Adenosine Deaminase Enzyme Activity Research Articles

Specific increase in enzymatic activity of adenosine deaminase 1 in rheumatoid synovial fibroblasts.

Adenosine deaminase (ADA; EC 3.5.4.4) activity is elevated in the synovial fluid (SF) of patients with rheumatoid arthritis (RA). Since the antiinflammatory effect of methotrexate is reportedly associated with increased levels of extracellular adenosine, the present study was undertaken to clarify the role of 2 ADA isozymes, ADA1 and ADA2, in the pathogenesis of RA. The activities of ADA1 and ADA2 were measured in SF from RA and osteoarthritis (OA) patients, in sera from RA patients, and in lysates prepared from mononuclear and polymorphonuclear cells from SF from RA patients, peripheral blood from RA patients, and fibroblast-like synoviocytes (FLS) from RA and OA patients. Also measured were the effects of proinflammatory cytokines on ADA1 activity and ADA messenger RNA (mRNA) expression in RA FLS, as determined using real-time polymerase chain reaction. The adenosine concentration in RA SF was measured by radioimmunoassay. The adenosine concentration in RA SF ranged from 0.027 microM to 0.508 microM (mean +/- SD 0.156 +/- 0.132 microM). At those concentrations, ADA1 would be expected to be functionally dominant due to its higher affinity for adenosine. ADA1 activity in RA SF (mean +/- SD 14.4 +/- 8.5 IU/liter) was significantly higher than that in OA SF (3.0 +/- 1.1 IU/liter) or RA sera (3.0 +/- 0.6 IU/liter); moreover, ADA1 activity in RA FLS lysate was the highest among the cell lysates tested. Proinflammatory cytokines did not affect ADA1 activity or ADA mRNA expression in RA FLS. Elevated ADA1 activity is an intrinsic characteristic of RA FLS, which likely contributes to the pathogenesis of RA by neutralizing the antirheumatic properties of endogenous adenosine.

Adenosine deaminase enzyme activity is increased and negatively correlates with catalase, superoxide dismutase and glutathione peroxidase in patients with Behçet's disease: original contributions/clinical and laboratory investigations.

AIM: Behçet's disease (BD) is an inflammatory vasculitis with immunologic, endothelial and neutrophil alterations. Adenosine deaminase (AD) is a marker of T-cell activation and is related to the production of reactive oxygen species by neutrophils with the production of NO(*), O(2)(*-), H(2)O(2) and OH(*). We reported increased tumour necrosis factor-alpha, soluble interleukin-2 receptor, interleukin-6, interleukin-8 and NO(*) in active BD. As there is a relation between cytokines, T cells and oxidative stress in inflammatory diseases, this study further evaluated: (1) plasma AD activity and its correlation with acute phase reactants; (2) thiobarbituric acid-reactive substances (TBARS) as an indicator for lipid peroxidation; and (3) antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase in patients with BD. The effect of disease activity and correlations between the measured parameters were explored. METHODS: A total of 35 active (n=17) or inactive (n=18) patients with BD (16 men, 19 women) satisfying International Study Group criteria, and 20 age-matched and sex-matched controls (nine men, 11 women) were included in this cross-sectional case-control study. AD and TBARS were measured in plasma, catalase in red blood cells (RBC), and SOD and GSHPx in both plasma and RBC in both groups. Acute phase reactants (alpha(1)-antitrypsin, alpha(2)-macroglobulin, neutrophils, erythrocyte sedimentation rate) were used to classify patients as active or inactive. RESULTS: Plasma AD (mean+/-standard error of the mean, 36.1+/-0.7 U/l) and TBARS (4.2+/-0.1 nmol/ml) levels were significantly (for each, p<0.001) higher in BD than in controls (24.1+/-0.8 U/l and 1.6+/-0.1 nmol/ml, respectively). RBC catalase activity was significantly (p<0.001) lower in BD than in controls (120.9+/-3.8 versus 160.3+/-4.1 k/g haemoglobin). SOD and GSHPx activities were significantly lower in both plasma and erythrocytes of patients with BD than in controls (plasma SOD, 442.4+/-8.6 versus 636.4+/-9.2 U/ml, p<0.001; RBC SOD, 3719.2+/-66.0 versus 4849.7+/-49.0 U/g haemoglobin, p<0.001; plasma GSHPx, 73.1+/-1.5 versus 90.6+/-2.9 U/ml, p<0.001; RBC GSHPx, 600.7+/-8.0 versus 670.6+/-10.1 U/g haemoglobin, p<0.001). Active BD patients had significantly lower antioxidant enzymes (except RBC catalase) and higher AD and TBARS levels than inactive subjects (for each, p<0.01). When considering all BD patients, a significant positive correlation was present between AD and TBARS (p<0.001) whereas both AD and TBARS were negatively correlated with antioxidant enzymes (for each, p<0.05). CONCLUSIONS: AD and lipid peroxidation are increased and associated with defective antioxidants in BD, suggesting interactions between activated T cells and neutrophil hyperfunction. Measures of pro-oxidative stress and antioxidative defence with AD activity as an indicator of T-cell activation can be considered as significant supportive diagnostic indicators, especially in active disease. In addition, strengthening the antioxidant defence may contribute to treatment modalities.

Flow cytometry analysis of adenosine deaminase (ADA) expression: a simple and reliable tool for the assessment of ADA-deficient patients before and after gene therapy.

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.

Adenosine-dependent airway inflammation and hyperresponsiveness in partially adenosine deaminase-deficient mice.

Adenosine is a signaling nucleoside that is elevated in the lungs of asthmatics. We have engineered a mouse model that has elevated levels of adenosine as a result of the partial expression of the enzyme that metabolizes adenosine, adenosine deaminase (ADA). Mice with lowered levels of ADA enzymatic activity were generated by the ectopic expression of an ADA minigene in the gastrointestinal tract of otherwise ADA-deficient mice. These mice developed progressive lung inflammation and damage and died at 4-5 mo of age from respiratory distress. Associated with this phenotype was a progressive increase in lung adenosine levels. Examination of airway physiology at 6 wk of age revealed alterations in airway hyperresponsiveness. This was reversed following the lowering of adenosine levels using ADA enzyme therapy and also through the use of the adenosine receptor antagonist theophylline, implicating both the nucleoside and its receptors in airway physiological alterations. All four adenosine receptors were expressed in the lungs of both control and partially ADA-deficient mice. However, transcript levels for the A(1), A(2B), and A(3) adenosine receptors were significantly elevated in partially ADA-deficient lungs. There was a significant increase in alveolar macrophages, and monocyte chemoattractant protein-3 was found to be elevated in the bronchial epithelium of these mice, which may have important implications in the regulation of pulmonary inflammation and airway hyperresponsiveness. Collectively, these findings suggest that elevations in adenosine can directly impact lung inflammation and physiology.

T-cell lines from 2 patients with adenosine deaminase (ADA) deficiency showed the restoration of ADA activity resulted from the reversion of an inherited mutation

AbstractInherited deficiency of adenosine deaminase (ADA) results in one of the autosomal recessive forms of severe combined immunodeficiency. This report discusses 2 patients with ADA deficiency from different families, in whom a possible reverse mutation had occurred. The novel mutations were identified in the ADAgene from the patients, and both their parents were revealed to be carriers. Unexpectedly, established patient T-cell lines, not B-cell lines, showed half-normal levels of ADA enzyme activity. Reevaluation of the mutations in these T-cell lines indicated that one of the inherited ADA gene mutations was reverted in both patients. At least one of the patients seemed to possess the revertant cells in vivo; however, the mutant cells might have overcome the revertant after receiving ADA enzyme replacement therapy. These findings may have significant implications regarding the prospects for stem cell gene therapy for ADA deficiency.

The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277–285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyialongipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophilamelanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.

Adenosine deaminase‐deficient mice as models for adenosine‐mediated lung inflammation and damage

AbstractAdenosine has been implicated to play a role in the inflammatory lung disease, asthma; however, there are few in vivo model systems with which to study the role of adenosine in aspects of this disease. We recently generated mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) that controls the levels of adenosine in tissues and cells. Results presented here demonstrate that elevated adenosine levels in ADA‐deficient mice result in abnormal lung development and the promotion of lung inflammation and damage. ADA‐deficient mice exhibit alveolar defects that are overcome by biochemically restoring ADA enzymatic activity to these animals. In addition, lowering ADA substrates in the lung using enzyme therapy reverses lung eosinophilia and mucus production. These results suggest that elevated adenosine, and perhaps abnormal adenosine signaling, are implicated in abnormal lung development and lung inflammation and damage in ADA‐deficient mice. These mice will therefore serve as useful in vivo models in which to study the role of purinergic signaling in aspects of lung development and disease. Drug Dev. Res. 52:416–423, 2001. © 2001 Wiley‐Liss, Inc.

Expression of murine adenosine deaminase (ADA) in transgenic maize.

A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta or the adenosine analogue 2'-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41-kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Culltures expressing ADA grew in the presence of 200 mg/l 2'-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progency of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.

Development of improved adenosine deaminase retroviral vectors.

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a "simplified" vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA-) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA- severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.

The Effect of Paraquat on Adenosine Deaminase Activity in Different Tissues of Mice (Mus musculus - Swiss Albino}

In this study, the effects of a LD{_30} (20 mg/kg{^-l}) dose of paraquat, a herbiside, was applied intraperitoneally to Mus musculus swiss albino mice have been investigated on adenosine deaminase activity in the Iiver, kidney and Iung of mice after 2, 4, 8, 16, 32, 64 and 72 hours. Paraquat did not cause an important change on the adenosine deaminase activity in the Iiver and kidney. While adenosine deaminase activity of the Iivers constantly showed parallelism with the control group, some insignificant increase and decrease have been found on the adenosine deaminase Ievels of the kidney. Meatume in the Iung in the beginning, while there was a significant inhibition in adenosine deaminase enzyme activity after 16th hour this inhibition replaced by activation. And statistically significant increase was found in the 64th and 72nd hours.

Molecular Analysis of T Lymphocyte-Directed Gene Therapy for Adenosine Deaminase Deficiency: Long-Term ExpressionIn Vivoof Genes Introduced with a Retroviral Vector

Peripheral blood lymphocytes from a patient with adenosine deaminase (ADA) deficiency were transduced in vitro with a replication-defective retroviral vector containing a human ADA-cDNA. Eighteen months after the last of a series of infusions of autologous retroviral vector-treated cells, vector sequences were detectable in DNA isolated from peripheral blood mononuclear cells (PBMCs), with an average copy number approaching one per cell. Increased ADA enzyme activity reaching approximately one-quarter normal levels was found in this population of cells. Other evidence of long-term retroviral vector expression in vivo included neomycin phosphotransferase (NPT) activity and demonstration of persistent vector mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). No evidence of spontaneous reversion of either mutant endogenous ADA allele was found.

Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase.

The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA-specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA-binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site-directed mutations within the highly conserved CHAE sequence of the postulated C-terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD.

Prognostic significance of adenosine deaminase in children with malignancies.

In 33 children, 23 with acute lymphoblastic leukemia (ALL) and 10 with solid tumors, the phenotype of the enzyme adenosine deaminase (ADA) was detected in the erythrocytes by electrophoresis in cellulose acetate. In all children the ADA enzyme activity was also determined in the plasma by spectrophotometry at the onset of the disease, during remission, at the end of the therapy, and during relapse. The phenotype in all children was ADA1-1. There was a difference in enzyme activity between the mean values of children with ALI and those with solid tumors. There were also differences among the subtypes of ALL and also among the stages of ALL and the stages of solid tumors. In 23 children with ALL the mean value (MV) and the corresponding standard error (SEM) of enzyme activity at the onset of the disease were MV +/- SEM = 60.2 +/- 6.2 IU/L. This was higher than that of the control group (control group: MV +/- SEM = 27.8 +/- 3.3 IU/L). During remission the enzyme activity was lower than that of the control group (MV +/- SEM = 19.6 +/- 1.7 IU/L); at the end of the therapy it was MV +/- SEM = 24.0 +/- 1.3 IU/L, which is close to that of the control group; and during relapse it was much higher compared with the control group (MV +/- SEM = 73.1 +/- 4.6 IU/L). These values are discussed in connection to the leukaemic subtypes. In 10 children with solid tumors the mean value of enzyme activity at the onset of the disease was MV +/- SEM = 48.8 +/- 2.4 IU/L. During therapy it was MV +/- SEM = 32.4 +/- 1.9 IU/L and at the end of therapy MV +/- SEM = 22.1 +/- 2.5 IU/L. The aim of this work is to study the qualitative isoenzyme abnormalities to better understand the biological nature of the malignancies, to distinguish main groups and subsets of ALL and solid tumors on an enzymatic basis, and to identify possible sensitive key enzymes as targets for chemotherapy.

Long-Term Culture and T Cell Receptor Analysis of T Cell Clones Isolated from a Patient with Adenosine Deaminase Deficiency and Type I Diabetes

We performed limiting dilution culture of T cells from a patient affected by primary immunodeficiency as a result of complete lack of adenosine deaminase (ADA) activity and also affected by insulin-dependent diabetes mellitus (type I diabetes). Despite the occurrence of immunodeficiency, we were able to raise and grow T cell clones derived from this patient in long-term culture. These T cells displayed ADA enzymatic activity and produced interleukin-2 after engagement of their T cell receptor (TCR)/CD3 complex. We analyzed the TCR repertoire of such clones by nucleotide sequencing of TCR β chains. The results show that the T cell clones express different Vβ but similar J regions. However, the CDR3 regions which are implicated in antigen recognition were found to be heterogeneous.

Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

Transfer of the ADA Gene Into Human ADA-Deficient T Lymphocytes Reconstitutes Specific Immune Functions

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

Ontogeny of Adenosine Deaminase in the Mouse Decidua and Placenta: Immunolocalization and Embryo Transfer Studies1

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.

Cardiovascular purinoceptors

Cardiovascular purinoceptors

The regional distribution of adenosine-regulating enzymes in the left and right ventricle walls of control and hypertrophic heart

The transmural distribution of the adenosine-generating enzyme 5'-nucleotidase (5'N) and of the adenosine-degrading enzymes adenosine deaminase (ADA), AMP deaminase (AMP-D) and adenosine kinase (Ado-K) were determined across the walls of left and right ventricles of control and hypertrophic rat hearts. The enzyme distribution across the left ventricle wall (but not across the right wall) of normal hearts was not uniform: 5'N activity shows its highest levels in the subepicardial and in the subendocardial regions, whereas all the other enzyme activities show their lowest levels. A similar pattern of transmural distribution was also detected in other mammalian species (ox and pig). In the experimental cardiac hypertrophy, caused by two different types of chronic cardiac overload, the levels and the profiles of transmural distribution of 5'N and ADA enzyme activities may significantly change across the rat left ventricle wall.

Hypomethylation andADA gene expression in mouse CAK cells

The adenosine deaminase (ADA) locus appears to be under complex transcriptional control since levels of ADA enzyme activity vary greatly between different tissues and stages of development. Evidence that a trans-acting factor may be involved with the regulation of this locus came from previous experiments where fusion of ADA-negative human JEG cells and mouse ADA-positive cells led to the trans-activation of human ADA in a hybrid nucleus. Here, we demonstrate that the near euploid mouse embryo fibroblast cell line, CAK, also lacks detectable ADA enzyme activity due to altered gene regulation. We further demonstrate that ADA in CAK cells is not amenable to activation by somatic cell fusion. Following treatment with 5-azacytidine and Xyl-A selection (for ADA), however, CAK clones were obtained that stably express the ADA gene. Molecular analysis of the parental CAK cells and the ADA-positive derivative clones demonstrated that both 5' and 3' regions of the ADA gene had become hypomethylated in the ADA+ clones. We conclude that methylation is another element involved with the transcriptional control of the ADA gene and that ADA might serve as a useful model for studying the interaction of cis- and trans-acting regulational elements.