Induction of interleukin 2 production but not methionine adenosyltransferase activity or S-adenosylmethionine turnover in Jurkat T-cells.

We have recently reported that methionine adenosyltransferase (MAT) in resting human peripheral blood T-cells is primarily present in the form of a precursor which we named lambda. This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits of the enzyme increase. When resting cells are activated by phytohemagglutinin, the decrease in lambda and increase in alpha/alpha' occurs after interleukin 2 (IL-2) production and before DNA synthesis. The human T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in response to exogenous stimuli such as T-cell mitogens and therefore provides a convenient model for studying biochemical reactions involved in T-cell activation. In this study the regulation of MAT activity and S-adenosylmethionine (AdoMet) in resting and activated Jurkat cells was investigated. Here we report that MAT activity in unstimulated Jurkat cells is about 10- and 3-fold higher than the activity in resting and activated peripheral blood mononuclear cells, respectively. Activation of Jurkat cells with phytohemagglutinin resulted in increased IL-2-production, but not an increase in MAT activity. Identical results were obtained using freshly isolated cells from acute lymphoblastic leukemia patients. AdoMet utilization and pool size were approximately 3- and 10-fold higher, respectively, in Jurkat cells compared to peripheral blood mononuclear cells, and both parameters were unaffected by phytohemagglutinin stimulation. Jurkat MAT was determined to be structurally indistinguishable from enzyme from T- or B-leukemia cells but was different from resting, normal T-cells in that it lacked the lambda form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the alpha, alpha', and beta subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in Jurkat cells are constitutively high and that induction of IL-2 synthesis in these cells is independent of changes in AdoMet synthesis or turnover. The lack of the lambda form and the difference in MAT regulation between leukemic T-cells and peripheral blood mononuclear cells may be exploited in the design of specific chemotherapeutic agents.

Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells.

Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6-11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6-12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by alpha-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Characterization of distinct forms of methionine adenosyltransferase in nucleated, and mature human erythrocytes and erythroleukemic cells.

Two peaks of methionine adenosyltransferase (MAT) activity from human erythrocytes were partially purified on a DEAE-cellulose column. Using anti-MAT antibodies, a 60 kDa form of MAT, referred to as rho was identified in peak I. Although rho represented the major MAT protein in crude erythrocyte extracts, the enzyme was very labile and accounted for only 6% of the total MAT activity. Peak II enzyme was stable, and consisted of the previously described catalytic alpha (53 kDa) subunit and the beta subunit (38 kDa), both of which are found in activated human lymphocytes and leukemic cells of lymphoid origin. Mature normal and polycythemic erythrocytes contained predominantly rho as the major MAT protein, while nucleated erythrocytes and reticulocytes contained predominantly the lambda (68 kDa), the major form found in resting human lymphocytes. Human erythroleukemic cells (HEL 92.1.7) contained the alpha, alpha' and beta subunits of MAT, and in this regard was indistinguishable from MAT found in activated lymphocytes and leukemic cells of lymphoid origin (Jurkat). Since rho was generated during the incubation of extracts from resting lymphocytes, which contain predominantly lambda, in the absence of protease inhibitors; the rho form of MAT appears to be derived from the lambda form by proteolytic cleavage. The data indicate that distinct forms of MAT are present at different stages of erythrocyte maturation and reveal the presence of a new form of MAT with reduced activity compared to previously described forms.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Methionine adenosyltransferase: structure and function.

Methionine adenosyltransferase (MAT), a key enzyme in metabolism, catalyzes the synthesis of one of the most important and pivotal biological molecules, S-adenosyl-methionine. In every organism studied thus far, MAT exists in multiple forms; most are encoded by related, but distinct genes. Molecular and immunological studies revealed the presence of considerable conservation in the structure of MAT from different species; however, the various MAT isozymes differ in their physical and kinetic properties in ways that allow them to be regulated differently. Recent studies suggest that human MAT is composed of nonidentical subunits that can assume multiple states of aggregation, each with different kinetic characteristics. The tissue distribution of MAT isozymes and the ability of cells within the same tissue to switch between the different forms of MAT suggest that this mode of regulation is important for cellular function and differentiation. Therefore, understanding the regulation and structure-function relationship of this fascinating enzyme should help us clarify its role in biology and may provide us with tools to effectively manipulate its activity in clinical situations such as cancer, autoimmunity and organ transplantation.

Creation of a functional S-nitrosylation site in vitro by single point mutation.

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.

Behavioural assessment of mice lacking D1A dopamine receptors.

Dopamine D1A receptor-deficient mice were assessed in a wide variety of tasks chosen to reflect the diverse roles of this receptor subtype in behavioural regulation. The protocol included examination of exploration and locomotor activity in an open field, a test of sensorimotor orienting, both place and cue learning in the Morris water maze, and assessment of simple associative learning in an olfactory discrimination task. Homozygous mice showed broad-based impairments that were characterized by deficiencies in initiating movement and/or reactivity to external stimuli. Data obtained from flash evoked potentials indicated that these deficits did not reflect gross visual impairments. The partial reduction in D1A receptors in the heterozygous mice did not affect performance in most tasks, although circumscribed deficits in some tasks were observed (e.g., failure to develop a reliable spatial bias in the water maze). These findings extend previous behavioural studies of null mutant mice lacking D1A receptors and provide additional support for the idea that the D1A receptor participates in a wide variety of behavioural functions. The selective impairments of heterozygous mice in a spatial learning task suggest that the hippocampal/cortical dopaminergic system may be uniquely vulnerable to the partial loss of the D1A receptor.

The organophosphate pesticide chlorpyrifos affects form deprivation myopia.

PURPOSE: The effects of the anti-cholinesterase organophosphate pesticide chlorpyrifos (CPF) on the refractive development of the eye were examined. Form deprivation was used to induce eye growth to address the previously reported relationship between organophosphate pesticide use and the incidence of myopia. METHODS: Chickens, a well-established animal model for experimental myopia and organophosphate neurotoxicity, were dosed with chlorpyrifos (3 mg/kg per day, orally, from day 2 to day 9 after hatching) or corn oil vehicle (VEH) with or without monocular form deprivation (MFD) over the same period. The set of dependent measures included the refractive state of each eye measured using retinoscopy, axial dimensions determined with A-scan ultrasound, and intraocular pressure. RESULTS: Dosing with CPF yielded an inhibition of 35% butyrylcholinesterase in plasma and 45% acetylcholinesterase in brain. MFD resulted in a significant degree of myopia in form-deprived eyes resulting from significant lengthening of the vitreal chamber of the eye. CPF significantly reduced the effect of MFD, resulting in less myopic eyes (mean refraction: VEH-MFD = -16.2 +/- 2.3 diopters; CPF-MFD = -11.1 +/- 1.8 diopters) with significantly shorter vitreal chambers. Nonoccluded eyes were, on average, slightly hyperopic. Treatment with CPF for 1 week in the absence of MFD led to no significant change in ocular dimensions or refraction relative to controls. CONCLUSIONS: The use of form deprivation as a challenge suggests that CPF treatment interferes with the visual regulation of eye growth.

Chronic use of benzodiazepines and psychomotor and cognitive test performance.

The performance of 43 long-term users (average = 5 years) of benzodiazepine (BZ) medications was examined on a battery of behavioral tasks, cognitive tests, and subjective mood rating scales. The performance of the chronic BZ users did not differ significantly from age- and sex-matched anxious subjects, except that critical flicker fusion (CFF) thresholds were lower and subjective ratings of tranquilization were higher in the BZ users. Twenty-two subjects were reexamined in order to determine the acute effects of BZ medications in long-term users. The acute administration of BZ medications significantly increased CFF thresholds, improved digit-symbol substitution test performance, impaired the delayed recall of verbal material, increased subjective ratings of tranquilization, and reduced physical sedation. Motor performance tests were not impaired and subjective feelings of sedation were not increased after the acute administration of BZs by chronic users. During withdrawal from long-term BZ use (17 subjects), CFF thresholds were elevated, subjective ratings of physical sedation and anxiety were increased, but performance on other psychomotor and cognitive tests was not altered. The results suggest that tolerance develops selectively to different behavioral and subjective effects of BZ medications with their continued use. Tolerance failed to develop to the antianxiety effects, the reduction of CFF threshold, and to the impairment of short-term memory caused by BZs. However, chronic users of BZ medications failed to demonstrate psychomotor-impairing or sedating effects to BZ medications. The results have implications for evaluating the safety of the long-term use of BZ medications.

Gender-dependent behavioral and sensory effects of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) in rats.

Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with behavioral and cognitive deficits in humans and animal models. Perinatal exposure to PCBs has also been associated with sensory deficits in animal models. These effects were hypothesized to be mediated in part by ortho-substituted PCBs, which do not or weakly bind to the aryl hydrocarbon (Ah) receptor. The present studies were designed to determine whether perinatal exposure to Aroclor 1254, a commercial mixture of > 99% ortho-substituted PCBs, would affect cognitive and sensory function in Long-Evans rats. Adult male and female offspring of female rats fed Aroclor 1254 (Lot #124-191; doses of 0, 1, or 6 mg/kg/day; gestational day 6 through postnatal day 21; n = eight/group) were trained to perform a signal detection task capable of assessing sensory thresholds. Training included autoshaping and operant conditioning. Thresholds for detecting a 1-s light stimulus were determined under background illuminations ranging from 2 lux to complete darkness. Female rats exposed to Aroclor 1254 autoshaped more rapidly than control females, at a rate akin to control males. Control females had lower thresholds than control males at all levels of background illumination. These differences were abolished by Aroclor 1254, which reduced thresholds in males and increased thresholds in females. These data extend previous findings of gender-specific effects of PCBs on neurobehavioral development to measures of acquisition and sensory function.

Assessment of foveal cone photoreceptors in Stargardt's macular dystrophy using a small dot detection task.

We measured frequency-of-seeing curves for tiny (1.125 and 3.375 min arc) stimuli flashed briefly at absolute threshold to estimate the density of foveal cones in normals and in subjects with Stargardt's macular dystrophy. Foveal absolute thresholds for Stargardt's were elevated 1.5 log units over normal. Analysis using Poisson counting statistics indicated that the quantal absorption to stimulate individual cones was normal for Stargardt's but that effective optical density of individual cones was reduced by > 1 log. Numerical density of foveal cones was reduced 1 log unit for Stargardt's patients with acuities of 20/30-20/100.

A table of color distance scores for quantitative scoring of the Lanthony Desaturate color vision test.

The Lanthony Desaturate Panel D-15 (D-15d) color vision test is used in neurotoxicological testing to assess acquired color vision deficits. The original test design included a qualitative scoring method. Quantitative scoring requires mapping the colored objects used in the test into a color space describing perceptual distances. A table of these distances has previously been published for the saturated version of this color vision test, but not the desaturate test. This communication includes a table of color distances for the calculation of Bowman's Total Color Distance Score (TCDS) for the D-15d. This table should be useful for non-computerized scoring under field test conditions or for devising one's own computerized scoring methods using the tabulated color distances for a look-up table. Data analysis programs using SAS or Matlab are available from the author.

Critical issues in the use and analysis of the Lanthony Desaturate Color Vision test.

The Lanthony Desaturate Color Vision test (D-15d) has been used to demonstrate the incidence of acquired color vision defects resulting from toxic exposure. The D-15d is a sensitive test designed to grade color deficiencies, but results can be difficult to interpret beyond the qualitative level, and the high incidence of errors reported for controls in some toxicology studies raises questions about how to effectively use this test. This article reviews standard administration of the test, physical determinants of performance, classification of acquired color vision defects, and methods of analysis that have been used to quantify results. The basis for a new method of analysis is discussed, illustrating the source of some characteristic errors, and recommendations are made for test protocols to attempt to more closely identify the type of color vision loss with the goal of identifying the site of toxicological insult.

Behavioral and electrophysiological estimates of visual thresholds in awake rats treated with 3,3',4,4',5-pentachlorobiphenyl (PCB 126).

Visual thresholds for luminance increments were obtained behaviorally and electrophysiologically from rats exposed to a polychlorinated biphenyl (PCB) during development. Male Long-Evans rats exposed to 0, 0.25, or 1.0 microg/kg/day of 3,3',4,4', 5-pentachlorobiphenyl (PCB 126) through gestation and weaning were trained as adults to perform a signal detection task. Estimates of threshold were derived from psychometric functions for each animal relating the proportion of hits to signal intensity. Thresholds derived under three luminance conditions did not differ significantly among the PCB-treated groups. After behavioral testing was completed, flash-evoked potentials were recorded from dark-adapted awake animals. Peak amplitudes increased linearly over approximately 3 log units of intensity. Extrapolations to 0 amplitude along the linear portion of the amplitude-log intensity functions produced estimates of absolute threshold of -5.44 to -5.53 log cd/m(2)-s. Waveforms recorded from awake animals had a large late negative component that was absent in previously reported anesthetized preparations. Developmental exposure to PCB 126 had no significant effect on absolute threshold or peak amplitudes and latencies.

A binding assay for serine hydroxymethyltransferase.

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.