phosphatase that can be found in the supernatant from sonicated rat liver mitochondria.     

Characterization of a stabilizing factor of rat liver and of the nature of its interaction with ATP-citrate lyase

A stabilizing factor, previously reported to protect phosphofructokinase (EC2.7.1.11) from thermal or lysosomal inactivation, has been shown to stabilize ATP-citrate lyase (EC 4.1.3.8) from thermal inactivation (B. Osterlund and W. A. Bridger, 1977, Biochem. Biophys. Res. Commun., 76, 1–8). We now report that this factor protects ATP-citrate lyase from inactivation by proteases extracted from lysosomes. While it has been suggested that the stabilizing factor may play a role in the turnover of other lipogenic enzymes, we have found that the factor has no stabilizing or other effects on NADP⁺-malic enzyme (EC 1.1.1.40). In order to assess the properties and mode of action of the stabilizing factor with regard to interaction with its target enzyme(s), the factor has been extensively purified from rat liver and characterized as to its composition. Although glutathione appears to copurify with the factor, and glutathione exerts some stabilizing effects on ATP-citrate lyase, the factor is clearly distinguishable from glutathione on the basis of its mode of action and its concentration dependence. Several other biological compounds have been tested in attempts to identify the chemical nature of the stabilizing factor. Thus, biotin, pyridoxal phosphate, glucose tolerance factor, substrates for ATP-citrate lyase, and oxidized glutathione have been eliminated as possible identities for the stabilizing factor. In contrast to results reported by other workers (who investigated stabilization of phosphofructokinase) we find this factor to be insensitive to treatment by proteases or sulfhydryl reagents when tested by its ability to protect ATP-citrate lyase from inactivation.     

DNA methylation of liver and HTC cells during corticosteroid induction

The status of DNA methylation, as measured by m⁵C² content of nuclear DNA, was examined during corticosteroid mediated TAT induction in rat liver and in dividing and nondividing HTC cells. The m⁵C content, determined by HPLC, was not significantly altered in HTC cells during TAT induction whether the cells were in the logarithmic or stationary growth phase. In the liver of adrenalectomized rats where the range of corticosteroid effects is greater than in HTC cells, a small but significant decline in genomic levels of m⁵C was detected between 1 to 8 hr post-induction. The alterations in DNA methylation did not fluctuate during induction by more than 8% in the liver or 7.5% in HTC cells. These results demonstrate that no gross change or elevation in m⁵C content is detected in two, different, hormonally responsive hepatocellular systems during gene activation.     

Similarity of the primary sequences of ribosomal RNAs isolated from vegetative and developing cells of Dictyostelium discoideum

Differentiating cell aggregates of Dictyostelium discoideum exhibit a pattern of rRNA metabolism quite different from that observed in the single-celled vegetative amoebas of this organism. We have examined whether the differences are related to a requirement for the production of new types of ribosomal RNA during development. Oligonucleotide maps and supplementary sequence data for 25 S, 17 S, and 5 S rRNAs from vegetative and developing cells have revealed no detectable alterations in primary sequence distinguishing any species of rRNA in developing cells from its vegetative cell counterpart.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent K_t values of the saturable systems were 64 ± 40 nM (n =3) and 1085 ± 313 nM (n =12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluor-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35–45%. In the presence of cortisone, cortisol, dexamethasone, aldosteron, testosterone, estradiol-17β and estrone (2 μM each) the uptake decreased 30–50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Culture-generated suppressor cells: evidence for an adherent cell component

Cells which suppress mixed lymphocyte reaction (MLR) and cytotoxic T-lymphocyte (CTL) generation can be demonstrated after culture for 3 to 5 days in the absence of added antigen. Such precultured suppressors are resistant to the cytotoxic effects of commercial rabbit anti-mouse brain serum, congenic anti-Thy-1.2 serum, and monoclonal anti-Thy-1.2 reagent. In addition, these cells, which adhere to both nylon wool and Sephadex G-10, ingest carbonyl iron. These properties suggest that precultured suppressors may not be thymus-processed. Precultured suppressor cells, when irradiated, are able to suppress only the MLR, and not the generation of allo-CTL. This finding, taken with our previously published work on the differential ontogeny of cells which suppress the MLR or CTL generation in culture, suggests that at least two kinds of suppressors are generated. One suppressor acts on CTL generation, is radiosensitive, and develops late in ontogeny; the other suppressor acts on proliferative cells in the MLR, is radioresistant, and develops early in ontogeny. Both kinds of suppressors, however, are adherent to nylon wool and Sephadex G-10, ingest carbonyl iron, and are resistant to anti-T-cell sera.     

Human teratoma cells share antigens with mouse teratoma cells.

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Prolongation of cardiac allograft survival by pretreatment of recipient mice with donor blood or spleen cells plus cyclophosphamide.

Using six mouse strain combinations, we attempted to prolong cardiac allograft survival by pretreatment of recipients with a single iv injection of donor-specific whole blood or spleen cells plus a single ip injection of cyclophosphamide (Cy). Significant prolongation of cardiac allograft survival occurred in a small proportion of pretreated mice of some strain combinations, with some grafts surviving for periods longer than 6–9 months. Cy injected alone did not influence the normal cardiac allograft rejection time of between 1 and 2 weeks. Depending upon the strain combination, accelerated rejection of all or some of the grafts occurred in mice pretreated with blood or spleen cells or myocardial cells alone.