Lutein associated with a transthyretin indicates carotenoid derivation and novel multiplicity of transthyretin ligands.

Transthyretins isolated from different species bind hydrophobic compounds and are often obtained in a yellow form. Such a transthyretin from chicken serum was purified by chromatography using Sepharose-coupled human retinol-binding protein. The yellow chromophore was extracted with methanol and purified by reverse phase HPLC followed by normal-phase chromatography on a nitrile column. Ultraviolet-visible absorbance and mass spectrometry identified the yellow compound as lutein, i.e. xanthophyll, (all-trans)-beta, epsilon-carotene-3,3'-diol, estimated to constitute 10-30% of associated colourless compounds. These components are different from the yellow component isolated from human transthyretin and establish that carotenoid-derived pigments can be associated with transthyretins.

Rescue of thymocytes from cell death by vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) has previously been shown to increase survival of cultured neurons and to prevent the neurotoxic effect of the envelope glycoprotein 120 of human immune deficiency virus (HIV). The present report shows that VIP also protects mouse and human thymocytes exposed to a cytolytic dose of prednisolone in vitro. The activity of VIP is dose-dependent, and specific, since the structurally related secretin has no effect. The effective concentration of VIP is within the physiological range, suggesting that VIP released from nerve terminals may modulate cell death in the thymic cortex. Results with an N-terminal and a C-terminal fragment of VIP implied that the complete VIP molecule is required for optimal protection against cytolysis.

A yellow component associated with human transthyretin has properties like a pterin derivative, 7,8-dihydropterin-6-carboxaldehyde.

Transthyretin (TTR) in plasma is associated with yellow compounds. Their properties differ, and in the chicken protein a major yellow compound has recently been identified as a carotenoid, lutein, also called xanthophyll. We now show that the major yellow component extracted from human TTR has properties like a pterin derivative, 7,8-dihydropterin-6-carboxyaldehyde (2-amino-4-hydroxy-6-formyl-7,8-dihydropteridine). The human TTR derivative has chromatographic and spectral properties identical to a yellow photochemical degradation product of biopterin and a spectrum like that of the pterin aldehyde.

Identification of a mammalian growth factor as a ribofolate peptide.

Thymocyte growth peptide (TGP) promotes DNA synthesis of immature thymocytes. TGP has been purified from sheep, human and calf thymus and recently characterized as an N-terminally blocked nonapeptide. Evidence is presented here that the blocking moiety consists of a formylpteroyl group bound to the N-terminal glutamyl residue of the nonapeptide. The pterin part of the TGP molecule has a ribityl substituent in analogy with riboflavin, which explains the pronounced hydrophilic property of TGP in contrast to unsubstituted and unconjugated folates. The compound can be classified as a ribofolate peptide, a novel class of growth factor. Zn2+ counteracts degradation of the molecule and is required for full biological activity; mass spectrometric data confirm that native TGP contains zinc.

In vivo stimulation of thymocyte proliferation by thymocyte growth peptide (TGP).

Chromatographically pure thymocyte growth peptide (TGP) was injected into one of the two thymus lobes of guinea pigs. Pulse labelling of DNA-synthesizing cells was obtained by an intracardial injection of bromodeoxyuridine (BrdUrd). After 24 and 48 h, the frequency of labelled cells was increased in the TGP-treated lobe in comparison with the control lobe. At 24 h, the increase was found in non-rosetting, large, low-density cells, the population containing the highest frequency of DNA-synthesizing cells. At 48 h, labelled rosette-forming, small, high-density cells appeared in an increased frequency in the TGP-treated lobe. Serum thymic factor (FTS), which is structurally related to TGP, did not produce similar effects. The results show that TGP is active in vivo and stimulates proliferation of cycling thymic precursor cells which then are transformed into small thymocytes.

Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin.

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270-280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl2 to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn2+.

Monosialoganglioside GM1 increases survival of thymocytes.

Gangliosides are normal constituents of the plasma membrane. Exogenous gangliosides can be incorporated into the membrane and extensive research in nervous tissue has demonstrated a beneficial effect of gangliosides on the functional recovery of lesioned neurons and protection against neurotoxins. This paper shows that the effect of gangliosides is not restricted to neurons. The monosialoganglioside GM1 efficiently increases the survival of thymocytes and protects them against both the lytic effect of the glucocorticoid prednisolone and the effect of a thymocytotoxic serum. The protective effect of GM1 was achieved both in vitro and in vivo.

Isolation of a thymocyte growth peptide from human thymus.

A thymocyte growth peptide (TGP), recruiting immature thymocytes into the S phase without affecting immunologically mature thymocytes or peripheral T cells, has previously been purified from bovine and rodent thymus. We now report the isolation of a corresponding human activity. Aqueous extracts of human thymus were found to stimulate the DNA synthesis of human thymocytes in vitro. Homogenates of suspended thymocytes displayed higher activity than extracts of intact thymus, whereas extracts of thymic stroma depleted of thymocytes contained much weaker activity. The results indicate the existence of a human TGP originating from the thymocytes and not from the stroma, implying an autocrine or paracrine growth regulation. Human TGP was purified from a formic acid extract of human thymus and found to have a molecular ratio between 1,000 and 2,000 and an isoelectric point of 4.5-4.9. The results demonstrate the existence of a human TGP similar but not identical to bovine TGP and it is proposed that this peptide acts as a progression factor for the intensely proliferating immature cortical thymocytes.

Autocrine growth stimulation of thymocyte precursors in the thymus.

A thymocyte specific growth peptide (TGP) has previously been purified from bovine thymus. In the present study TGP activity is demonstrated in the conditioned medium from a murine thymic leukemia cell line with prothymocyte phenotype, but is absent from media of cultured thymic epithelium. This murine TGP shows specificity for immature thymocytes and growth-stimulating kinetics identical to bovine TGP. Murine and bovine TGP show different chromatographic behaviour which may indicate biochemical differences. The results suggest that the immature thymocyte population includes both TGP-producing and TGP-responding cells. Our finding of TGP activity in fresh homogenates of normal mouse thymocytes supports this interpretation. We propose that TGP acts as an autocrine growth factor for rapidly cycling immature cortical thymocytes.

Enhanced mitogenic reactivity of human T cells after passage through the thymus. Response to phytohemagglutinin and mercuric chloride.

Lymphocytes in blood samples taken from the thymic, brachiocephalic and internal jugular vein of children undergoing thoracic surgery were compared regarding their phenotypic markers and their proliferative response to mercuric chloride (HgCl2) and phytohemagglutinin (PHA). Of these two mitogens, PHA stimulated the DNA synthesis of the lymphocytes from the thymic vein to a significantly higher degree than those taken from the brachiocephalic or internal jugular vein. The response to HgCl2 resulted in the highest stimulation of the DNA synthesis in cells from the thymic and the brachiocephalic veins, though the differences were not significant. The higher reactivity to PHA and the tendency to higher reactivity to HgCl2 of lymphocytes taken from efferent thymic blood might be due to a higher frequency of T cells. This, however, could not be verified by immunohistochemical staining, as Leu4-positive cells occurred in the same proportion in all three venous samples. We therefore suggest that the passage through the thymus of circulating T cells may increase their immune reactivity by the influence of thymic maturation hormones.

Effect of growth factors on the proliferation of thymocytes in different functional and anatomical compartments.

The intrathymic location of cells responding to the thymocyte growth peptide (TGP) was investigated in guinea pigs. Thymus cells were labelled by topical application of a fluorescein isothiocyanate solution and the percentage of fluorescent cells analyzed by flow cytometry. Large cells forming rosettes with rabbit erythrocytes, known to respond to TGP, had a lower percentage of fluorescent cells than non-rosetting cells which do not respond to TGP. If the TGP-responding cells are anatomically segregated in the guinea pig thymus, this might indicate that they are preferentially located in the deep, juxtamedullary part of the cortex, where one major proliferating compartment is situated.

Purification to apparent homogeneity of a thymocyte specific growth factor from calf thymus.

A thymic peptide previously found to recruit thymocytes from G1 into S phase has been purified from a crude thymic extract by subsequent steps of gel exclusion chromatography and reverse phase high performance liquid chromatography (HPLC). The purified material, which appeared homogeneous on thin-layer chromatography and HPLC, stimulated the DNA synthesis of cultured guinea pig thymocytes in a nanomolar concentration range. The amino acid composition revealed a high content of acidic amino acids and no apparent homology to previously defined growth factors and thymus differentiation hormones.

Recruitment of thymocytes from G1 into S phase by a thymic factor.

A thymic factor previously shown to stimulate the DNA synthesis of immature thymocytes was found to recruit G1 cells synchronously into S phase within 1.5-2 h. The S + G2 + M duration of the cultured thymocytes was not affected. In the absence of the thymic factor, the responding thymocytes seemed to be blocked in G1 at a putative restriction point 1.5 h prior to S phase. When the addition of the thymic factor to cultured cells was delayed, the responsiveness rapidly declined, indicating that target cells were transferred into an unresponsive state. A stimulating effect of the DNA synthesis of cultured thymocytes was obtained also after a transient exposure to the thymic factor for 2-3 h. The stimulating activity was completely absorbed from the medium by incubation with thymocytes at both 4 and 37 degrees C, but not by lymph node lymphocytes which are unresponsive to the thymic factor. Our interpretation of the results is that the thymic factor acts on thymocytes in G1, triggering the responsive cells via membrane receptors to proceed from G1 into S phase. The thymic factor is proposed to be a progression factor for the rapidly cycling immature thymocytes and to be involved in the regulation of the intense thymic growth in vivo.

Characterization of the target cell for a thymocyte specific growth factor in guinea pigs.

A growth factor prepared from calf thymus has been described previously (Thymus 3, 289, 1981). The factor was shown to stimulate the DNA synthesis in thymic cells but not in peripheral lymphocytes in guinea pigs. We now present a further analysis of the target cell. Thymus cells were fractionated into subpopulations based on their different adherence to plastic, buoyant density and agglutinability by peanut agglutinin (PNA) and the influence of the growth factor on DNA synthesis was studied. The results showed that the responding cell was enriched in the population of non-adherent PNA-positive (PNA +) cells of low buoyant density. This cell population showed high spontaneous DNA synthesis in vitro, but did not contain the phytohemagglutinin-responsive thymocyte, which resided in the PNA-negative (PNA -) population of low density. The results strongly indicated that the target cell of the thymic growth factor is the immunologically immature proliferating thymocyte of the thymic cortex.

Search for thymocyte growth-stimulating activity in extracts of different tissues and among established growth factors and thymic maturation hormones.

A thymocyte specific growth factor has previously been prepared from calf thymus. Similar activity is now shown to be present in thymus and spleen of guinea pigs, but not in lymph nodes, liver and kidney. Purified thymic maturation hormones, interleukins and several growth factors for other cell types than lymphocytes were tested and found not to stimulate DNA synthesis of thymocytes. A slight activity was found in thymosin fraction 5, known to contain at least 35 polypeptides. Our factor lacked comitogenic effect when tested in combination with concanavalin A (Con A) and phytohemagglutinin, did not promote continuous growth of ConA-induced lymphoblasts and did not affect the cyclic AMP level of thymocytes. The data indicate that our factor is functionally distinct from the purified hormones and defined factors tested.

New preparative schedule and further characterization of a thymocyte specific growth factor from calf thymus.

Purification of a thymocyte specific growth factor is described. A formic acid extract of calf thymus was acetone precipitated, extracted with acidic methanol and precipitated with ether. With an assay based on the stimulation of DNA synthesis in thymocyte cultures the crude extract was purified by several steps of gel filtration and ion exchange chromatography. The stimulating activity had an apparent Mr of 1,600 and a pI of 5.6-5.9 and was devoid of reactive amino groups. It behaved amphoteric and was sensitive to pronase. We propose that the activity resides in an N-terminal-blocked peptide without biochemical relationship to defined thymic differentiation hormones or established growth factors.

Studies on thymocyte subpopulations in guinea pigs. III. Physical and functional characterization of six subpopulations separated by density gradient centrifugation and PNA binding.

Thymocytes were separated according to increasing buoyant density into the three subpopulations Ia (25% of recovered cells), Ib (20%) and II (55%), and according to binding to peanut agglutinin (PNA)into PNA+ (65%) and PNA- cells (35%). The frequency of PNA+ was 56% in Ia, 60% in Ib and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for Ia, 130 fl for Ib and 100 fl for population II. PNA+ and PNA- cells were very similar as regards cell volume. Thus, PNA+ and PNA- cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population Ia. The mitogen-responsive thymocytes also belonged to population Ia, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.

Chemical and biological properties of a thymocyte specific growth factor from calf thymus.

From an acetone precipitate of bovine thymus a factor was isolated which stimulated the proliferation of thymocytes in vitro, without affecting growth of peripheral lymphocytes, bone marrow or liver cells. The stimulating material was purified by ultrafiltration, ion-exchange chromatography, gel filtration and semipreparative thin-layer chromatography. The activity resided in an acidic, hydrophilic low molecular weight molecule, apparently devoid of reactive amino groups. The effect was abolished by acidic hydrolysis but resisted treatment with DNase, RNase and trypsin. The effect was independent of serum and choice of culture medium. A mean increase of [3H]thymidine incorporation of 64% (range 47--101%) was obtained in thymic cells after 5 h of culture. The effect was dose-dependent and declined after 10 h. The mitotic activity increased correspondingly. A possible role of the factor in the regulation of proliferation in the thymus in vivo is suggested. The early kinetics of spontaneously proliferating thymocytes in cultures is discussed and compared to the intense growth and cell death in the thymus in vitro.