Ulf Ernström

Cyclic amp in freshly prepared thymocyte suspensions. evidence for stimulation by endogenous adenosine

Abstract Thymocyte suspensions were prepared from guinea pig thymus by gentle mincing and repeated washings. The mincing resulted in a ten-fold increase of the cyclic AMP level, most of the cyclic AMP being intracellular. The level remained high during the washing procedures. During incubation at 37° the level of intracellular cyclic AMP gradually fell by a temperature dependent process. The cyclic AMP content was increased by non-methylxanthine phosphodiesterase inhibitors (papaverine, Ro 20-1724, ZK 62.711) but decreased in the presence of theophylline or isobutylmethylxanthine. Adenosine and phenylisopropyl adenosine acutely increased cyclic AMP by a theophylline inhibited mechanism. An increase was also obtained by inhibitors of adenosine uptake (dipyridamol, dilazep) and of adenosine deaminase (EHNA). The results were compatible with a release of adenosine during the isolation of thymocytes, and with an adenosine-induced increase of intracellular cyclic AMP. This interpretation was supported by in vivo labelling of purine nucleotides with [3H]adenine. A marked rapid fall of purine nucleotides was found during the preparation of the thymocytes and at the same time adenosine metabolites appeared in the medium.

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.

Quantitation of B and T Lymphocytes in Guinea Pigs with Evidence for a Release of both Cell Types from the Spleen into the Blood

B and T lymphocytes were quantitated in lymphoid organs and in blood of young normal guinea pigs by the use of EAC rosettes and rabbit erythrocyte (RE) rosettes as markers. Special attention was focused on the release of B and T cells from the spleen, estimated from the difference between the content of B and T cells in splenic efferent and afferent blood. The following frequencies of resette-forming cells (RFC) were found. Thymus: 0.3% EAC- and 88% RE-RFC; spleen: 44% EAC- and 46% RE-RFC; lymph nodes: 13% EAC- and 39% RE-RFC; arterial blood: 13% EAC- and 42% RE-RFC, and bone marrow: 2% EAC- and 7% RE-RFC. A fairly large number of cells in the lymph nodes and blood could not be identified by any of the two markers. Some possible explanations for this are discussed. The content of both EAC- and RE-RFC in splenic efferent blood significantly exceeded that in the afferent blood, indicating a release of both B and T cells from the spleen into the blood. The possibility of a release of a third type of mononuclear cell cannot be excluded from the present results.

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.

Mitotic Activity of Thymocytes in a Synthetic Tissue Culture Medium. Effect of L-AIanine

DNA synthesis in guinea pig thymocytes suspended in RPMI 1640 medium increased to a peak after 4-5 h in culture and was followed by increased mitotic activity, indicating that many thymocytes in S phase proceeded through G2 into mitosis. Addition of L-alanine to the medium markedly increased the DNA synthesis within 1 h and the mitotic frequency from 6 h. The increase in DNA synthesis when L-alanine was present in the medium was thus caused by an increased number of cells in S phase. Human thymocytes cultured in RPMI 1640 for 18 h had a low mitotic frequency. Addition of L-alanine immediately started DNA synthesis in the arrested thymocytes resulting in increased mitotic activity from 6 h later. The results show that L-alanine is a growth factor for guinea pig and human thymocytes and should be included in tissue culture media used for such cells. Growth of thymocytes in vitro was partly synchronized, and the mitotic studies indicated that many cells had entered S phase near the start of incubation.

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)‐β,ϵ‐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.

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+.

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.

L-alanine--an essential amino acid for growth of lymphocytes in vitro.

A factor an ultrafiltrated (UM 10) extracts of calf thymus, liver and spleen stimulated the uptake of 3H-thymidine in DNA of cultured lymphocytes. The factor was purified by ion-exchange chromatography and gel chromatography and identified as alanine, which is lacking in the culture medium, RPMI 1640. An optimal amount of L-alanine increased the uptake of 3H-thymidine in DNA of lymphocytes from different species by about 100%. L-alanine should be regarded as a growth factor for lymphocytes in vitro and should be added to RPMI 1640, when this tissue culture medium is used for lymphocytes.

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.