The inhibition of the mitogenic stimulation of B lymphocytes by a serine protease inhibitor: commitment to proliferation correlates with an enhanced expression of a cell-associated arginine-specific serine enzyme

By the use of diisopropylfluorophosphate (DFP) we have been able to show that the mitogenic stimulation of murine B lymphocytes can be maximally inhibited a few hours before commitment of the cells to DNA synthesis. This inhibition was shown to be specific for a serine enzyme(s). The results of experiments designed to determine the location of this enzyme indicated that the mitogens-linked serine enzyme is not a secreted extracellular factor but is cell-associated. Fluorographic analysis of electrophoretic gels of cell homogenates labeled with [3H]DFP revealed the presence of one major and three minor bands which were arginine-specific serine enzymes. In stimulated cells, there was a clear quantitative difference in the amount of [3H]DFP bound to the major band (approx. 23,000 m.w.) suggesting that this protein may be critical to the progression of the cells through the cell cycle into the S phase of DNA synthesis.     

The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes.

In this paper we bring evidence suggesting that there is activation of an esterase upon reaction of anti-immunoglobulin antibodies (anti-Ig) with murine B lymphocytes. B lymphocytes upon exposure to anti-Ig cap the ligand-receptor complexes and immediately afterward become briefly motile. It is this latter step which is inhibitable by exposure to di-isopropyl phosphofluoridate (DFP). Various experimental manipulations indicated that treatment with anti-Ig activates the cell for motility which, however, is not manifested until the temperature is raised to 37 degrees C. The cell incubated with anti-Ig at cold temperatures becomes susceptible to the effect of DFP, suggesting that the antibody-treated cells are activated up to but not beyond the DFP inhibitable step. Exposure of cells to DFP and removal of it before their treatment with anti-Ig does not affect the anti-Ig-induced response. Four lines of evidence indicate that the reduction of lymphocyte movement of DFP is due to the inhibition of an esterase activated by the combination of antibody and cell: 1) The inhibition by DFP is irreversible; once DFP has reacted it can be washed away and the antibody-treated cell is still inhibited. 2) The inhibition increases with time of contact of lymphocytes and DFP and with the concentration of DFP. 3) A very poorly phosphorylating phosphonate, phenyl ethyl pentylphosphonate is completely inactive under conditions where an excellent phosphorylating phosphonate, p nitrophenyl ethyl pentylphosphonate maximally inactivated the cells' movement. 4) The amino acid esters, tosyl L arginine methyl ester and benzoyl arginine methyl ester specifically prevent the inactivation by DFP. The last finding suggests that tosyl L arginine methyl ester and benzoyl L arginine methyl ester might be substrates for the putative antibody-induced lymphocyte esterase. Lymphocytes incubated with antibody in the cold for more than 30 min lose their ability to move when the temperature is raised, suggesting that there is a time-dependent deactivation of the cell.     

PHA stimulation of human lymphocytes during amino acid deprivation. Protein,RNA and DNA synthesis

Resting lymphocytes are in the G₀ phase of the cell cycle. Upon activation by PHA, they progress into G₁ with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine-deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine). Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan- or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation. Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.     

Interrelationships in experiments in vitro between the migration activity of leukocytes and RNA and DNA synthesis]

The following correlations were revealed in the parallel study of leukocyte migration in vitro in the presence of a specific antigen and of spontaneous RNA and DNA synthesis in the cultured lymphocytes: 1) a direct correlation between the RNA and DNA synthesis in lymphocytes; 2) a close correlation between the antigen-induced migration and the levels of RNA and DNA synthesis. The effect of the antigen was evidenced by the inhibition or stimulation of leukocyte migration. A high ratio of RNA synthesis to DNA synthesis corresponded to the migration inhibition and a low one--to the migration stimulation. The ratio value varied mainly on account of the changes in the level of DNA synthesis. Participation of T and B cells in the regulation of the antigen-induced leukocyte mobility is discussed.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

"Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes

To detect nuclear proteins that might be involved in induction of cellular mitogenesis, we examined the effect of various mitogens on early changes in synthesis of nuclear proteins in murine B lymphocytes. Using two-dimensional gel electrophoresis, we found that activation of B cells by mitogens (anti-immunoglobulin antibody, lipopolysaccharide, phorbol 12-myristate 13-acetate (PMA)/A23187) was associated with a rapid and prominent (5-20-fold) increase in the synthesis of a 40-kDa/pI 5.0 nuclear protein, here termed numatrin. Numatrin was found to be absent from the cytosol (soluble fraction) of resting as well as activated B cells and was markedly resistant to DNase/RNase digestion and 2 N NaCl extraction, indicating that this protein is tightly bound to the nuclear matrix. Kinetic studies showed that the increase in synthesis of numatrin was detected 60-120 min following mitogen activation, reached a peak at 16 h, and declined to almost control level by 48 h, correlating with the peak of cellular DNA synthesis. The increase in synthesis of numatrin in normal B cells was found to be associated exclusively with cellular commitment for mitogenesis because activation of B cells by stimuli such as B cell stimulating factor 1, PMA alone, and calcium ionophore A23187, which do not stimulate an increase in DNA synthesis, also failed to induce an increase in the synthesis of numatrin. Inhibition of anti-Ig-induced proliferation (by PMA pretreatment) was associated with a 63% inhibition in the synthesis of numatrin. Addition of 8-mercaptoguanosine to these PMA-treated cells was associated with restoration of the increase in synthesis of numatrin, concomitant with induction of proliferation. Elevated synthesis of numatrin was also detected in the malignant B lymphoma cells: Raji, BAL-17, and WEHI-231. Taken collectively, these results suggest that numatrin, a tightly bound nuclear matrix protein, is a growth-regulated protein which might have an important role in regulation of cellular mitogenesis in normal and malignant B lymphocytes.     

Protease activity of normal and PHA stimulated human lymphocytes

An assay measuring the release of TCA soluble radioactive peptides from ³H acetylated casein or hemoglobin has been used to demonstrate that human peripheral blood lymphocytes contain a number of proteases, including cathepsin D, a neutral serine protease(s) inhibited by DFP and TLCK and probably a thiol protease(s) as well. We have also found a neutral protease activity bound to the surface of the lymphocyte, but not secreted into the medium which is not inhibited by TLCK. TLCK inhibits blast transformation to PHA under conditions that do not profoundly affect protein synthesis and inhibits the total extractable proteolytic activity of lymphocytes by approximately 25%. Lymphocytes contain one or more proteases that may play a role in blast transformation and other lymphocyte functions.     

Persistent effects of ouabain treatment on human lymphocytes: synthesis of DNA, RNA and protein in stimulated and unstimulated cells.

Pretreatment of human lymphocytes for 2 days in 2 X 10(-6)M ouabain caused irreversible loss of their subsequent capacity to stimulate in the mixed lymphocyte reaction (MLR). Pretreatment for the same period with 10(-7)M ouabain resulted in an enhanced incorporation of thymidine into DNA of the responding cells in the MLR; this effect was also on the stimulating cells, as previously reported by Christen et al. (Cell, Immunol. 19, 137-142 (1975)). Pretreatment of stimulating lymphocytes with 10(-7)M ouabain caused a persistent but reversible inhibition of the synthesis of RNA and protein in the MLR; peak incorporation of labelled uridine or alanine reached the same level as that of the control cultures, but 24 h later. Exactly the same persistent but reversible inhibition was found in the case of DNA syntheis of cells pretreated with 10(-7)M ouabain and then stimulated by antigens (streptolysin-O and varidase) or by mitogens (phytohemagglutinin and concanavalin A); the same level of incorporation of labelled thymidine occurred but 24-48 h later than in the case of the controls. Pretreatment with the cardiotonic steroid under these conditions also resulted in a pronounced inhibition of the basal, unstimulated levels of RNA and protein synthesis in the case of both control lymphocytes and those which had been treated with mitomycin C. The effects of ouabain pretreatment on basal RNA and protein synthesis were identical for both 2 X 10(-6)M and for 10(-7)M; the effect of pretreatment of stimulating cells with these two concentrations was completely opposite: irreversible inhibition of the proliferative response of allogeneic responding cells at the former concentration and delayed activation at the latter.     

Effect of interferons on protein synthesis in human lymphocytes: enhanced synthesis of eight specific peptides in T cells and activation-dependent inhibition of overall protein synthesis

Lymphoblastoid and fibroblast IFN inhibited PHA stimulation of overall protein synthesis in human lymphocytes by ca. 30%. Inhibition occurred within the first 6 hr of PHA treatment and was not progressive. DNA synthesis at 48 hr was inhibited to the same extent. Overall protein synthesis in resting lymphocytes was not detectably inhibited by IFN concentrations up to 1000 U/ml. Thus, inhibition of protein synthesis and subsequent reduction of cell proliferation by IFN require certain early events in mitogen activation. Resting lymphocytes were not unresponsive to IFN treatment, however. Two-dimensional electrophoretic analysis of newly synthesized proteins after IFN treatment showed enhanced synthesis of a specific set of eight peptides (I-peptides), which were shown to be synthesized in T lymphocytes. This enhancement was produced by both IFN-alpha and IFN-beta after 4 to 6 hr of exposure and was identical for all lymphocyte donors studied. After growth stimulation, IFN treatment produced no enhancements of additional peptides, although the original eight I-peptides were enhanced as usual. It is concluded that the biochemical activities of the I-peptides, which remain to be determined, cannot inhibit protein synthesis in resting lymphocytes, but may do so after mitogen activation, when the major physiologic restriction of lymphocyte protein synthesis is released. Alternatively, the I-peptides may be unrelated to regulation of protein synthesis but may be involved in viral protection or enhancement of NK activity.     

Anti-mitogenic function of interferon-induced (2'-5')oligo(adenylate) and growth-related variations in enzymes that synthesize and degrade this oligonucleotide

Addition of (2'5')ApApA to concanavalin-A-stimulated mouse spleen lymphocytes strongly inhibits the large increase in RNA and protein synthesis which takes place 24-48 h after stimulation. The inhibitory effect on protein synthesis precedes the effect on RNA synthesis and takes at least 6 h to be detected. Histone synthesis is preferentially inhibited at 48 h. No effect on protein synthesis was detected in unstimulated resting lymphocytes, or in stimulated lymphocytes during the first 24 h after concanavalin A treatment. The anti-mitogenic effect of the (2'-5')oligo(adenylate) seems to result, therefore, from inhibition of protein synthesis taking place before initiation of DNA replication. The mitogenic stimulus produced by the lectin enhances, in lymphocytes, the level of the 2'-phosphodiesterase which degrades (2'-5')oligo(adenylate). Enhancement of the 2'-phosphodiesterase was also observed after serum stimulation of confluent monkey kidney cells. Furthermore, the ratio of (2'-5')oligo(adenylate) synthetase to 2'-phosphodiesterase is ten-times lower in fast-growing kidney cells than in quiescent serum-starved cells. A model for the role of (2'-5')oligo(adenylate) synthesis and degradation in the regulation of cell proliferation by interferon and by mitogens is presented.     

The protein synthetic surge in response to mitogen triggers high glycolytic enzyme levels in human lymphocytes and occurs prior to DNA synthesis

A simultaneous increase is found in the level of protein synthesis and the major regulatory glycolytic enzyme, phosphofructokinase (PFK), in early phytohemagglutinin exposure of human lymphocytes. The induction of DNA synthesis is demonstrated to be a much later event. This indicates that the increase of glycolysis in mitogen-stimulated cells precedes cell proliferation, but occurs simultaneously with a general increase in protein synthesis. Chemical inhibitors are used to clarify the interrelationship of protein synthesis, glycolytic enzymes levels, and DNA synthesis. Inhibition of protein synthesis with cycloheximide in the mitogen-exposed lymphocytes prevents any increase in PFK levels, implicating protein synthesis as a cause for the increased glycolysis. Cycloheximide also prevents entry into S phase in mitogen-stimulated lymphocytes which may be due to inhibition of the synthesis of enzymes necessary for DNA synthesis, such as DNA polymerase. Aphidicolin, a specific DNA polymerase inhibitor, is found to have no effect on the increase in protein synthesis and PFK levels that precedes DNA synthesis. The increase in glycolysis in mitogen-stimulated lymphocytes occurs simultaneously with, and is dependent upon, increased protein synthesis, and precedes DNA synthesis and lymphocyte proliferation; thus, the high glycolytic rate of mitogen-stimulated cells is not merely a secondary manifestation of rapid cell proliferation as has been previously reported.     

Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B.

Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column. The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B. The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B. The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations. The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A.     

Neutrophil-assisted DNA synthesis by human lymphocytes in response to mevalonic acid; enhancement by cytochalasin B

Mevalonic acid is capable of initiating DNA synthesis, morphologic transformation, and cell division in cultures of human peripheral blood lymphocytes. Pure populations of lymphocytes respond poorly to mevalonic acid, but their response can be enhanced by peripheral blood neutrophils. Addition of cytochalasin B (0.5-1.0 micrograms/ml), but not cytochalasin A, to mixed neutrophil-lymphocyte cultures enhances the response of lymphocytes to both Con A and mevalonate, but the increment in mevalonate-induced DNA synthesis (+343%) far exceeds the enhancement which cytochalasin B produces in the Con A response (+24%). As a consequence, the DNA synthetic response in mevalonate (10(-2) M) containing cultures averages 39% of the response to an optimal dose of Con A. The cytochalasin B-enhanced response of mixed neutrophil-lymphocyte cultures to mevalonate is abolished by prior X irradiation of the lymphocytes, or their pretreatment with mitomycin C, as well as by the addition of hydroxyurea to the cultures but is not altered by prior X irradiation or mitomycin C pretreatment of the neutrophil helper population. These experiments suggest that the enhancing effect of cytochalasin B in the response of mixed neutrophil-lymphocyte cultures to mevalonic acid results from its ability to potentiate a time-dependent conditioning effect on lymphocytes which neutrophils exert. The conditioning effect of neutrophils cannot be achieved by cell-free neutrophil lysosomal enzymes released by exocytosis, and reactive oxygen species potentially generated by neutrophils are not involved. Attempts to demonstrate the production by neutrophils of a soluble mediator induced by mevalonate in the presence of cytochalasin B were without success. In the presence of cytochalasin B, only the metabolically active R(-) enantiomeric form of mevalonate is capable of initiating DNA synthesis in mixed neutrophil-lymphocyte cultures. The cytochalasin B-potentiated response of mixed neutrophil-lymphocyte cultures to mevalonic acid is preferentially displayed in cultures containing E-rosette-negative, as opposed to E-rosette-positive, lymphocytes. These observations add to the growing knowledge about the relationship between mevalonate metabolism, DNA synthesis, and cell replication.     

Effects of beta-lapachone, a peroxide-generating quinone, on macromolecule synthesis and degradation in Trypanosoma cruzi

Incubation of Trypanosoma cruzi epimastigotes with beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), a lipophilic o-quinone, produced inhibition of [3H]thymidine, [3H]uridine, and L-[3H]leucine incorporation into DNA, RNA, and protein, respectively. With 1.6 microM beta-lapachone, DNA synthesis was preferentially inhibited. The inhibition was irreversible, and time and concentration dependent. Other effects of beta-lapachone were (a) inhibition of 3H precursor uptake into epimastigotes, (b) exaggerated degradation of DNA, RNA, and protein, (c) increased unscheduled synthesis of DNA, and (d) increased number of strand breaks in nuclear and kinetoplast DNA. DNA damage by 1.6 microM beta-lapachone was repaired by reincubating the drug-treated epimastigotes in fresh medium for 24 h, but with 7.8 microM beta-lapachone DNA damage was irreversible. The p-quinone isomer alpha-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione), was less effective than beta-lapachone, especially on DNA and RNA synthesis, and did not stimulate unscheduled DNA synthesis. Since beta-lapachone redox cycling in T. cruzi generates oxygen radicals while alpha-lapachone does not (A. Boveris, R. Docampo, J. F. Turrens, and A. O. M. Stoppani (1978) Biochem. J. 175, 431-439), the summarized results support the hypothesis that oxygen radicals contribute to beta-lapachone toxicity in T. cruzi.     

Excretion of DNA by purified human lymphocyte subpopulations.

A large proportion of DNA synthesized in vitro by human lymphocytes stimulated with plant mitogens or specific antigens is selectively excreted from the cells. To determine if DNA excretion differs among various types of lymphocytes, we examined purified human lymphocyte subpopulations for DNA synthesis and excretion in response to stimulation by L-PHA. The relative proportion of newly synthesized DNA that is excreted by unseparated mononuclear cells, macrophage-depleted cells, T, and B lymphocytes is identical despite great differences in the magnitude of their responses. Low levels of both DNA synthesis and excretion by macrophage-depleted cells and B cells can be increased by reconstitution with macrophages and T cells, respectively. These data indicate that DNA exretion is a general property of lymphocytes stimulated to undergo DNA synthesis by plant mitogens.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Aflatoxin B1, a selective inhibitor of DNA synthesis in mammalian cells.

Aflatoxin B1 in concentrations between 0.01 and 0.1 microgram/ml inhibits DNA synthesis in African green monkey cells in culture, but has little effect on RNA synthesis and no effect on protein synthesis. The drug even at concentrations up to 1.0 microgram/ml does not interfere with DNA repair promoted by ultraviolet irradiation nor does it induce DNA repair. The inhibition of DNA synthesis attains maximum values 3 h after addition of aflatoxin B1 and is irreversible upon removal of the drug. Profiles of pulselabeled DNA sedimented in alkaline sucrose gradients indicate that aflatoxin B1 blocks initiation of replication rather than elongation.     

The determination of the radiochemical purity of phosphorus-32 and tritium-labeled diisopropylphosphorofluoridate (DFP).

A method is described for the determination of the radiochemical purity of labeled diisopropylphosphorofluoridate (DFP), based on the irreversible inhibition reaction with the enzyme α-chymotrypsin. The nature of the impurities in commercially available ³²P- and ³H-labeled DFP is discussed.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

Biogenesis of chromatin in animal cells. Stimulation of nuclear protein and DNA synthesis in hepatocytes after brief inhibition of translation by cycloheximide]

A drastic and brief inhibition of protein synthesis (up to 95% for 3--6 hrs) by cycloheximide in the liver of rats starved for 24 hrs results in a recovery and subsequent marked stimulation of non-histone proteins, histone chromosomal proteins and DNA. The stimulation of non-histone protein synthesis was observed after 1 hr (inhibition) 12--24 hrs (recovery and stimulation of protein synthesis) and 48--60 hrs (stimulation of DNA synthesis) following the administration of cycloheximide. Two periods of histone biosynthesis were observed. The first one (24--36 hrs) was not coupled and the second one (48--60 hrs) was coupled with DNA replication. During the recovery and stimulation of protein synthesis acetylation of the histone and non-histone proteins proceeds at an increased rate. Possible applicability of the model in question for investigations of chromatin biogenesis is discussed.