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PubMed1980

Characterization of facteur thymique sérique (FTS) in the thymus. I. Fixation of anti-FTS antibodies on thymic reticulo-epithelial cells.

Facteur thymique sérique (FTS) is a circulating nonapeptide inducing T cell differentiation. Its strict thymus dependency, already shown by its disappearance after thymectomy and its presence in thymus extracts, was confirmed using indirect immunofluorescence or immunoperoxidase by the binding to reticulo-epithelial cells of an antibody produced against synthetic FTS. The specificity of the reaction was demonstrated by the inhibition of binding observed after preincubating the anti-FTS antibody with synthetic FTS.

Animals↗

PubMed1987

Improved detection of anti-Jo-1 antibody, a marker for myositis, using purified histidyl-tRNA synthetase.

The increased detection of anti-Jo-1 antibody afforded by the use of the purified antigen, histidyl-tRNA synthetase, in counterimmunoelectrophoresis is demonstrated. Using purified antigen, anti-Jo-1 antibody was detected in the sera of 16/33 (48.5%) patients with confirmed myositis and in 20/45 (44.5%) patients with confirmed or possible myositis. This rate is approximately double that obtained with commercial thymus extracts both in this study and seven others reported in the literature. The presence of antibody shows marked correlation with the activity of myositis at the time of serum sampling and with the presence of interstitial lung disease. Detection rates are similar in patients with polymyositis and dermatomyositis both with and without additional connective tissue diseases.

Adult↗

PubMedDate not supplied

Effect of the thymus storage factor (TSF) on the blood sugar and insulin level.

The rat thymus extract prepared with physiological NaCl solution causes--by its TSF component--an increase of the blood sugar level. After sugar loading in TSF-treated animals, the blood sugar level becomes gradually higher and the insulin secretion diminishes as compared to the control animals. Thymectomy moderates but does not inhibit the TSF decreasing effect on the insulin level. Consequently, TSF primarily causes a decrease of the blood insulin level which correlates with a rise of the blood sugar level.

Animals↗

PubMedDate not supplied

[Action of thymosin on the ultrastructure of the bone marrow in the rat].

A preparation isolated from thymus extract and possessing thymosin-like activity was studied for its influence on the ultrastructure of the rat bone marrow cells. A dose of the preparation adequate to therapeutic one (7 mg/kg) causes a transitory "irritation" (dystrophy and stimulation) in cellular elements of bone marrow. A supertherapeutic dose (70 mg/kg) has a definitely toxic effect on its cells and is characterized by an expressed tropism to the endothelium of sinusoids and capillaries.

Animals↗

PubMed1999

ATP depletion, purine riboside triphosphate accumulation and rat thymocyte death induced by purine riboside.

Purine riboside (purine-1-D-ribofuranoside, nebularine), an adenosine analog, exerts cytotoxic effect both in vivo and in vitro. However, exact biochemical mechanism for its toxicity and sensitivity of lymphoid cells remains unknown. The present experiments have examined the sequential metabolic changes leading to cell death, induced in cultured rat thymocytes during incubation with purine riboside. Among 22 analogs tested, purine-riboside and tubercidin were most toxic as determined by trypan blue exclusion and lactate dehydrogenase leakage from the cells. 2-Chloroadenosine and 2'-deoxyadenosine were only moderately toxic, whereas other analogs tested were without effect on cell viability. In the presence of purine riboside, more than 90% of ATP was lost after 2 h of incubation. Hypoxanthine accumulated in the medium and the formation of purine-riboside triphosphate exceeded 4-fold the physiological concentration of ATP in the cell. Inhibition of adenosine kinase by 5-iodotubercidin reversed the cytotoxic effect of purine riboside. Interestingly, cells virtually deprived of ATP after 2 h of incubation with purine riboside maintained high nucleotide energy charge value and high viability. Purine riboside triphosphate was capable to replace ATP in stimulation of glycolysis in cell-free thymus extract. We conclude that for a short time (a few hours) purine riboside triphosphate formed in the cell may serve in the absence of ATP as an intermediate of cellular energy metabolism in rat thymocytes. However, possibly due to toxic effects of purine-riboside triphosphate, cells were finally dying. Thus, ATP depletion and adenosine kinase mediated purine riboside phosphates formation are the principle causes of rat thymocytes death exposed to purine riboside.

Adenosine↗

PubMed1992

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.

Animals↗

PubMed2002

Distinct expression of cyclooxygenase-1 and -2 in the human thymus.

Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins (PG). Given the role of COX and PG during intrathymic T cell development in the mouse, we investigated the expression and localization of these isozymes in the human thymus. mRNA and proteins correspondent to COX-1 and -2 were observed from whole thymus extracts. By immunohistochemistry, COX-2 was selectively localized in the medulla and it was predominant in a subset of stromal cells. By contrast, COX-1 was diffusely and exclusively present in the cortex, both in thymocytes at early stages of differentiation and in cytokeratin-positive epithelial cells, as demonstrated by double immunostaining and flow cytometry analysis. COX-2-positive cells in the medulla expressed cytokeratin and HLA-DR molecules, but they were negative for dendritic or macrophagic antigens. In addition, COX-2-positive cells expressed both the epidermal growth factor receptor and its ligand, the transforming growth factor-alpha. The inducible isoform of the PGE(2) synthase was also present in the same cells, while was absent from COX-1-expressing cells of the cortex. Finally, electron microscopy confirmed that COX-2 was mainly localized in the cytoplasm of cytokeratin-positive cells, along the rough endoplasmic reticulum. In conclusion, COX-2 and the inducible isoform of PGE(2) synthase appear to be constitutively and selectively present in medullary epithelial cells of the human thymus, whereas COX-1 is predominantly present in the thymic cortex, both in the stroma and in developing thymocytes.

Child↗

PubMed1995

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.

Adenosine Deaminase↗

PubMed1992

T-cell receptors of man and mouse studied with antibodies against synthetic peptides.

We used polyclonal rabbit antibodies directed against synthetic peptides predicted from the gene sequence of the human T-cell receptor (TCR) beta-chain YT35 to study the antigen receptor on human helper T-cell leukemia lines and on normal mouse thymocytes. Antibodies were raised to peptides corresponding to joining segment (J beta) and to a conserved stretch of sequence around the first cysteine in the constant region (C beta). These peptides were selected on the basis of homology with corresponding segments of immunoglobulin light chains. The specificity of the antibodies was established using synthetic overlapping peptides that modelled the complete TCR beta-chain. Western blot analysis was performed against detergent lysates of T cells. Both of the antibodies reacted strongly with 2-3 polypeptides in the mass range 40-45 kDa in mouse and human cells. Clearance experiments using monoclonal antibodies against murine TCR alpha- and beta-chains and against human TCR beta-chain and immunoprecipitations with monoclonal antibody to the murine T3 complex established that these components represented the alpha/beta heterodimer. An additional component around 31 kDa was detected by anti-J beta antibodies in murine thymus extracts. The use of the affinity-purified antipeptide antibody in two-dimensional Western blot analyses allows the clear discrimination between the characteristic individual receptors of monoclonal neoplastic T cells and the polydisperse patterns representative of heterogeneous normal populations. Antigenic cross-reactions between T-cell receptor beta-chains of man and mouse observed with monoclonal antibodies and rabbit antisera to peptides are consistent with the homology in gene sequence between the two species.

Amino Acid Sequence↗

PubMed1980

Heterogeneity of precipitating antibodies in polymyositis and dermatomyositis. Characterization of the Jo-1 antibody system.

Precipitating antibodies occurring in 26 "pure" polymyositis (PM) patients and 22 "pure" dermatomyositis (DM) patients were studied by double immunodiffusion using calf thymus nuclear extract and whole calf thymus extract. Seventeen of 26 (65%) PM sera and 13 of 22 (59%) DM sera showed positive reactions, and 18, 10, and 2 sera exhibited 1, 2, and 3 precipitin lines, respectively. Common specificities were established by immunodiffusion for the 11 PM and 6 DM sera with strong precipitin lines. These were classed into three groups of shared specificity and six reactions specific for individual sera. These 9 calf thymus antigens were shown to be different from the known soluble tissue antigens Sm, nuclear RNP, Ro, and La. A 600-fold purification of the most frequently appearing system designated Jo-1 was achieved and its clinical specificity for patients with PM syndromes was established.

Antibodies↗

PubMed2002

The TREX2 3'-->5' exonuclease physically interacts with DNA polymerase delta and increases its accuracy.

Proofreading function by the 3'-->5' exonuclease of DNA polymerase delta (pol delta) is consistent with the observation that delta efficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol delta from isotonic (pol delta(i)) and detergent (pol delta(d)) calf thymus extracts. Pol delta had a 20-fold higher ratio of exonuclease to DNA polymerase than pol deltai. This was due to the physical association of the TREX2 3'-->5' exonuclease to pol delta(d), which was missing from pol delta(i). Pol delta(d) was fivefold more accurate than pol i under error-prone conditions (1 mM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate than pol delta(i) under error-prone conditions (1 mM dGTP and 20 microM dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 microM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3' --> 5' exonucleases, such as TREX2, through its association with pol delta can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Animals↗

PubMed2001

Gene disruption of tissue transglutaminase.

Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either epsilon-(gamma-glutamyl)lysine or N(1), N(8)-(gamma-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2(-/-) mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2(-/-) mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2(-/-) thymocytes (by CD95, dexamethasone, etoposide, and H(2)O(2)) and in vitro on TGase 2(-/-) mouse embryonal fibroblasts (by retinoids, UV, and H(2)O(2)) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.

Animals↗

PubMed1984

Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus.

A polypeptide containing approximately equal to 112 amino acid residues, with the thymosin alpha 1 sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin alpha 1. The new polypeptide, named "prothymosin alpha," was found to be the major substance crossreacting with thymosin alpha 1 antiserum in rat thymus extracts; peptides corresponding to thymosin alpha 1 or thymosin alpha 11 were not detected. In gel filtration at pH 2.8, prothymosin alpha emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. On the same gel filtration columns, synthetic thymosin alpha 1 (calculated Mr = 3108) emerged at a position corresponding to a molecular weight of 10,000-11,000. Thus, both prothymosin alpha and thymosin alpha 1 appear to exist in solution as oligomers, possibly as trimers. Prothymosin alpha and synthetic thymosin alpha 1 also were separated readily in reverse-phase HPLC and in isoelectric focusing; the isoelectric point of prothymosin alpha determined by the latter procedure was found to be 3.55, consistent with an unusually high content of glutamic and aspartic acids based on amino acid analyses. Prothymosin alpha appears to represent the native polypeptide from which thymosin alpha 1 and other fragments are generated during the isolation of thymosin fraction 5.

Amino Acid Sequence↗

PubMed1997

Identification of DNA replication and cell cycle proteins that interact with PCNA.

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.

Animals↗

PubMed1984

Evidence for the presence of immunoreactive acetylcholine receptors on human thymus cells.

Triton extracts from thymuses and thymomas from either normal subjects or MG patients specifically bind [125I] alpha-bungarotoxin (alpha Bgt). This binding is saturable (apparent Ks = 5.9 X 10(-9) M) and can be inhibited by cold alpha Bgt, acetylcholine or nicotinic agonists. These results demonstrate the presence on human thymus cells of acetylcholine receptor (ACh.R), 2.8% for normal human thymus in regard of ACh.R concentration in human muscle. Normal human thymic extracts were used as an antigen source in the radioimmunoassay for the detection of anti-ACh.R antibodies in positive MG patients' sera. 72% of these sera were also positive with the thymic antigen. The demonstration of ACh.R in human thymus brings further support to the hypothesis of intrathymic autosensitivity against ACh.R as a major factor in MG pathogenesis.

Antibodies↗

PubMed1978

10-S DNA polymerase from calf thymus which copies both poly(rA) . oligo(dT) and activated DNA.

A novel DNA polymerase, which could use both poly(rA) . oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocussing (pI = 6.5--7.0) or sucrose rate-zonal centrifugation (10--10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) . oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) . oligo(dT)-dependent activity differed from the previously described DNA polymerase gamma from other sources in the following ways: 1. The activity was inhibited by 100--300 mM KCl and and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26 degrees C than at 37 degrees C. 3. The Km value for dTTP was 2.6--3.0 . 10(-4) M, which is several hundred-fold greater than that of DNA polymerase gamma. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase alpha, except that it was less sensitive to N-ethylmaleimide and anti-alpha polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.

Animals↗

PubMed1986

Activation of human monocyte and natural killer cell-mediated tumour cell killing by two dialysable thymic factors.

Components of calf thymus extract dialysable under acid conditions contained two natural killer (NK) cytotoxicity-stimulating factors, CSFa and CSFb, which could be separated by ion exchange chromatography. NK cytotoxicity of human peripheral blood mononuclear cells (PBMC) against human K562 tumour cells was strongly enhanced after 72 h pre-incubation with the factors. The CSFa/b-specific stimulation of PBMC required the presence of monocytes. The cytotoxic effector cells activated during pre-incubation of PBMC with the CSF were identified as monocytes and as NK cells present in the fraction of large granular lymphocytes (LGL). Selective cell depletion studies with LGL-containing subpopulations (free of monocytes) allowed factor-specific discrimination of the activated LGL. Pre-incubation of PBMC with CSFa stimulated NK cytotoxicity of LGL (Leu 7+11-; T8-), whereas pre-incubation with CSFb resulted in stimulation of LGL (Leu 7+11-; T8+). The biological effects of CSFa and CSFb could be further distinguished by analysis of surface marker expression during incubation of PBMC. CSFb scarcely influenced T4 expression, but strongly enhanced the expression of T8 and that of transferrin receptor, whereas CSFa had no significant influence on the expression of these three surface markers. Both factors induced a drastic reduction of tumour take incidence or tumour development in mice when applied before and after tumour challenge.

Animals↗

PubMed1991

VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies.

To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response.

Animals↗