Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells

Methionine adenosyltransferase (MAT) catalyzes the formation of S-adenosylmethionine (SAM), the principal methyl donor, and is essential to normal cell function. The two forms of MAT, liver specific and non-liver specific, are products of two genes, MAT1A and MAT2A, respectively. We have reported a switch from MAT1A to MAT2A gene expression in human liver cancer cells. In the current work, we examined whether the type of MAT expressed by the cell influences cell growth. HuH-7 cells were stably transfected with MAT1A and were subsequently treated with antisense oligonucleotides directed against MAT2A. MAT2A antisense treatment reduced the amount of MAT2A mRNA by 99% but had no effect on MAT1A mRNA. Cell growth and DNA synthesis rates were reduced by approximately 20-25% after transfection with MAT1A and by an additional 30-40% after MAT2A antisense treatment. SAM level and SAM:S-adenosylhomocysteine (SAH) ratio increased by 50-75% after MAT1A transfection and by an additional 60-80% after MAT2A antisense treatment. DNA methylation changed in parallel to changes in SAM level and SAM:SAH ratio. Supplementing untransfected HuH-7 cells with SAM in the culture medium increased SAM level, SAM:SAH ratio, and DNA methylation and decreased cell growth and DNA synthesis. In conclusion, cell growth is influenced by the type of MAT expressed. The mechanism likely involves changes in SAM:SAH ratio and DNA methylation.     

The cell wall and membrane of Cryptococcus neoformans possess a mitogen for human T lymphocytes

The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 +/- 2, 270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense.     

Interaction of liver methionine adenosyltransferase with hydroxyl radical

Liver methionine adenosyltransferase (MAT) plays a critical role in the metabolism of methionine converting this amino acid, in the presence of ATP, into S-adenosylmethionine. Here we report that hydrogen peroxide (H2O2), via generation of hydroxyl radical, inactivates liver MAT by reversibly and covalently oxidizing an enzyme site. In vitro studies using pure liver recombinant enzyme and mutants of MAT, where each of the 10 cysteine residues of the enzyme subunit were individually changed to serine by site-directed mutagenesis, identified cysteine 121 as the site of molecular interaction between H2O2 and liver MAT. Cysteine 121 is specific to the hepatic enzyme and is localized at a "flexible loop" over the active site cleft of MAT. In vivo studies, using wild-type Chinese hamster ovary (CHO) cells and CHO cells stably expressing liver MAT, demonstrate that the inactivation of MAT by H2O2 is specific to the hepatic enzyme, resulting from the modification of the cysteine residue 121, and that this effect is mediated by the generation of the hydroxyl radical. Our results suggest that H2O2-induced MAT inactivation might be the cause of reduced MAT activity and abnormal methionine metabolism observed in patients with alcoholic liver disease.     

Erythrocyte and brain methionine adenosyltransferase activities in patients with schizophrenia

The activity of methionine adenosyltransferase (MAT) was investigated in erythrocytes and postmortem brain specimens (cortex gyrus frontalis, hippocampus and thalamus) of patients with schizophrenia treated with neuroleptics. In comparison with the control group, abnormally low values of MAT Vmax and an increased MAT affinity towards methionine (lower Km values) were found in erythrocytes. In the brain, a regionally selective decrease of MAT Km was found in cortex gyrus frontalis but the Vmax values were however, unchanged. In the regions of cortex gyrus frontalis and hippocampus, but not in thalamus, the values of Vmax and Km were inversely correlated with the duration of schizophrenia. In rats treated for 28 days with the typical neuroleptic haloperidol and the atypical clozapine, a significant increase of MAT activity was found in the corpus striatum. There is the possibility that the changes observed in MAT activity in patients with schizophrenia are attributed to the neuroleptic medication.     

Cloning and functional characterization of the 5''-flanking region of human methionine adenosyltransferase 2A gene

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.     

Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin

The Gal beta(1-3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4+ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4+ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-gamma and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3- Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4- Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars.     

Iron-containing proteins augment responses of human lymphocytes to phytohemagglutinin and pokeweek mitogen in serum-free medium

The effects of various iron-containing compounds on the responses of human peripheral lymphocytes to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were studied in serum-free medium supplemented with bovine serum albumin. Hemoglobin, transferrin and ferritin enhanced the incorporation of 3H-thymidine into DNA after PHA-stimulation of lymphocytes, while hemin, iron metal powder, ferrous sulfate, chromium powder, and zinc sulfate have little effect. The response to PWM, measured by plaque formation, was enhanced only by transferrin. Desferrioxamine, a chelating agent specific for ferric iron, completely removed these augmentative effects. The results indicate that iron-containing proteins influenced the responses of lymphocytes to stimulation by PHA and PWM in serum-free medium.     

Differential induction of tumor necrosis factor alpha in murine and human leukocytes by Mycoplasma arthritidis-derived superantigen

Mycoplasma arthritidis-derived superantigen (MAS) is exclusively produced by M. arthritidis, which is the only known mycoplasma to produce a superantigen. As a superantigen, MAS shows properties similar to those of the staphylococcal enterotoxins and related substances, such as binding to major histocompatibility complex (MHC) class II and V beta-specific stimulation of T cells. In this series of experiments, we demonstrate some differences between MAS and other superantigens. MAS induced the production of tumor necrosis factor alpha (TNF-alpha) mRNA in human as well as in murine leukocytes. However, only in murine leukocytes was the mRNA adequately translated into the protein. In human peripheral blood mononuclear cells, we found only small amounts of TNF, whereas in murine spleen cells we detected levels more than three times higher. The proliferative response to MAS has been shown to be restricted to I-E alpha in the murine MHC. Furthermore, TNF was induced in I-E alpha+ bone marrow-derived macrophages by MAS. In these cells, MAS rapidly induced very high levels of TNF and the amounts of mRNA detected correlated to the amount of protein produced. In comparison with other superantigens, including the staphylococcal enterotoxins, toxic shock syndrome toxin 1, and exfoliative toxin A, the failure of MAS to induce TNF-alpha in human peripheral blood mononuclear cells is specific for MAS and not common to all superantigens. The direct activation of bone marrow-derived macrophages also seems to be specific for MAS. These data suggest that the induction of TNF-alpha by MAS is dependent on the strength of binding to the MHC class II molecule.     

Expression of the superantigen Mycoplasma arthritidis mitogen in Escherichia coli and characterization of the recombinant protein

Mycoplasma arthritidis mitogen (MAM), is a soluble protein with classical superantigenic properties and is produced by an organism that causes an acute and chronic proliferative arthritis. Unfortunately, the process of obtaining purified MAM from M. arthritidis culture supernatants is extremely time-consuming and costly, and very little material is recovered. Thus, our laboratory has expressed MAM in Escherichia coli by using a protein fusion expression system. The construction and expression of recombinant MAM (rMAM), as well as a comparison of the biological properties of rMAM to those of native MAM, are discussed. Briefly, conversion of the three UGA codons to UGG codons was required to obtain full-length expression and mitogenic activity of rMAM. Antisera to native MAM recognized both rMAM and the fusion protein. The T-cell receptor Vbeta and major histocompatibility complex class II receptor usages by rMAM and the fusion protein were identical to that of native MAM. In addition, the ability to induce suppression and form the superantigen bridge could also be demonstrated with rMAM. Importantly, dose-response experiments indicated that homogeneous native MAM and rMAM were of equal potency. Thus, MAM has been successfully expressed in E. coli, thereby creating a viable alternative to native MAM.     

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.     

Dengue virus infection of human T lymphocytes

A 39-year old male patient was admitted to the University Hospital of the Faculty of Medicine of Ribeir?o, Preto with signs and symptoms of sudden dyspnea, generalized myalgia and behavioral disorders. The initial suspicion was alcohol abstinence syndrome and the patient was referred for psychiatric and neurologic care. The evolution of the patient with a worsening of signs and symptoms, presence of crises of tachypnea, agitation, difficulty to swallow, irritability and hydrophobia, and his report of having been bitten by a suspected dog raised the hypothesis of rabies. The diagnosis was confirmed by examination of a corneal impression, biological tests in the cerebrospinal fluid (CSF) and saliva and visualization of Negri bodies in nervous tissue (direct immunofluorescence). The patient evolved with agitation, aggressiveness, and worsening tachypnea intercalating with apnea, and died on the 4th day after admission.     

Tricyclic antidepressants induce apoptosis in human T lymphocytes

Apoptosis is a form of programmed cell death that is involved in cell turnover. In the present study we show that the tricyclic antidepressants (TCAS) imipramine, clomipramine and citalopram induce apoptosis in human peripheral lymphocytes. Lymphocytes were incubated with these three drugs for up to 48 h. Apoptosis was characterized by typical nucleosomal DNA fragmentation on agarose gel, as well as quantitated using 4'-6-diamidino-2-phenylindole (DAPI) staining and 3'-OH end-labeling of fragmented DNA at the single cell level. Apoptosis induced by TCAs was shown to be dose-dependent and could be detected after a 24 h incubation. The optimal concentrations of the three TCAs found to induce apoptosis were 50 microM imipramine, 20 microM clomipramine and 180 microM citalopram. Furthermore, immunofluorescence and three-color flow cytometry were used to identify the phenotype of apoptotic cells. TCA-induced apoptosis was shown to involve exclusively T-lymphocytes. Cytotoxic T-lymphocytes were more prone to undergo apoptosis than were T-helper cells. In conclusion, the present investigation clearly demonstrates that TCAs exert cell biological effects upon human T-lymphocytes. Further studies are required to determine the possible clinical relevance of these findings.     

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in normal pregnancy

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleukin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T "helper/suppressor" (CD4+CD45RA+) and "suppressor"/effector T cells (CD8+S6F1-), and decreased numbers of T "helper/inducer" (CD4+CD29+), T "helper/memory" (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE2 mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.     

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.     

The effects of L-dopa on the activity of methionine adenosyltransferase: relevance to L-dopa therapy and tolerance

L-dopa, the major treatment for Parkinson's disease (PD), depletes S-adenosyl-L-methionine (SAM). Since SAM causes PD-like symptoms in rodents, the decreased efficacy of chronic L-dopa administered to PD patients may result from a rebound increase in SAM via methionine adenosyl transferase (MAT), which produces SAM from methionine and ATP. This was tested by administering intraperitoneally saline, or L-dopa to mice and assaying for brain MAT activity. As compared to controls, L-dopa (100 mg/kg) treatments of 1 and 2 times per day for 4 days did not significantly increase MAT activity. However, treatments of 3 times per day for 4 and 8 days did significantly increase the activity of MAT by 21.38% and 28.37%, respectively. These results show that short interval, chronic L-dopa treatments significantly increases MAT activity, which increases the production of SAM. SAM may physiologically antagonize the effects of L-dopa and biochemically decrease the concentrations of L-dopa and dopamine. Thus, an increase in MAT may be related to the decreased efficacy of chronic L-dopa therapy in PD.     

Induction of telomerase activity in stimulated human lymphocytes precedes expression of topoisomerase II alpha

Telomerase activity has been demonstrated in human immortal cell lines and in tumors, whereas it is generally absent from normal tissues, with the exception of germ cells. Low levels have also been detected in blood and skin cells. In this report we describe up-regulation of telomerase activity in normal human blood lymphocytes by mitogen stimulation. After 24 h of mitogen treatment a strong induction was detectable using the PCR-based telomeric repeat amplification protocol. The level of activity remained almost constant when the cultivation lasted 72 h. By contrast, topoisomerase II alpha was induced later with a maximum expression after 48-72 h. Our data show that telomerase can be induced in normal peripheral lymphocytes prior to expression of the S-phase typical protein topoisomerase II alpha indicating that telomere elongation might be initiated before DNA replication.