Structure and differential mechanisms of regulation of expression of a serine esterase gene in activated human T lymphocytes.

A cDNA clone encoding a polypeptide resembling proteolytic serine esterases (from cytotoxic T-cells; SECT) was isolated from human peripheral blood lymphocytes which had been cultured in the presence of the T-cell mitogen phytohemagglutinin for 72 h. The cDNA encodes a polypeptide of 247 amino acids which show homology of 99% with the protein sequence encoded by a cDNA clone (1-3E) isolated from staphylococcal enterotoxin A-stimulated human peripheral blood lymphocytes and 68% with the protein sequence (cytotoxic cell protease type I) derived from a cDNA clone (C11) encoding a serine esterase isolated from a murine cytotoxic T-cell line. The overall nucleotide sequence homology between the SECT cDNA and 1-3E was 99% and 73% between SECT and C11. Comparing the coding regions of SECT and C11 showed 75% homology, whereas the 5'- and 3'-untranslated regions showed 67% homology. Phytohemagglutinin stimulation results in 30-, 60-, and 370-fold increases in cytoplasmic SECT mRNA with respect to unstimulated cells after 6-, 24-, and 72-h cultures, respectively. At 6 h, the increase in SECT mRNA occurs in the absence of increases in SECT gene transcription and cytoplasmic mRNA stabilization. A 5-fold increase in SECT nuclear RNA seen at this time suggests that stabilization of SECT nuclear RNA transcript is responsible for early increases in SECT mRNA levels. At 24 and 72 h, the increased cytoplasmic SECT mRNA levels can be accounted for by increased transcriptional activity of the SECT gene.

Growth-related changes in specific mRNAs upon lectin activation of human lymphocytes.

A cDNA library in lambda gt10 was constructed from the cytoplasmic poly(A) +RNA of human peripheral blood lymphocytes after 72 hr of phytohemagglutinin stimulation, with the aim of assessing selective gene expression as a result of lymphocyte activation. Thirteen recombinants were isolated by the use of an enriched probe and differential screening. These clones were categorized into two groups with respect to their hybridization to mRNA. In the first group three recombinants were isolated, which hybridized to single discrete mRNAs in the size range 0.7-1.7 kb. The mRNAs corresponding to these clones were present at elevated levels in activated lymphocytes, but the kinetics of increase differed. The 0.7-kb mRNA coded for by clone p1L1 increased two-fold at 6 hr and remained elevated over 72 hr, as did beta-actin mRNA. The 1.7-kb mRNA coded for by clone p9L2 increased two- to three-fold after 6 hr and was maximally expressed after 24 hr exposure to phytohemagglutinin, coincident with the onset of DNA replication, and maintained this level up to 72 hr. The 1.0-kb mRNA coded by p10L2F which was rare in resting cells increased 25- to 30-fold after 6 hr, prior to overall transcriptional increases and reached peak levels after 72 hr when a substantial proportion of the cells were in the S and G2 + M phases of the cell cycle. This clone was undetectable or very rare in the leukemic T-lymphoblast cell line CCRF-CEM. The second group of clones, consisting of the remaining 10 recombinants, did not hybridize to discrete bands, but to a smear on RNA blots.(ABSTRACT TRUNCATED AT 250 WORDS)

One-carbon metabolism in lectin-activated human lymphocytes.

Serine is an essential amino acid for the lectin-mediated transformation of human peripheral blood lymphocytes due to the inability of this cell to synthesize sufficient quantities via either the phosphorylated pathway or by reversal of the serine hydroxymethyltransferase reaction to meet the metabolic demands. The level of intracellular serine is tightly regulated, and the culture medium concentration for optimum cellular transformation falls within a relatively narrow range. The three-carbon atom of serine is the major source of one-carbon units required for purine and pyrimidine nucleotide biosynthesis, but the key effect of both serine deprivation and of high medium serine levels would appear to be on protein synthesis. Although an alternative source of one-carbon units, as provided by high levels of formate in the culture medium, can partially reverse the effects of serine deprivation, the only other demonstrable source of one-carbon units, tryptophan, requires serine for its incorporation and subsequent metabolism. Methionine is also essential for lymphocyte transformation and is involved in the synthesis of a small amount of phosphatidylcholine, although most of this phospholipid is provided by choline and lysophosphatidylcholine from the serum-supplemented culture medium.

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.

Profiles of urinary volatiles from metabolic disorders characterized by unusual odors.

The profiles of urinary volatiles from patients with phenylketonuria, maple syrup urine disease, isovaleric acidemia, or trimethylaminuria (fish-odor syndrome) were in each case vastly different from the normal urinary volatiles profile. In the maple syrup urine disease case, metabolites that occur distal to the block were found and a mechanism for their formation is suggested. A new major metabolite in isovaleric acidemia was also found. As well as providing a reliable diagnostic tool for diseases characterized by odors, the analysis of urinary volatiles may provide information to help our understanding of still unexplained aspects of the diseases.

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.

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.

The effects of lectin transformation on cytoplasmic polyadenylated RNA from human lymphocytes.

The translational activity of cytoplasmic poly(A)+ RNA from resting human lymphocytes was approximately 20% of that from phytohemagglutinin-transformed lymphocytes in a rabbit reticulocyte lysate assay. Translation assays in the presence of cap analogues suggested that the mRNA from resting cells was relatively deficient in functional 5'-terminal cap structures. Neither mRNA fraction inhibited the translation of globin mRNA in the cell-free assay, and both preparations were essentially pure as shown by hybridisation with [3H]poly(U). The size distribution and poly(A) tail length of poly(A)+ RNA was similar in the resting and transformed cell and both preparations directed the synthesis of peptides of molecular weight 15 000 to 90 000. Two dimensional gels of total proteins from resting and transformed lymphocytes showed predominantly quantitative changes. However cross-hydridising cDNA and mRNA from resting and transformed cells after the common sequences have been removed by hydroxylapatite chromatography showed that about 4% of the cytoplasmic poly(A)+ RNA from transformed lymphocytes was not present in resting cells. This difference may result from transformation-specific gene expression.

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.).

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N(2) per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N(2) per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.

New metabolites in isovaleric acidemia.

Two metabolites, 4-hydroxyisovaleric acid and mesaconic acid, have been identified and quantified in the urine of a patient with isovaleric acidemia. These compounds do not appear to have been reported previously as being components of human metabolism. In addition, large quantities of 3-methylbutyrolactone, the lactone of 4-hydroxyisovaleric acid, were observed in the volatile profile obtained by headspace chromatography. The demonstration of 4-hydroxyisovaleric acid supports the contention that urinary methylsuccinic acid seen in patients with isovaleric acidemia has arisen by omega-oxidation of isovaleric acid. The identification of mesaconic acid may indicate that the methylsuccinic acid formed in these patients is subject to further metabolism.

Two new sulphur-containing amino acids in man.

Two unusual sulphur-containing amino acids have been isolated from urine of a baby who died with major physical malformations and failure of growth and development. Sensitive mass spectrometric methods were used to identify the nanomole quantities of the compounds available as S-(2-carboxypropyl)-cysteine and S-(2-carboxypropyl)-cysteamine. Incubation of fibroblasts in either [14C]Valine or [35S]cysteine resulted in radioactive labelling of the compounds, suggesting their origin from conjugation of methacrylic acid with cysteine and subsequent decarboxylation of the cysteine conjugate. Specific assay of methacrylyl-CoA hydratase is needed for final proof that this is a new inborn error of that enzyme, but these findings and parental consanguinity make this very likely. It seems possible that methacrylic acid or one of its derivatives may have caused the malformations present in the baby.

De novo purine synthesis in avian liver. Co-purification of the enzymes and properties of the pathway.

The enzymes of the de novo purine biosynthetic pathway have been partially co-purified from pigeon liver by a method dependent upon the use of the nonionic polymer polyethylene glycol for enzyme stabilization and cofractionation. Although the enzymes did not appear to constitute a large macromolecular complex it was evident that some particular inter-relationship between them was preserved during the purification procedure. Analysis of the end products and pathway intermediates was carried out primarily by sensitive high pressure liquid chromatographic techniques. Substrate and cofactor requirements were confirmed and optimal conditions of pH, temperature, and K+ ion activation established. At phosphoribosyl pyrophosphate (PP-ribose-P) concentrations below 0.3 mM the activity of the first pathway enzyme amidophosphoribosyltransferase was rate-limiting, and the inhibition of this enzyme by AMP regulated the rate of purine ring synthesis. At higher concentrations of PP-ribose-P, aminoimidazole ribonucleotide synthetase, the fifth enzyme of the pathway became rate limiting and was subject to inhibition by added AMP. It was evident that the regulation of purine synthesis was quite complex and that AMP inhibition (perhaps reflected in a low adenylate energy charge) can be effected at different points on the purine pathway.