Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.

Terminal incorporation of 2'-deoxyadenosine into polyadenylate segments of polyadenylated RNA in G1-phase-arrested human T-lymphoblasts.

In the presence of the adenosine deaminase inhibitor erythro-9-[3(2-hydroxynonyl)]adenine microM concentrations of 2'-deoxyadenosine (dAdo) are toxic to nondividing human lymphoid cells and induce G1-phase arrest in T-leukemic lymphoblasts, effects which appear to be independent of ribonucleotide reductase inhibition by accumulated 2'-deoxyadenosine 5'-triphosphate. We sought to determine if 2'-deoxyadenosine 5'-triphosphate had effects similar to those of other cytotoxic adenosine analogues which are incorporated into polyadenylated RNA [poly(A)+ RNA]. In the presence of erythro-9-[3-(2-hydroxynonyl)]adenine, 8-14C]dAdo, at minimal cytostatic concentrations, was incorporated into the polyadenylate segments of cytoplasmic poly(A)+ RNA in the human T-leukemic lymphoblast line CCRF-CEM, and 70% of incorporated dAdo was in the 3'-terminal position. No DAdo was found in enzyme hydrolysates of nonpolyadenylated regions of poly(A)+ RNA or of poly(A)-RNA. Enzymic hydrolysis of polyadenylated segments from labeled poly(A)+ RNA yielded adenosine:dAdo ratios of approximately 55:1.

Both myeloproliferative disease and leukemia are induced by transplantation of bone marrow cells expressing v-myc.

An in vivo system has been established to investigate v-myc-induced hematopoietic neoplasia in mice. A Moloney murine leukemia virus (Mo-MLV)-derived recombinant retrovirus containing v-myc was used to infect immature bone marrow cells, and these cells were then transplanted into lethally irradiated recipients. All provirus-positive reconstituted mice were found to develop hematopoietic proliferative disorders and, in certain cases, overt leukemia--myeloblastic, myelomonocytic and T lymphocytic. In all cases expression of v-myc was high and the disease type did not correlate with the level of expression. We have isolated immortalized monocytes, myeloid progenitors and T lymphocytes from several of these mice and shown tumorigenicity in secondary syngeneic recipients. This system provides a model for investigating the progression from a pre-leukemic disease to malignancy. In addition, we describe a recombinant v-myc-containing retrovirus that directs high-level v-myc expression from the Mo-MLV promoter in all the hematopoietic cell types examined.

Tumor progression following transformation of murine monocytes by v-myc: acquisition of immortalization and tumorigenicity.

Monocyte transformation by the v-myc oncogene has been used to study myelomonocytic tumor progression in vitro. Murine monocytes transformed by a recombinant retrovirus containing MC29 v-myc were found to exhibit a proliferative burst to day 28-40 post-infection. There-after growth slowed and cell number remained relatively static to day 80-90 post-infection. During both the proliferative and quiescent periods, the cells were dependent on the myelomonocytic growth factor CSF-1 for growth and viability. Analysis of this transformation revealed that the initial transformants were polyclonal, non-immortal, and non-tumorigenic in syngeneic mice. At day 80-90 post infection, a fresh round of cellular proliferation occurred and, in contrast to the initial burst, growth was sustained allowing the establishment of cell lines. These lines were found to be monoclonal, immortal, growth factor independent and, in certain cases, tumorigenic in syngeneic mice. Associated with the establishment of growth factor independent cell lines was the constitutive synthesis of the myelomonocytic growth factor, CSF-1. Proto-oncogene screening of the initial transformants and the cell lines also revealed the expression of c-raf and the CSF-1 receptor, c-fms. Our results indicate that, following transformation by v-myc, monocytes can progress in vitro to become growth factor independent and immortal and that both monocyte transformation and immortalization can be dissociated from tumorigenicity.

In vitro transformation of Li-Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.

Transfection of SV40 early region DNA into normal human diploid fibroblasts (NHDFs) increases their proliferative potential to a limited extent. We have investigated the roles of the SV40 large T antigen (LTAg) regions responsible for binding to the protein products of the retinoblastoma (Rb) and p53 genes in this temporary escape from senescence. Patients encoding LTAg mutants were transfected into NHDFs and into Li-Fraumeni syndrome (LFS) fibroblasts which are heterozygous wild-type (wt)/null-mutant for p53. A LTAg mutated in the p53-binding region (T402DE) had greatly reduced efficiency of focus formation, and a p110Rb-binding mutant was unable to induce any foci. T402DE-induced NHDF foci senesced at the same time as untransfected cells, but the equivalent LFS foci all had increased proliferative potentials, with the greatest increase being seen in clones that lost the wt p53 allele. One LFS clone expressed the T402DE mutant during focus formation, but later lost both the T402DE DNA and the wt p53 allele. We conclude that SV40-induced focus formation in NHDFs requires the LTAg p110Rb-binding region, and is enhanced by loss of normal p53 function. In contrast, increased proliferative potential is primarily due to loss of p53 function.

Inhibition of human immunodeficiency virus type 1 replication by the K10-K42 peptide of GAP31 is due to induction of rapid but nonspecific precipitation of viral and nonviral proteins.

The 33-amino acid peptide K10-K42 has previously been described as having potent anti-HIV-1 activity, and antiviral efficacy against hepatitis B and human cytomegalovirus in vitro. Although the exact mechanism of antiviral activity was unknown, it was hypothesised that the K10-K42 peptide inhibited HIV-1 by interfering with one or more of the intracellular processes of reverse transcription, integration, and/or viral gene expression. We performed a series of experiments to identify and characterize the inhibitory mechanism, and to determine whether intracellular expression of the K10-K42 peptide would potentiate its antiviral efficacy in vitro. Surprisingly, our results revealed that the antiviral activity of the K10-K42 peptide could be explained without implicating intracellular inhibition of HIV-1 replication. The activity appeared to be due to an extraordinary capacity of the K10-K42 peptide to precipitate viral and nonviral proteins in vitro. The protein-precipitating capacity of the K10-K42 peptide was sequence specific and a scrambled version of the 33-amino acid peptide did not retain the activity. Although the unusual biochemical properties of the K10-K42 peptide probably negate a number of potential therapeutic applications, they do merit further investigation. Moreover, these findings provide a plausible explanation of the mechanism by which the K10-K42 peptide can inhibit replication of viruses from families as genetically and functionally diverse as Retroviridae, Hepadnaviridae, and Herpesviridae.

Flow cytometric DNA analyses of frozen samples from children's solid tumors.

Cell ploidy and proliferative activity may be useful to clinicians in treating children with solid tumors. Frozen specimens from children with malignant solid tumors were tested at the time of diagnosis for ploidy and proliferative activity. The flow cytometric DNA histograms were examined to assess the sensitivity of the tumor cell preparation and staining method. All the frozen tumors received (n = 58), from children aged 1 mth to 17 yrs, were analyzed and included in this study. The more common tumors were neuroblastomas (n = 21) and Wilms' tumors (n = 16). The majority of tumors (91%) exhibited a diploid peak from the significant proportion of stromal/epithelial cells in the tumor specimen. The tightness of the diploid G0G1 peaks as measured by their coefficient of variations (CV) produced a mean CV of 2.43% +/- SD 0.62, range 1.3% to 4.6%. The G0G1 CVs from the aneuploid peaks had a mean of 2.34% +/- SD 0.68 with the range 0.5% to 3.7%. There was, however, a proportion of tumors (9%) which showed no normal "diploid" peak on the histogram. These tumor cell suspensions had insignificant numbers of diploid cells. Unless the position of the diploid peak could be identified on the DNA histogram the ploidy result from such tumors would be incorrect. Reference diploid cells can be added to cell suspensions prepared from frozen tumors in order to identify the position of the G0G1 peak. Reference diploid cells cannot be added to single cell preparations from paraffin embedded specimens, therefore making it impossible to define the diploid peak position in these tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Serine metabolism in rat embryos undergoing organogenesis.

Rat embryos (9.5 days gestation) were cultured for 48 h in heat-inactivated homologous serum containing [3-14C] serine. Analysis of the distribution of the radioactive label in the conceptus demonstrated that almost one half of the incorporated serine was cleaved to provide one-carbon units for the synthesis of purine and pyrimidine nucleotides. Analysis of the free amino acids in the serum, the exocoelomic fluid and the cells of the yolk sac and the embryo showed that there was a variably selective increase in the concentration of amino acids in the exocoelomic fluid compared with the serum and a significant decrease in the specific radioactivity of the free serine within the conceptus which was the highest in the yolk sac and lowest in the embryo. These findings would support the concept of yolk sac lysosomal degradation of medium serum protein as a major source of amino acids supplying almost 86% of that required during this phase of embryonic development. The specific radioactivity of purine bases in cellular nucleotides, RNA and DNA was similar to that of the free serine in both yolk sac and embryo. This indicated that an alternative, as yet unidentified source of one-carbon units was available for purine nucleotide biosynthesis. Analysis of the cellular purine nucleotides revealed low ATP/GTP ratios in both the embryo and its yolk sac and this may be related to the undifferentiated state of many of the cells of the conceptus.

De novo purine synthesis in cultured rat embryos undergoing organogenesis.

The cultured rat embryo undergoing organogenesis (9.5-11.5 days of gestation) together with its associated yolk sac synthesize purine nucleotides via the de novo synthetic pathway. Although both the embryo and its yolk sac contain significant levels of the purine base salvage enzymes adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the culture medium that consists largely of rat serum contains no measurable quantities of salvageable purine bases or nucleosides but high activity levels of purine catabolic enzymes. Short-term pulse-chase experiments with adenine and guanine, carried out under virtually serum-free conditions, confirmed that purine base salvage mechanisms were active and that there was no significant net transfer of purines between the embryo and its yolk sac. A comparison between the specific radioactivities of the [14C]glycine added to the culture medium for the studies of the de novo synthetic pathway and the purine bases in both the cellular nucleotides and the nucleic acids indicated the existence of a large glycine pool, which almost certainly was derived from the degradation of medium serum proteins by the yolk sac. Although there are no clear-cut data available on the in vivo plasma levels of purines that could be potentially utilized to meet the demands of the embryo, it is evident that the de novo pathway is adequately developed to meet these needs.

Computer simulation modeling of abnormal behavior: a program approach.

A need for modeling abnormal behavior on a comprehensive, systematic basis exists. Computer modeling and simulation tools offer especially good opportunities to establish such a program of studies. Issues concern deciding which modeling tools to use, how to relate models to behavioral data, what level of modeling to employ, and how to articulate theory to facilitate such modeling. Four levels or types of modeling, two qualitative and two quantitative, are identified. Their properties are examined and interrelated to include illustrative applications to the study of abnormal behavior, with an emphasis on schizophrenia.

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene.

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.

De novo purine synthesis in human lymphocytes. Partial co-purification of the enzymes and some properties of the pathway.

A partially purified enzyme extract from lectin-transformed human peripheral blood lymphocytes synthesized purine nucleotides de novo. Although the relatively lower specific activity of the pathway compared with that in the avian liver preparation previously described (Rowe, P. B., McCairns, E., Madsen, G., Sauer, D., and Elliott, H. (1978) J. Biol. Chem. 253, 7711-7721) limited the extent of purification, a number of properties were established: (i) Ammonia could be utilized as readily as glutamine for the synthesis of phosphoribosylamine but only glutamine provided N-3 of the purine ring; (ii) in the presence of either GTP or NAD, AMP or GMP were synthesized; (iii) purine synthesis was inhibited at the level of phosphoribosylamine synthesis by both AMP and GMP, irrespective of whether ammonia or glutamine was the N donor; (iv) while the synthesis of AMP and GMP from IMP was self-regulated, GTP also appeared to be an inhibitor of the synthesis of GMP from IMP; (v) amidophosphoribosyltransferase was isolated from both transformed and nontransformed cells in a low molecular weight form which was converted to a high molecular weight form in the presence of GMP; and (vi) no evidence was obtained for the existence of a classical multienzyme complex for purine synthesis.

Gamma-glutamyl hydrolase conjugase). Purification and properties of the bovine hepatic enzyme.

Bovine hepatic gamma-glutamyl hydrolase (conjugase) has been purified to homogeneity. A feature of the purification procedure was the use of high affinity macromolecular polyanion enzyme inhibitors which formed tight complexes with the enzyme altering its solubility, gel filtration, and ion exchange properties. The enzyme, which cleaves the gamma-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with its lysosomal localization. Zinc is essential for enzyme stability. The presence of highly reactive sulfhydryl groups was evident from the extreme sensitivity to oxidizing agents and organomercurials. Very little thermal denaturation occurs below 65 degrees, but the enzyme is extremely sensitive to 0uffer anions, in keeping with the polyanionic nature of the substrate. In order to study the mechanism of action of the enzyme, a wide range of pteroylpolyglutamates, N-t-Boc polyglutamates and free polyglutamates were synthesized containing L-[U-14C]glutamic acid residues in different positions. Two pteroyltriglutamate derivatives were also synthesized in which an alpha bond replaced one of the two available gamma bonds. Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at gamma-glutamyl bonds and the presence of a COOH-terminal gamma bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should therefore be more appropriately classified as an acid hydrolase; (c) longer chain gamma-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of gamma bonds was independent of the NH2-terminal pteroyl moiety. Studies with polyanions such as the glycosaminoglycans and dextran sulfate supported the concept that the polyanion structure of the substrate was a major factor in substrate-active site interaction.