Liquid chromatographic analysis of amino and imino acids in protein hydrolysates by post-column derivatization with o-phthalaldehyde and 3-mercaptopropionic acid.

A method for the simultaneous determination of imino and amino acids is reported. The method, based on the derivatization of amino acids, separated by ion-exchange chromatography, by reaction with alkaline hypochlorite and o-phthalaldehyde-3-mercaptopropionic acid, increases the sensitivity towards imino acids to the picomole level. The use of 3-mercaptopropionic acid gives intensely fluorescent derivatives and improves the stability of primary and secondary amino acid fluorophores towards oxidation. The accuracy and reproducibility of the method are increased by using three internal standards. The determination of all amino and imino acids present in protein hydrolysates, including tryptophan, in a single run is demonstrated.

Thymostimulin: an immunohistochemical and immunological evaluation.

The immunohistochemical and immunological properties of a rabbit serum raised against the calf thymic hormonal factor "Thymostimulin" were studied on mammalian and human thymuses, discussing the problem of species-specificity. The serum was also injected in chick embryos to study the possible interrelationship between avian thymus and Bursa of Fabricius.

A comparative study on the immunological effects of bovine and porcine thymic extracts: induction of lymphoproliferative response and enhancement of interleukin-2, gamma-interferon and tumor necrotic factor production in vitro on cord blood lymphocytes.

Forty samples of cord blood lymphocytes were isolated from 40 normal healthy full-term newborns. The initial 20 samples were used to determine the dose-response curve of three different thymic extracts (TP-1, bovine thymic extract; TG-15-I and TG-15-II, both porcine thymic extracts) and one of renal origin (KG-1) as a control of non-lymphoid organ extract, by measuring the E-rosette T cells. Results showed that E-rosette T cells increased significantly when the thymic extract concentration was increased to 12.5 micrograms/ml. However, there was no statistical difference between TP-1, TG-15-I and TG-15-II in the increase of E-rosette-forming cells. The remaining 20 samples were preincubated with 0, 12.5, 25 or 50 micrograms/ml of thymic extracts. It was observed that the lymphoproliferation, interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and tumor necrotic factor (TNF) production were all significantly increased after thymic extract treatment. No statistical difference between these three thymic preparations in the stimulation of lymphoproliferative response was found. However, among the three thymic extracts, TP-1 appears to induce the highest amounts of IL-2, IFN-gamma and TNF. Of the TG-15-I and TG-15-II, the former stimulates higher IL-2 production whereas the latter enhances IFN-gamma and TNF production. The different immunostimulating effects and potencies that these three thymic extracts showed may reflect not only the species difference but also the difference in preparation procedures. Different components in these thymic extracts may be responsible for different biological activities. Results from these comparative studies may provide useful information in future clinical trials for the treatment of the primary immunodeficiency diseases according to their pathogenesis and may also indicate a possible beneficial effect of the combination of chemotherapy and thymic extracts.

Migration of putative progenitor T cells in response to thymus-derived chemotactic factors.

Progenitor T cells reach the thymus through the circulation from hematopoietic organs and then migrate toward the site of differentiation in the thymus. The mechanism that regulates such intrathymic migration is not well understood. In order to clarify this mechanism, in vitro chemotactic activity for murine thymocytes was assayed in the extracts and culture supernatants of thymic tissue elements. A potent thymocyte chemotactic activity was found in the extract and culture supernatant from Ig-, Ia- thymic stromal cells. Peanut agglutinin-positive (PNA+1), Thy 1+, TL-, Lyt 1+2-, L3T4- thymocytes, Ig-, Thy 1- bone marrow cells, and mononuclear cells of spleen and peripheral blood, but neither B cells nor lymph node cells, were chemotactically attracted by the factor(s). The chemotactic activity was found in none of the following materials tested: the extract and culture supernatant of thymocytes, culture supernatant of lymph node stromal cells, normal mouse serum, and zymosan-activated serum. The chemotactic activity was found in three molecular fractions by gel chromatography. The activity in all three fractions was destroyed by trypsin digestion or by heating at 56 degrees C for 30 min. These results suggest that Ig-, Ia- thymic stromal cells but not thymocytes secrete a chemotactic factor(s) for progenitor T cells with three molecular species. The factor is considered to play an important role in the migration of intrathymic progenitor T cells into the site of differentiation.

Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone.

The endocrine system displays highly complex interactions among its components. Excesses or deficiencies of hormone production in one gland may alter the production of hormones by others. Several physiological functions are affected by a balance among hormones acting either together or in sequence. For example, FSH secretion has been demonstrated to be affected by hypothalamic influences upon the anterior pituitary through a specific releasing factor, the decapeptide LRF. This decapeptide stimulates the release of both LH and FSH by the pituitary, and these gonadotropins cause the production of steroids by the testes and the ovaries. Gonadal steroids in the blood act directly upon the anterior pituitary to regulate the output of gonadotropins as originally proposed by Moore and Price in 1932 (3), or act indirectly upon the hypothalamus to adjust the output of pituitary hormones in accordance with the needs of the reproductive system. However, such a simple negative feedback of steroids on the hypothalamic-hypophysial axis cannot account for the differential secretion of FSH observed during the estrus cycle. Therefore, the concept that a gonadal protein, inhibin, specifically regulates FSH secretion was proposed. This concept has now been validated by the isolation and characterization of two forms of inhibin that exert their effects on the pituitary to suppress FSH secretion both in vitro and probably in vivo. Furthermore, the production of inhibin is stimulated by FSH, thus establishing a reciprocal relationship between the release of FSH and inhibin. Since hormones in the body are controlled through interlocking complexes of factors, a variety of secondary factors, in one way or another, may also exert influence on the regulation of FSH secretion. As an example, TGF beta, a protein growth factor found in all tissues, promotes the basal secretion of FSH by the pituitary and enhances FSH-mediated estrogen production by the granulosa cells. It is therefore not surprising that two forms of a novel protein, activin and activin A, isolated from the same FF from which inhibins were isolated, show bioactivities similar to those of TGF beta. These activins are formed as dimers of the two beta-subunits of inhibin, probably as a result of the rearrangement of the gene products. This novel observation that different arrangements of gene products can result in opposite biological activities may thus reflect a wholly different level of control of FSH secretion. If such a phenomenon occurs in other biosystems, it would represent an important form of homeostatic mechanism for controlling biologically active substances.(ABSTRACT TRUNCATED AT 400 WORDS)

[Immunochemical identification and study of interspecies thymus antigen-2]. / Immunokhimicheskaia identifikatsiia i izuchenie mezhvidovogo antigena-2 timusa.

A new interspecific animal thymus antigen (AgT-2) was identified. It was shown that AgT-2 is a microglobulin with a molecular mass about 12 kDa, electrophoretic mobility of alpha 1-globulins and isoelectric point of 4.6. Heating of the protein to 80 degrees C for 30 min did not lead to the loss of its immunochemical activity. AgT-2 was identified in extracts of bovine fetal thymus, spleen and liver. It was discovered in the extracts of the lung and colon of adult animals. Cross-reactions were found between bovine, walrus, deer and chicken AgT-2. This fact confirms that AgT-2 is interspecies-specific. AgT-2 was not identified in men, dogs, mice, rats and rabbits. Immunochemical research of immunocorrecting preparations revealed that AgT-2 is a constant component of T-activin, thymalin, thymosin and TP1-Serono and its concentration varies from 1.6 to 3.2%. It has been stated that immunochemical test for AgT-2 can be used as a marker in the production and standardization of active thymus factors.

Cancer and thymus extract.

Different types of malignant epithelial tumours were found to contain very large amounts of substance immunoreactive with the anti TEX globulin, when studied by direct immunofluorescence method.

Stimulation of lymphocyte proliferation to monitor fractionation of thymus extracts.

Multi-step fractionations by solvent extractions, gel filtrations, ion-exchange chromatography, etc., of bovine thymus extracts were monitored by a standardized assay, in vitro, which led to the described peptides, thymones A, B and C. The assay uses spleen cells from neonatally thymectomized mice as tissue relevant to immunoregulation. Assay of incorporation of [3H]-thymidine into DNA located peaks of activities. Calf thymus outer fraction ("CTO") showed activity, but Fraction 5, synthetic thymosin alpha 1, [G1n1]-FTS, and glutathione, were inactive in this assay.

Nuclear antigens: characterization of the acidic nuclear ribonucleoprotein-Sm-antigen complex by analytical and preparative isoelectric focusing.

The soluble ribonucleoprotein nuclear antigen (RNP), associated with the Sm antigen and present in extracts of rabbit thymus acetone powder, was purified by ion-exchange chromatography. Analytical isoelectric focussing (IEF) showed two bands at pH 5.2 and two bands at pH 5.4. After treatment by RNAse these 4 bands disappeared, and a new band with a pI of 6.1, representing the intact protein moiety, appeared. During preparative IEF in granulated gels the antigenicity of RNP was lost. Only the Sm antigen could be detected by counter-immunoelectrophoresis. Fractions with Sm antigenicity refocussed as two intense bands (pI 6.8 and 7.2) and one or two fainter bands (pI 6.1). On reducing SDS-PAGE the SM antigen revealed two bands of apparent molecular weights of about 110,000 and 28,500, indicating a total molecular weight of 138,500.

Enrichment of in vitro and in vivo immunologic activity of purified fractions of calf thymic hormone.

Thymus humoral factor (THF), a thymus hormone which participates in the processes leading to acquisition of immunocompetence of lymphoid cells has been isolated in our laboratory by a stepwise gel filtration through various Sephadex columns. THF so isolated appears to be a polypeptide of 3,000 mol wt which contains approximately 30 amino acid residues. Here we have tested the biological activity of THF fractions of successive degrees of purity upon lymphoid cells from both intact and neonatally thymectomized mice. The lymphoid cell populations were treated with the various THF fractions by in vitro incubation for a short time and by repeated injection in vivo. The treated cells evidenced increased ability to react in the graft-versus-host assay in vivo and in mixed lymphocyte cultures in vitro concomitantly with the rise of intracellular cAMP. On the other hand no activity whatsoever was shown by any of the control materials tested. These bioassays permitted isolation of fractions progressively more active than the original crude dialyzate of thymus extract tested. Thus the active peptide component of THF eluted from DEAE Sephadex A-25 column was estimated to be 2 X 10(4)-fold more active than the crude dialyzate of thymus extract which served as a starting material.

Endogenous thymic factors regulating cell proliferation and analysis of their mechanism of action.

Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. An extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G1 leads to S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. greater than 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. The active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4-4) inhibits [3H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.

Properties of TFX-A product obtained from calf thymus.

Properties of TFX--a product obtained from calf thymus by Pharmaceutical Comp. in Jelenia Góra--are described. The preparate is a mixture of polypeptides and peptides of various molecular weights with various biological activities. Attempts of isolation of active fractions, devoid of balast proteins are described.

Is there acetylcholine receptor in human thymus?

Recent studies have suggested that thymus tissue of the calf may bear nicotinic acetylcholine receptors. The presence of similar receptors in thymus tissue of man could thereby serve as a source of antigen for the production of antibody to the acetylcholine receptor in patients with myasthenia gravis. In the present experiments, human thymus tissue was examined for the presence of acetylcholine receptor. Both [125I] alpha-bungarotoxin binding and antiserum to the human acetylcholine receptor were used in tests for acetylcholine receptor in thymus glands from normal individuals and from patients with myasthenia gravis. Neither normal nor myasthenic thymus tissue were found to possess the [125I] alpha-bungarotoxin binding or the antigenic properties of the acetylcholine receptor.

Biologic activities of various thymus preparations.

Thymosin and THF were prepared from total normal calf thymus (TT), from calf thymus depleted of thymocytes by whole-body irradiation (TE), and from calf thymus lymphocytes (TL). The activities of these products were compared in four different in vitro assays that monitored for various T-cell functions. The results suggest that the preparations obtained from thymus epithelium are more active in inducing T helper cell function in nude/nude mouse spleen cells than substances isolated from thymus lymphocytes. The latter seem to act on a more advanced step in the T cell line differentiation.

Stimulation of anti-influenza serum antibody formation in the rat using bovine thymus polypeptide extract.

The authors show that a thymus polypeptide extract, TP1, prepared by them has the effedt of stimulating the formation of anti-influenza serum antibodies and do not show antigenic properties. The extract causes increase of serum lymphocytes. Electrophoretic analysis in polyacrylamide gel reveals a number of 11 fraction, while paper chromatography for amino acids, a number of 12 spots.

[The role of thymic microenvironment in T-cell maturation]. / Maturation des lymphocytes T: rôle du microenvironnement thymique

The role played by different components of thymic microenvironment in T-cell-maturation is examined: antibodies to a soluble thymic factor (STF) were thus shown to inhibit T-cell maturation in the chicken while incubation of bursal "null" cells with STF could induce the differentiation of some of them into T cells. In addition to STF, whose properties resemble those of other soluble thymic factors, an insoluble thymic factor (ITF) is described: it is localized mainly in membranes associated with medullary blood vessels, and its injection to mice provokes an influx of marrow stem cells into the thymus.