Effects of serum on membrane transport. I. Separation and preliminary characterization of factors which depress lysine or stimulate adenosine transport in rabbit alveolar macrophages.

The effects of normal rabbit serum (NRS) on two transport systems in rabbit lung macrophages have been examined. A 20 min preincubation with serum was required for the effects, which were retained for at least 40 min after serum was removed. No serum was present during the transport studies. (a) Preincubation with 0.5 or 1.0% NRS resulted in depression of lysine transport to 59 +/- 2.6% (SE, 31 observations) of control levels. The activity was heat stable to 100 degrees C for 30 min and lost after dialysis. Pretreatment with serum did not alter the intracellular concentration of lysine attained when cells were then incubated with 10 mM lysine for 30 min. The relative depression of lysine transport by serum was unaltered by preloading with such high concentrations of lysine. (b) Preincubation with 5% NRS resulted in enhancement of adenosine transport by 35 +/- 2.3% (SE, 60 observations). Activity was stable to heating at 65 degrees C for 40 min but lost at 100 degrees C for 20 min. It was nondialyzable. Total radioactivity accumulated after 30 min incubation with 1 mM adenosine was unaffected by serum pretreatment. The two activities were separable by passage over Sephadex G25.

Membrane transport by murine lymphocytes. I. A rapid sampling technique as applied to the adenosine and thymidine systems.

We have developed a rapid sampling technique for animal cells in suspension for the purpose of measuring membrane transport in lymphocytes. The method involves rapid centrifugation of cells through a layer of silicone oil into perchloric acid after incubation periods as short as 4 s. Using this method we have described the uptake of thymidine and the uptake and transport systems of adenosine by murine bulk nonadherent spleen cells. The two uptake systems are markedly different. Adenosine was shown to be taken up by classical carrier-mediated diffusion, while thymidine was not. In addition we have explored the metabolism of the two nucleosides under the conditions we employed for measuring transport or uptake; Both nucleosides are phosphorylated extensively. We also investigated the uptake and metabolism of thymidine over a 2-h interval the standard time used to measure DNA synthesis in lymphocytes. Unless cells were separated from medium by centrifugation through oil before TCA addition, the TCA precipitable counts exceeded the total radioactive uptake. Hence the standard method for measuring thymidine utilization yields estimates under these conditions which can be as much as 100% too high.

Effects of serum on membrane transport. II. Serum and the stimulation of adenosine transport, a possible mechanism.

When monolayers of freshly obtained rabbit lung macrophages are exposed to the nucleoside analogue, showdomycin (sho), adenosine transport, measured over a 45 s interval, is irreversibly inhibited. Low doses of the drug or short periods of exposure, however, do not result in decreased transport, while higher concentrations or longer exposures result in exponential decline. The initial lag is not due to a long reaction time of sho with the transport carrier or to nonspecific sites absorbing the drug. Previously it was shown that preincubation of monolayers with normal rabbit serum (NRS) results in increased adenosine transport. When monolayers are first exposed to sho so as to inhibit transport to varying degrees and then incubated with NRS, transport is increased over the inhibited level. Several experiments make it unlikely that serum removes the drug from the cell surface in a nonspecific fashion. Moreover, serum given before, during, or after sho alters the dose response curve so that no shoulder is seen. One way to explain these results makes use of target theory: the adenosine transport system could be comprised mainly of "coupled" or "clustered" sites of which only one is active at any time as well as "hidden" sites which are inactive. When a site in a group is irreversibly inactivated by sho, another in the group becomes activated. Serum might activate or uncouple all sites and also cause the appearance of hidden sites, which previously neither transported nor bound sho.

Acriflavin resistance in the hemoflagellate, Leishmania tarentolae.

The accumulation, metabolism, and distribution of acriflavin (acr) in two culture strains of Leishmania tarentolae were studied. One strain, reported previously, was sensitive to the dye, i.e. became dyskinetoplastic and could not be subcultured in the presence of 470 ng/ml acr, and one was resistant. Accumulation was studied by fluorescence of the dye within cells and by uptake of acr-(3)H by cells. Metabolism was studied by paper chromatography of aqueous extracts from cells grown with acr-(3)H, and distribution was examined by fluorescence and quantitative electron microscope radioautography. Substances affecting the response to acr included hemin and an acr-sensitizing factor initially obtained from red cells but here shown to be distinct from hemoglobin. In the presence of the sensitizing factor or in the absence of hemin, the resistant strain became dyskinetoplastic and could not be subcultured. Acr fluorescence appeared in the nucleus of the resistant strain, and the percentage of radioautography grains appearing in the nucleus increased. Under these conditions the distribution of radioactivity from chromatographed extracts was altered from the normal in a similar fashion. Because sensitization of the resistant strain is associated with increased amounts of acr in the nucleus, that organelle may be implicated in the mode of action of acr. In general, the two strains behaved alike except for (a) the response to acr, (b) the arginine requirement for optimal growth, and (c) the sensitivity to cycloheximide. Thus, one cannot exclude the wider possibility that acr may act on the cytoplasm and the nucleus as well as on the mitochondrion.

The human Jurkat (FHCRC-11) cell line is heterogeneous in ploidy and cell size and releases detergent-soluble DNA.

Jurkat (FHCRC-11) cells, a human lymphoblastic leukemic line, were characterized as being hypotetraploid with a characteristic deletion in the short arm of chromosome 2 from the terminus to band 24. Although Jurkat cells were size heterogeneous, variability in ploidy was not correlated with density and size differences observed when cells were fractionated by means of gradient centrifugation using Nycodenz as the supporting medium. Also no difference was seen in the chromosome distribution of cells cultured from different portions of the gradient. During cell division Jurkat cells incorporated [3H]thymidine ([3H]TdR) into newly made DNA, including a small percentage that was released into the soluble fraction upon detergent lysis. Small light cells from the top portion of the gradient were more efficient on a per cell basis in incorporating [3H]TdR into DNA from both the detergent-soluble and detergent-insoluble fractions. However, due to the hypotetraploid nature of these cells a definitive assignment to a specific stage in the cell cycle was not possible.

The TOP2 gene of Trypanosoma brucei: a single-copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II.

A mixed oligonucleotide probe containing sequences encoding a septapeptide found in yeast, Drosophila and human DNA topoisomerase II was used to screen a genomic library of Trypanosoma brucei. A positive was obtained, and nucleotide sequencing shows that the entire gene encoding DNA topoisomerase II of this organism, TbrTOP2, resides within the T. brucei insert of the clone. A single open reading frame of 1221 triplet codons starting from the first ATG was identified; the amino acid sequence deduced from it is highly homologous to other eukaryotic DNA topoisomerase II and corresponds to a 137-kDa polypeptide. Analysis of restriction endonuclease digests of T. brucei DNA by blot hybridization following gel electrophoresis indicates that TbrTOP2 is a single-copy gene.

Use of Filtron Mini-ultrasette tangential flow device and Filtron Microsep centrifugal concentrators in the early stages of purification of DNA polymerases.

Isolation and initial partial purification of several DNA polymerases from Trypanosoma brucei were achieved. Concentration of relatively large volumes necessary in the early stages of the protocol was accomplished by means of the Filtron Mini-ultrasette tangential flow devices. Subsequently, different size classes of polymerases were obtained by means of Filtron Microsep concentrators, which utilize dead-end filtration and are ideal for concentrating small volumes (several mL) to even smaller volumes (microL). The use of the Filtron systems allowed us to determine relative amounts of different polymerase species quickly and efficiently and to overcome routine problems with concentrating labile macromolecules.

Identification of novel mRNA isoforms for human DNA polymerase beta.

Recently, we reported the organization of the thirteen exons of the human DNA polymerase beta (beta-pol) gene and the sequences of the exon-intron junctions. Splice variants of human beta-pol mRNA have been postulated to be related to cancer development. Here, we report the characterization of isoforms of human beta-pol mRNA in different cells by reverse transcription polymerase chain reaction (RT-PCR). DNA sequence analysis of RT-PCR products revealed eight alternative splicing mRNA isoforms in the brain cancer cell line, SK-N-MC. These various isoforms were consistent with alternative splicing of four exons (II, IV, V, and VI) and with a 105-nucleotide insertion (exon alpha) between exons VI and VII. We also found an isoform with a 19-nucleotide sequence inserted into the exon IV and V junction, which resulted from usage of a different 3' splice site. Seven of the isoforms resulted in truncated open reading frame (ORF); five corresponded to deduced peptide of amino acids 1-20 of beta-pol and two corresponded to amino acids 1-60 of beta-pol. Only one of the right mRNA isoforms, that with the exon alpha insertion, was in-frame with the entire wild-type ORF resulting in a deduced protein of 370 residues, compared with the wild-type protein of 335 residues and 39 kD. This longer ORF was shown to be capable of encoding a beta-pol protein, larger than wild-type beta-pol, that cross-reacted with beta-pol antibody and exhibited beta-pol enzymatic activity. The mRNA isoform with the exon alpha insertion was not tumor specific because it as detected in low abundance in all cells tested, except the colon cell line CCD18 Co where the isoform was absent. The genomic location of exon alpha is in intron VI, 990 bp upstream of exon VII and flanked by consensus splice sites. Thus, this 105-bp genomic sequence is a beta-pol exon present in a low-abundance beta-pol mRNA isoform capable of encoding an approximately 42-kD beta-pol.

Effects of serum on membrane transport. III. Serum and inhibition of lysine transport.

Rabbit lung macrophages have been shown to transport lysine by means of a single carrier-mediated system that is depressed after relatively short incubations with 1% normal rabbit serum (NRS). Our present results show that three amino acids, glutamine, phenylalanine and tyrosine, at or near the concentration found in 1% NRS, are involved intracellularly in regulating lysine influx across the plasma membrane. The means by which they do so requires a 20- to 30-min preincubation, the effect is maintained for 45 min in their absence, and both maximal velocity and affinity of lysine for its carrier are altered in an apparently uncompetitive fashion. The kinetic data are unlike those reported for other trans phenomena involved in amino acid transport. We propose that the effect of internal inhibitor on lysine influx probably does not involve the direct interaction of the transamino acid on the lysine carrier.

Incorporation of 3H-labeled nucleosides and 3H-labeled deoxynucleosides into detergent soluble DNA.

Detergent soluble DNA from splenocytes of immunologically stimulated mice has been shown to incorporate [3H]dThd more rapidly than detergent insoluble DNA. In this report we compare the incorporation of other 3H-labeled nucleosides and 3H-labeled deoxynucleosides and the distribution of 3H in the different size classes of detergent soluble DNA. The order of incorporation into DS DNA is [3H]dThd greater than [3H]dCyd greater than [3H]Ado greater than [3H]dGuo approximately equal to [3H]Cyd greater than [3H]dAdo greater than [3H]Guo. We also show that the previously reported slight enrichment in Gua + Cyt content is not due to preferential incorporation of dGuo or of dCyd into any one size class.

Membrane transport by macrophages in suspension and adherent to glass.

We have compared membrane transport of lysine and adenosine by rabbit lung macrophages in suspension and adherent to glass over slips. The rapid sampling techniques employed permitted measurements to be made over intervals as short as 10 seconds. Suspended cells transported both nutrients at a faster rate, although the Km values for the two transport systems remained unchanged. Colchicine, cytochalasin B and adenosine did not interfere with the enhancement.

Domain mapping of human apurinic/apyrimidinic endonuclease. Structural and functional evidence for a disordered amino terminus and a tight globular carboxyl domain.

We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity.

Oligonucleotides with bistranded abasic sites interfere with substrate binding and catalysis by human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in oxidative damage DNA repair. The enzyme, which repairs abasic sites, makes a single nick 5' to the phosphodeoxyribose, leaving a free 3'-hydroxyl. We recently described single turnover kinetics for human recombinant AP endo acting on an oligonucleotide with a single abasic site. We hypothesized that the structural changes induced by the presence of a second abasic site might provide insight into how AP endo recognizes the first abasic site. Here we performed steady state and single turnover experiments using bistranded abasic site substrates, with the second site located on the complementary strand to the one being followed and either opposite to the first or displaced in the 5' direction. All sites on the complementary strand were within half a helical turn of the first. The catalytic efficiency was reduced 80 to 96% and the Kd for substrate binding and dissociation was elevated 40- to 125-fold. The smaller changes occurred when the second site was opposite the first site or displaced by four nucleotides. In addition, if the second abasic site was directly across the helix or displaced by 1 or 3 nucleotides from the first abasic site, cleavage of the first abasic site was subject to apparent substrate inhibition, which did not occur if the second abasic site was displaced by four nucleotides from the first. While a substrate containing a nick without a phosphodeoxyribose on the contralateral strand abasic site did not inhibit nicking of the first strand, a substrate with a nicked abasic site on the contralateral strand was an even stronger inhibitor of enzyme action than an oligonucleotide containing the corresponding abasic site on each strand. Consequently, the inhibitory effect of the second abasic site is probably the result of prior cleavage of the abasic site on the contralateral strand with resulting distortions to the DNA helix that interfere with enzyme binding and/or cleavage.

Human apurinic/apyrimidinic endonuclease is processive.

Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m) and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro.

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.

Plasma membrane mediated thymidine transport in AKR spleen cells.

In this paper we characterize the thymidine transport systems in nonadherent spleen cells from normal leukemic (AKR) mice and from AKR mice which have been stimulated in vivo with concanavalin A (Con A). We have shown that splenic lymphocytes from normal AKR mice transport thymidine (two kinetic components, Km values of 34 microM and 1.6 mM) whereas lymphoid cells from C57L/J and outbred (CD-1) mice do not. Following Con A stimulation of AKR mice, three components (Km values of 6 microM, 212 microM, and millimolar range) were observed. The current data should be compared with previously published results for splenocytes from Con A stimulated CD-1 mice. Although those cells transport thymidine with two kinetic components (Km values of 160 microM), they lacked the lowest Km system present in AKR splenocytes. Thymidine transport was also examined in lymphocytes from several AK x L recombinant inbred mouse strains derived from the cross AKR/J x C57L/J. Two strains which lacked MuLV did not show time-dependent thymidine translocation whereas two strains which possessed MuLV demonstrated time-dependent thymidine translocation. Moreover, cells from the congenic strain L.AKR-Akv-2, which carried the Akv-2 genome on a C57L background, also showed thymidine transport. Thus a unique ability to transport thymidine can be correlated with the presence of the murine leukemia virus genome.

Membrane transport by murine lymphocytes. II. The appearance of thymidine transport in cells from concanavalin A-stimulated mice.

We have investigated membrane transport of thymidine and adenosine by bulk murine nonadherent spleen cells from animals treated with the lectin concanavalin A. Separation of cells from radioactive medium was achieved by means of the oil microfuge technique which permits incubation periods as short as 4 sec. We were able to distinguish between the membrane-linked function of transport and uptake which includes subsequent accumulation and metabolism of the transport solute. Previously we reported that cells derived from untreated mice could not be shown to translocate thymidine in a carrier-mediated fashion. In contrast, cells from concanavalin A-treated mice showed two membrane transport systems for thymidine, a high affinity system (Km = 160 micronM) and one with low affinity (Km = 4 mM). The transport of thymidine conformed to the usual criteria for membrane carrier-linked function: increased uptake with time, substrate saturability and chemical specificity. In contrast to thymidine uptake, the transport of adenosine by cells from lectin-treated mice was similar to that shown by cells from untreated animals. Thus a specific membrane-linked activity is altered differentially in the course of mitogen-effected lymphocyte stimulation. We have found that determination of DNA synthesis by the standard method gave values which were as much as 700% too high, since the counts obtained by direct precipitation with TCA of cells incubated with radio-labeled thymidine exceed the cell-associated radiolabel obtained by the rapid sampling technique. With lectin-stimulated cells, the discrepancy observed was up to three times greater. Hence the validity of the standard assay for blastogenesis must be viewed with caution.

Contractile proteins participate in release of erythroid growth regulators from mononuclear cells.

We have investigated the role of contractile proteins of circulating mononuclear cells in generation of membrane-associated, erythroid growth regulatory molecules. Lymphocytes and monocytes were incubated under serum-free conditions without and with cytochalasin B, cytochalasin D, or colchicine, and effects on positive and negative erythropoietic activities were determined in cell membranes and in surface membrane vesicle-rich pellets and supernatants of dialyzed medium conditioned by the cells. In serum-free cultures of human bone marrow, plasma membranes and exfoliated membrane-derived vesicles from cytochalasin-treated lymphocytes lost their capacity to support the formation of erythroid bursts, while monocyte membrane-associated inhibitory activity was abolished by preincubation with cytochalasin. In contrast, membrane-associated activities of colchicine-treated cells were unaffected. Cytochalasin-induced alterations of membrane regulatory molecules were observed in a dose-dependent fashion over a wide range of concentrations (1 to 100 micrograms/mL) tested. However, the capacity of membrane vesicle-free supernatants of medium conditioned by lymphocytes or monocytes was unaffected by cytochalasins, regardless of drug concentration used. Lysates of cytochalasin B-treated cells inhibited the activity of deoxyribonuclease I to a greater degree than did lysates of untreated cells, suggesting that the relative amount of monomeric actin is increased in the cytoplasm of treated cells. Furthermore, results of experiments with D-glucose and with cytochalasin D suggest that cytochalasin effects are independent of alterations in glucose metabolism. The data indicate that expression of plasma membrane-associated regulators is sensitive to agents that block polymerization of actin. They raise the possibility that changes in distribution of actin between unpolymerized and filamentous pools may influence the organization and/or function of mononuclear cell surface-associated erythroid regulatory molecules.

Kinetics of thymidine incorporation into detergent-soluble DNA of mouse lymphocytes.

We reported recently that splenocytes from concanavalin A-stimulated mice rapidly incorporated [3H]thymidine into non-mitochondrial DNA that was detergent soluble and distributed in size classes between 200 and 5000 base pairs. In this report we show that small [3H]thymidine-labeled oligonucleotides (less than 100 base pairs long) appear by 15 min. Subsequently, [3H]thymidine in small oligonucleotides diminishes as incorporation into larger size classes of detergent-soluble DNA occurs in a pattern that is stable for at least 3 hr. Although incorporation of [3H]thymidine into the oligonucleotides is not sensitive to aphidicolin or hydroxyurea, the appearance of [3H]thymidine in larger species is blocked by both drugs. These results indicate that the enzymatic process involved in synthesis of oligonucleotides is somehow distinct from the process involved in synthesis of larger detergent-soluble size classes. Synthesis of the latter may be replication related.