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Molecular cloning of human plasma glutathione peroxidase gene and its expression in the kidney.

A genomic clone containing the human plasma glutathione peroxidase (GSH-Px) gene has been isolated using a rat plasma GSH-Px cDNA as a probe. The partial nucleotide sequence of the clone completely matched the sequence of the human plasma GSH-Px cDNA. The results of Southern blot hybridization indicate that the human plasma GSH-Px gene consists of at least 4 exons and 3 introns, and spans about 12 kb. RNA blot analysis demonstrated that the human plasma GSH-Px gene is expressed in the kidney.

Animals↗

[Development of thymic tumor in HTLV-I transgenic rats under control of a lymphoid tissue specific promoter].

The p40tax protein of Human T lymphocyte virus type I (HTLV-I) is known to be a transactivator not only for the own viral gene but also for the various cellular genes (host genes), including a number of cytokine genes and genes for cell proliferation. Its tumorigenicity was also reported in vivo, such as developments of mesenchymal tumor, neurofibroma, mammary carcinoma and large granular lymphocytic leukemia, using the transgenic technique. To investigate the tumorigenicity in lymphoid tissues, a series of HTLV-I pX transgenic rats (lck-pX rats) which expressed the pX gene under control of lymphocyte specific protein tyrosine kinase (p56 lck) gene type 1 promoter was produced. In some lines of lck-pX rats, pX mRNA was expressed selectively in the thymus, lymph nodes and spleen as expected. However, pX mRNA expression was also detected in other tissues from other lines of lck-pX rats. Thymic tumor with epithelial markers developed in lck-pX rats as early as 8 weeks of age and high expression of pX mRNA was detected in the tumor tissue. This is the demonstration that HTLV-I pX gene causes thymic tumor in the transgenic rat and an lck-pX rat may be a suitable animal model for elucidating pathogenetic role of HTLV-I pX gene in lymphoid tissue neoplasia.

Animals↗

Rat cytotoxic T lymphocytes against human T-lymphotropic virus type 1-infected cells recognize gag gene and env gene encoded antigens.

T cell immune responses in syngeneic WKA/H rats were analyzed by using lymphoid cell lines, TARS-1, TART-1, and TARL-2, infected with human T-lymphotropic virus type 1 (HTLV-1). Spleen cells of rats in which these cell lines had been rejected were sensitized in vitro with the same cell lines, and cells cytotoxic to these HTLV-1+ cell lines, and cells cytotoxic to these HTLV-1+ cell lines were generated. The effector cells were CTL of the CD5+ CD8+ phenotype and showed restriction of MHC class I Ag. Direct tests as well as cold target cell inhibition tests with an array of cell populations showed that these CTL reacted only with syngeneic HTLV-1+ cell lines. When xenogeneic HTLV-1+ cell lines were similarly utilized for in vitro sensitization, rat CTL specific for syngeneic HTLV-1+ cells were generated. They were not, however, reactive with xenogeneic HTLV-1+ cells used for sensitization. Syngeneic rat cells selectively expressing gag, env, or pX gene coded Ag were prepared by infection of recombinant vaccinia viruses. In cold target cell inhibition tests of anti-HTLV-1 CTL with thus prepared cells, cytotoxicity against the syngeneic HTLV-1+ cells line, TARS-1, was inhibited by syngeneic cells expressing gag gene or env gene coded Ag. Inhibition was, however, more consistent and more dominant by cells with gag gene than those with env gene. Syngeneic cells with pX gene and MHC class I incompatible cells with gag, env, or pX gene did not inhibit cytotoxicity.

Animals↗

Active transcription of the selenium-dependent glutathione peroxidase gene in selenium-deficient rats.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, Ec.1.11.1.9) is the best characterized selenoenzyme in higher animals. However, neither the mechanism whereby selenium (Se) becomes incorporated into the enzyme nor the level at which the expression of Se-GSH-Px gene is regulated by Se is fully understood. In the current investigation, we have determined the relative rates of the transcription of the Se-GSH-Px gene in purified liver nuclei isolated from rats fed on Se-supplemented or Se-deficient diets. No significant difference in the transcription rates appeared in these two groups. These results are consistent with the previous observations that active message for Se-GSH-Px- that is, translatable mRNA for Se-GSH-Px- is present in Se-deficient tissues (Li et al., J. Biol. Chem., 265, 108-113, 1990). The data also suggest that the alteration of Se-GSH-Px activity and the corresponding protein and mRNA levels in rats subjected to dietary Se manipulation can be attributed only to post-transcriptional regulation.

Animals↗

Synthesis in Escherichia coli of the HTLV-I trans-acting protein p40x.

The pX gene of human T-cell leukemia virus type I (HTLV-I) encodes a protein that activates the expression of viral genes in trans. Plasmid constructs designed to express the pX gene under the control of either the temperature-inducible lambda PL promoter or the trp promoter were used to transform several Escherichia coli strains, including murein-lipoprotein and Ion mutant strains. Upon induction it was possible to detect the synthesis of a new polypeptide of approximately 40 kDa which reacted specifically with serum from an ATL patient.

Cloning, Molecular↗

Expression of calpain II gene in human hematopoietic system cells infected with human T-cell leukemia virus type I.

We examined the distribution of calpains I and II in human hematopoietic system cell lines by Western and Northern blot analyses and enzyme activity assay. Expression of calpain I, a low Ca(2+)-requiring cysteine protease, was observed in all human T-cell lines tested. By contrast, expression of calpain II, a high Ca(2+)-requiring form, in human T-cells was closely correlated with human T-cell leukemia virus type I (HTLV-I) infection, which is known to result in the expression of adult T-cell leukemia-associated antigens, interleukin-2 (IL-2) receptor alpha, and Ca(2+)-dependent cell proliferation. Specific expression of calpain II in HTLV-I-infected cells occurred at the mRNA level. Furthermore, expression of calpain II in human natural killer-like cells was augmented by HTLV-I pX gene transfection. In HTLV-I-infected cells, the trans-acting transcriptional activation of the long terminal repeat and control elements for the IL-2 receptor alpha, c-fos, and granulocyte-macrophage colony-stimulating factor genes by the Tax from the pX gene is already known. Our results suggest that the similar trans-activation occurs to the calpain II gene in HTLV-I-infected hematopoietic system cells.

Blotting, Western↗

HTLV-I env-pX transgenic rats: prototype animal model for collagen vascular diseases.

To evaluate the function of HTLV-I env-pX gene in vivo, we developed two lines of transgenic rats (env-pX rats) that expressed env-pX gene products, under control of own LTR promotor. In various tissues of the rats, env and pX mRNAs were constitutively expressed, irrespective of age. At age 5 weeks, swelling of the bilateral ankle joints histologically showing synovial lining hyperplasia, severe chronic inflammation, erosion of the joint cartilage, and bone destruction with pannus formation began to develop in these env-pX rats. These histologic features resemble those of rheumatoid arthritis (RA) in man. High titered rheumatoid factors and low anti-dsDNA antibodies and hyper-gamma globulinemia were detected. Necrotizing arteritis resembling polyarteritis nodosa, polymyositis, myocarditis and Sjögren syndrome-like sialoadenitis developed, together with RA-like arthritis even in one individual animal. Thymic atrophy with low body weight was also observed. The evidence indicates that env-pX rats appear to be suitable animal models for elucidating pathogenetic mechanisms involved in not only HTLV-I related diseases but also various collegen vascular and autoimmune diseases of unknown etiology in man.

Aging↗

Models of HTLV-I-induced diseases. Infectious transmission of HTLV-I in inbred rats and HTVL-I env-pX transgenic rats.

To examine the pathogenic roles of HTLV-I in HTLV-I-induced diseases, we developed two models; namely HTLV-I carrier rats and HTLV-I env-pX transgenic rats. Among life long HTLV-I carriers in seven rat strains, only WKAH rats with the RT1k haplotype developed chronic progressive myeloneuropathy, resembling HAM/TSP clinically and histologically in humans, designated as HAM rat disease and after long incubation periods. Apoptosis of myelin forming cells, oligodendrocytes and Schwann cells associated with HTLV-I infection appears to be the primary cause of HAM rat disease. Local activation of the pX gene and TNF alpha gene was evident in these rats. WKAH rats transgenic for HTLV-I env-pX gene were established and at age 5 weeks, swelling of the bilateral ankle joints began to develop and histological features of the affected joints resembled findings in cases of rheumatoid arthritis (RA): high-titers of rheumatoid factors were present in these rats. A series of vascular collagen diseases such as polyarteritis nodosa-like angiitis, polymyositis, myocarditis, and Sjögren's syndrome-like sialodenitis together with RA were present, even in one individual animal. These transgenic rats as well as HAM rats appear to be suitable animal models for elucidating pathogenic mechanisms implicated in HTLV-I-induced diseases and also various demyelinating vascular collagen diseases of unknown etiology.

Animals↗

Chronic graft-versus-host disease-like autoimmune disorders spontaneously occurred in rats with neonatal thymus atrophy.

We earlier reported that the human T cell leukemia virus type-1 pX gene transduced into rat thymic epithelial cells had an impact on biology of the cells. We report here that FW-pX rats born by mating of F344 transgenic rats expressing the pX gene without tissue specificity with nontransgenic Wistar rats developed disorders, including atrophy of the thymus, lymphocytopenia, and inflammatory cell infiltration into multiple organs, similar to events in chronic graft-vs.-host disease (GVHD). Vanishment of thymic epithelial cells especially in the cortex and marked depletion of CD4 CD8 double-positive thymocytes were evident in the neonatal thymus in these rats. The relative abundance of CD8 compared to CD4 T cells may be related to dominant infiltration of CD8 T cells into the affected organs. Additionally, adoptive transfer of FW-pX splenocytes could induce lymphocytic infiltration into sublethally irradiated wild-type syngeneic recipients. Analysis of the expression level of the Foxp3 gene in peripheral blood mononuclear cells revealed that the numbers of immunoregulatory T cells were less in FW-pX rats than in wild-type rats. The collective evidence suggested that the FW-pX rats spontaneously developed chronic GVHD-like autoimmune diseases, following abortive differentiation of T cells in the thymus in early days of the newborn. This rat model may shed light on the pathogenesis of chronic GVHD and also other systemic autoimmune diseases, the etiology of which is unknown.

Animals↗

Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation.

We have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL-60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.

Cell Differentiation↗

Regulation of glutathione peroxidase mRNA level by dietary selenium manipulation.

Glutathione peroxidase (GSH-Px) contains selenium at its active site as a selenocysteine moiety. We have shown that feeding mice a selenium-deficient diet for a long period caused a large decrease in the GSH-Px mRNA level as well as in GSH-Px activity both in the liver and kidneys (Toyoda, H., Himeno, S. and Imura, N. (1989) Biochim. Biophys. Acta 1008, 301-308). In the present study, the transcription rate of the GSH-Px gene was determined by a nuclear run-on assay using liver nuclei of mice fed a selenium-deficient or selenium-adequate diet. The results clearly demonstrate that the transcription rate of the GSH-Px gene was not changed by dietary selenium manipulation, indicating that dietary selenium regulates the level of GSH-Px mRNA in the post-transcriptional step.

Actins↗

Aberrant gene expression by CD25+CD4+ immunoregulatory T cells in autoimmune-prone rats carrying the human T cell leukemia virus type-I gene.

Transgenic rats expressing the env-pX gene of human T cell leukemia virus type-I under the control of the viral long terminal repeat promoter (env-pX rats) developed systemic autoimmune diseases. Prior to disease manifestation, the immunosuppressive function of CD25(+)CD4(+) T (T-reg) cells was impaired in these rats. Since T cell differentiation appeared to be disordered in env-pX rats, we assumed that the impairment of T-reg cells might be caused by an abortive differentiation in the thymus. However, reciprocal bone marrow transfers between env-pX and wild-type rats revealed that direct effects of the transgene unrelated to the thymus framework induced the abnormality of T-reg cells. To identify molecular changes, comparative analyses were done between env-pX and wild-type T-reg cells. Expression of the Foxp3 gene and cell-surface markers supported a naive phenotype for env-pX T-reg cells. Array analyses of gene expression showed some interesting profiles, e.g. up-regulation of genes associated with the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in env-pX T-reg cells. Additionally, expression of the suppressor of cytokine signaling (SOCS) family genes, which inhibit the JAK/STAT signals, was extremely low in env-pX T-reg cells. These findings suggest that the transgene may mediate the down-regulation of the SOCS family genes and that subsequent excess signals through the JAK/STAT pathways may result in the loss of function of env-pX T-reg cells. We suggest that investigation of the pathology of T-reg cells in our autoimmune-prone rat model may aid in understanding the roles of T-reg cells in human autoimmune diseases.

Animals↗

High levels of soluble interleukin 2 receptors released from human retrovirus-infected cells.

Soluble interleukin 2 receptors (IL 2R) from human retrovirus-infected cells were analyzed. All the T cell lines integrated with human T cell lymphotropic virus (HTLV)-I and -II, or transfected with HTLV-I gag-pX gene, were found to release high levels of IL 2R constitutively. In contrast, this was not found in T cell lines in which HTLV-I was integrated but not expressed, human immunodeficiency virus (HIV)-infected T cell lines, or T cell, B cell, granulocyte and macrophage cell lines which were HTLV-I negative. These results raise the possibility that the pX-gene product might stimulate the generation of soluble IL 2R. In the sera from patients with adult T cell leukemia, large amounts of IL 2R were released, in contrast to sera from healthy carriers of HTLV-I. The molecular weight of IL 2R was determined to be about 50 KD by size-exclusion HPLC.

Cell Line↗

Immunological hyperresponsiveness in HTLV-I LTR-env-pX transgenic rats: a prototype animal model for collagen vascular and HTLV-I-related inflammatory diseases.

We have earlier reported that diverse collagen vascular diseases, including arthritis, arteritis, thrombosis, myocarditis, myositis, sialo-/dacryoadenitis and dermatitis develop with the advent of autoantibodies in transgenic rats carrying the LTR-env-pX gene of human T lymphocyte virus type I (LTR-env-pX rats). To clarify the pathogenesis of these collagen vascular diseases, immunological features of LTR-env-pX rats were examined. In LTR-env-pX rats affected with these diseases, expression of CD80/86 on both tissue-infiltrating and peripheral T cells increased, compared with findings in non-transgenic rats with experimental inflammatory diseases. CD80/86 was also upregulated on peripheral T cells in LTR-env-pX rats prior to the development of diseases. Lymphocytes from LTR-env-pX rats showed an increase in autologous proliferation and were hyperreactive against several mitogens, including concanavalin A, immobilized anti-CD3 antibodies, and superantigens in vitro. Antigen-specific immune response was also enhanced in LTR-env-pX rats. The collective evidence indicates that lymphocytes of LTR-env-pX rats constitutively express surface molecules related to T cell activation and are immunologically hyperresponsive. Bone marrow cell transfer from LTR-env-pX rats to lethally irradiated non-transgenic rats revealed that these immunologically pre-activated and hyperresponsive lymphocytes play a critical role in the pathogenesis of several collagen vascular diseases, especially of dermatitis in LTR-env-pX rats.

Animals↗

[Isolation, sequencing and ultrastructure of HTLV-I and HTLV-II retroviruses. Presence of the HTLV-II subtype b among Spanish intravenous drug addicts].

BACKGROUND: Six cases of HTLV-I/II infection were selected for isolation and characterization of these retrovirus. METHODS: Detection of anti-HTLV antibodies was carried out by enzyme immunoassay (EIA), immunofluorescence (IFI), and Western blot (WB). Analysis of proviral DNA was performed by PCR. Viral culture and partial sequencing of the pol and pX genes were carried out. Electron microscopy morphologically characterized the viral particles. RESULTS: Serologic study demonstrated four cases of HTLV-II, one of HTLV-I, and one non-typeable HTLV infections. This last case was confirmed as positive for HTLV-II by PCR. Five new HTLV-II and one HTLV-I infected cell lines have been established by co-culture. Electron microscopy allowed morphologic characterization of the viral particles found in the infected cells. The sequence of the five strains of HTLV-II was identical demonstrating a divergence of 0.49% in the pX region and of 4.5% in the pol region compared with the HTLV-II Mo prototype. Comparison of these sequences with those corresponding to different strains of HTLV-II isolates from American Indians (subtypes b) suggest that these Spanish strains are more closely related with the subtype b than with the subtype a (HTLV-II Mo). Genetic variability study did not reveal any change in the sequence of these stains suggesting that the variability of these retroviruses in very infrequent in the regions studied. The analysis of the pol region of the HTLV-I strain demonstrated a divergence of 3.4% with respect to the sequence of the ATK-1 prototype (Japan) and of 1.7% of the strain HS-35 (Caribbean) showing a greater relation with the Caribbean strains than with those from Japan. CONCLUSIONS: The presence of HTLV-II subtype has been confirmed among intravenous drug addicts in Spain. Isolation and characterization of the HTLV-I strain demonstrated that this also circulating around Spain despite its South American origin.

Base Sequence↗

Malignant transformation of thymoma in recipient rats by heterotopic thymus transplantation from HTLV-I transgenic rats.

Transgenic rats expressing the pX gene of human T lymphocyte virus type-I (HTLV-I) under control of the rat lymphocyte-specific protein tyrosine kinase type-I promoter (lck-pX rats) developed benign epithelial thymomas. When the thymuses of newborn lck-pX rats were transplanted into the subcapsular space of the kidney in other thymectomized lck-pX rats, similar tumors developed in the transplanted thymuses. Following the tumor growth, dissemination in the abdominal cavity and distant metastasis occurred. The tumors were histopathologically similar to the original thymomas, but prominent nuclear atypia and high mitotic activity were present. The Ki-67 index was twice as high as that in the originals. The tumors were transplantable into the subcutis of lck-pX rats, although transplantation of the originals never succeeded. All evidence indicated that malignant transformation of thymoma was induced by the heterotopic transplantation. Expression of the pX transgene in the transformed tumors were significantly reduced. Among host genes, the expression of p16ink4a/ARF, which was significantly upregulated in the originals, was never detected in the transformed tumors. Genomic Southern blots and PCR suggest that homozygous deletion of the p16ink4a/ARF gene may play important roles in malignant transformation in this model. Our model described here is a useful unique model for in vivo malignant transformation.

Animals↗

[Higher expression levels of alternatively spliced pX mRNA in human T lymphotropic virus type I (HTLV-I) asymptomatic carriers positive for antibodies to p40tax protein].

We cloned and sequenced complementary DNA (cDNA) of human T lymphotropic virus type I (HTLV-I) pX gene mRNA expressed in peripheral blood lymphocytes of asymptomatic carriers and searched for a possible correlation between HTLV-I pX mRNA expression levels to viral load and anti-p40tax status. Nine cDNA clones had four alternatively spliced forms of mRNA, three forms were identical to the reported cDNA sequences. One cDNA clone contained a novel splicing acceptor site, thereby indicating an unidentified form of pX mRNA, termed as pX delta 17 delta 37. These results, together with previous observations, suggest that HTLV-I has complex pattern of splicing. The expression levels of the five pX mRNAs, pX, pX delta 17, p21rex, orf II, and pX delta 17 delta 37, were examined in twenty-one asymptomatic carriers. Alternatively spliced pX mRNA species were detected in all the carriers. Semiquantitation of pX mRNAs revealed that pX and pX delta 17 were the dominant mRNA species among alternatively spliced pX mRNA. All pX mRNAs but the orf II message correlated with amounts of proviral DNA significantly (p < 0.05). The levels of proviral DNA and mRNAs were significantly higher in anti-p40tax positive carriers than in negative ones. These observations suggest that the alternatively spliced pX mRNAs are expressed ubiquitously in the peripheral blood lymphocytes of HTLV-I asymptomatic carriers with complex pattern of splicing and that the presence of anti-p40tax may be a marker for a higher viral load and viral replication levels in HTLV-I asymptomatic carriers. Our observations may explain previous finding that the carriers rate is higher in the offspring of anti-p40tax positive mothers than those of anti-p40tax negative mothers.

Aged↗

Tissue specific expression of the plasma glutathione peroxidase gene in rat kidney.

Rat plasma glutathione peroxidase (GSH-Px) was purified 1,400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1,529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5 kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.

Amino Acid Sequence↗