Tapan Audhya

Thymopoietin to thymopentin: experimental studies

SummaryThymopoietin is a polypeptide hormone of the thymus consisting of 49 amino acids. The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to amino acids 32-36 of thymopoietin, appears to represent the active site of thymopoietin in that it has all the biological activities of the native hormone. Thymopoietin is secreted by epithelial cells of the thymus and is pleiotropic in action, affecting neuromuscular transmission, induction of early T cell differentiation and immune regulation. The immuno-regulatory actions of thymopentin on peripheral T cells are mediated by intracellular cyclic GMP elevations in contrast to the intracellular cyclic AMP elevations induced in precursor T cells that trigger their further differentiation to T cells. Thymopoietin and thymopentin have the biological characteristics of being immunonormalizing in a number of animal model systems of immune dysbalance. These include immune dysbalances induced by thymectomy or the thymic involution associated with aging or by other procedures in thymus-intact animals. The normalizing action of thymopentin, whether the immune dysbalance be in the direction of hyper- or hyporesponsiveness points to its potential utility in human diseases characterized by immune dysbalance.RésuméLa thymopoïétine est une hormone polypeptidique se composant de 49 amino-acides. La thymopentine (TP-5) Arg-Lys-Val-Tyr, un pentapeptide correspondant aux amino-acides 32–36 de la thymopoïétine, paraît représenter le site actif de la thymopoïétine en ce sens qu’elle a toutes les activités biologiques de rhormone naturelle. La thymopoïétine est sécrétée par les cellules épithéliales du thymus et a une action pléiotrope, affectant la transmission neuromusculaire, l’induction de la première phase de la différenciation des cellules T et l’immuno-régulation. Les effets immunorégulateurs de la thymopentine sur les cellules T périphériques sont médiatisés par des hausses intracellulaires du GMP cyclique, alors que ce sont des hausses intracellulaires de l’AMP cyclique induites dans les cellules T précurseurs qui déclenchent leur différenciation plus poussée en cellules T. La thymopoïétine et la thymopentine présentent les caractéristiques biologiques d’une action immunonormalisante dans un certain nombre de systèmes-modèles animaux de déséquilibre immunologique. Ceux-ci englobent les déséquilibres immunologiques induits par la thymectomie ou Pinvolution thymique associée à l’âge ou autres processus affectant des animaux au thymus intact. L’action immunonormalisatrice de la thymopentine lors de déséquilibre immunologique, qu’il aille dans le sens d’une hyper- ou d’une hyporéactivité, souligne son utilité potentielle dans la pathologie humaine caractérisée par un déséquilibre immunologique.ZusammenfassungThymopoietin ist ein Polypeptid-Thymushormon aus 49 Aminosäuren. Das Pentapeptid Thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, welches den Aminosäuren 32–36 des Thymopoietin entspricht, stellt insofern den aktiven Bereich von Thymopoietin dar, als es alle biologischen Wirkungen des Nativhormons besitzt. Thymopoietin wird von den Epithelzellen des Thymus abgesondert und wirkt pleiotrop; es beeinflusst die neuromuskuläre Öbertragung, die Induktion früher T-Zellen-Differenzierung und die Immunregulation. Die immunregulatorische Wirkung von Thymopentin auf die peripheren T-Zellen wird vermittelt durch intrazellulären zyklischen GMP-Anstieg im Gegensatz zum intrazellulären zyklischen AMP-Anstieg; dieser wird von T-Zellenvorläufern induziert und löst deren weitere Differenzierung zu T-Zellen aus. Thymopoietin und Thymopentin haben die biologische Eigenschaft, in einer Reihe von Tiermodellen mit gestörtem immunologischem Gleichgewicht den immunologischen Normalzustand wieder herzustellen, z.B. bei gestörtem immunologischem Gleichgewicht infolge von Thymektomie ebenso wie bei Involution des Thymus infolge von Alterung oder anderen Prozessen in Tieren mit intaktem Thymus. Díe normalisierende Wirkung von Thymopentin, gleich ob sich das gestörte immunologische Gleichgewicht als Hyper- oder Hyporeaktivität äussert, weist auf eventuellen Nutzen bei der Behandlung menschlicher Krankheiten hin. sofern solche durch gestörtes immunologisches Gleichgewicht geprägt sind.Riassunto La timopoietina è un ormone polipeptide del timo e consiste in 49 amminoacidi. II pentapeptide denominato timopentina (TP-5) Arg-Lys-Asp-Val-Tyr, che corrisponde agli amminoacidi 32–36 della timopentina, sembra rappresentare la regione attiva dell’ultirna nel senso ehe possède tutte le attività biologiche dell’ormone naturale. La timopoietina viene secernata da cellule epiteliali del timo ed esplica un’azione pleiotropica, influenzando la trasmissione neuromuscolare, l’induzione della differenziazione precoce delie cellule T, nonché la regolazione immunitaria. Le azioni immunoregolatrici della timopentina sulle cellule T periferiche vengono mediate da elevazioni GMP cicliche intracellulari, a differenza alle elevazioni AMP cicliche intracellulari indotte nelle cellule T precursori che determinano la loro ulteriore differenziazione alle cellule T. La timopoietina e la timopentina posseggono le caratteristiche biologiche di avere un’azione immunonormalizzante in una serie di sistemi modelli nell’animale di disequilibrio immunitario. Questi comprendono disequilibri causati dalla timectomia o dalla involuzione timica associata con l’invecchiamento o da altri processi in animali timointatti. L’azione normalizzante della timopentina, essendo il disequilibrio immunitario nel senso o dell’ipersensibilità o dell’iposensibilità, indica la sua utilità Potenziale nelle malattie umane caratterizzate da disequilibrio immunitaria.ResumenLa timopoietina es una hormona polipéptida del timo consistente en 49 aminoácidos. El pentapéptido timopentina (TP-5) Arg-Lis-Asp-Val-Tir, correspondiente a los aminoácidos 32-36 de la timopoietina, representa el centro activo de la timopoietina en que posee todas las actividades biológicas de la hormona original. La timopoietina es segregada por las células epiteliales del timo y su acción es pleitrópica, influyendo en la transmisión neuromuscular, en la inducción temprana de la diferenciación de las células T y en la regulación inmune. Las acciones inmunoregu-ladoras de la timopentina sobre las células T periféricas son mediadas por elevaciones intracelulares del GMP cíclico al contrario de las elevaciones intracelulares del AMP cíclico inducidas en las células T precursoras que causan su ulterior diferenciación en células T. La timopoietina y la timopentina poseen las características biológicas de ser inmunonormalizadoras en un número de sistemas modelo de animales de desequilibrio inmune. Estos incluyen desquilibrios inmunes inducidos por timectomía o la involución timica asociada con la edad o por otros procedimientos en animales con timo intacto. La acciôn normalizadora de la timopentina, ya esté el desequilibrio inmune orientado hacia una hipero- o hiporespuesta, señala su utilidad potencial en las enfermedades humanas caracterizadas por desequilibrios immunes.

Cleavage of esters using carbonates and bicarbonates of alkali metals: synthesis of thymopentin

Abstract A novel method for hydrolysis of primary esters using aqueous alkali carbonates or bicarbonates and an alcohol as cosolvent is described. Several peptide esters, including a Cbz-Arg-Lys(Cbz)-Asp(OBzl)-Val-Tyr-OBzl (sequence corresponding to the active site of thymic immunoregulatory hormone, thymopoietin), were hydrolyzed to demonstrate the utility of this method.

Biologically active analogs of thymopentin with enhanced enzymatic stability

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.

Structural requirements for the biological activity of thymopentin analogs

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.

Thymopoietin radioreceptor assay utilizing lectin-purified glycoprotein from a biologically responsive T cell line

Radiolabeled thymopoietin that was biologically active and of high specific activity was prepared by a novel technique involving protection of free amino groups, selective excision of the protected N-terminal prolyl group with post-proline cleaving enzyme, reaction of the newly exposed alpha-amino group with a highly radioiodinated compound, and deprotection and purification of the polypeptide. Binding of this radiolabeled thymopoietin was not demonstrable by conventional techniques with cells, cell membranes, or solubilized cell membranes, apparently due to the presence of active proteases in these preparations. A glycoprotein with thymopoietin binding properties was prepared by lectin purification from the detergent-solubilized membranes of CEM cells, a human T cell line that responds to thymopoietin in vitro with increases in intracellular cyclic GMP. Presumably this procedure separated the thymopoietin binding protein from membrane proteases, thus permitting the development of a radioreceptor assay. Evidence is presented that the thymopoietin binding protein represents a thymopoietin receptor that is probably related to the mediation of immunoregulatory actions of thymopoietin on a subset of peripheral T cells.

Highly purified neurophysin proteins: Method of isolation based on their acidic properties☆

Abstract The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m ). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m ). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent pI value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.

Cooperativity of thymopoietin 32–36 (the active site) and thymopoietin 38–45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.

Isocratic resolution of amino acid thiohydantoins by high-performance liquid chromatography

Abstract Sixteen amino acid thiohydantoins encountered during COOH-terminal degradation of peptides and proteins with ammonium thiocyanate have been separated and identified by reverse phase high-performance liquid chromatography using a DuPont Zorbax ODS column and a novel procedure for preparation of thiohydantoin derivative of serine and threonine is described. Three isocratic systems were employed: System 1, buffer A alone (0.01 n sodium acetate, pH 4.5); system 2, 10% acetonitrile in buffer A; system 3, 24% acetonitrile in buffer A. All 16 thiohydantoins could be resolved using a combination of systems 1, 2, and 3. The method is rapid and sensitive (1 nmol) and constitutes a step toward automation of the solid-phase COOH-terminal sequencing method.

Immunohistochemical localization of thymopoietin with an antiserum to synthetic Cys-thymopoietin28–39

Thymopoietin-containing cells in the thymus were identified immunohistochemically using murine antiserum generated by immunization with synthetic Cys-thymopoietin28-39 (Cys-TP28-39). human thymopoietin, This antiserum, previously shown to react with both bovine and human thymopoietin, gave reactivity restricted to cortical and medullary epithelial cells of bovine and human thymus. Monoclonal antibodies with reactivity restricted to native bovine thymopoietin did not react with tissue sections of bovine thymus; most likely the epitopes recognized by monoclonal antibodies are not expressed on the inactive precursor forms of thymopoietin within thymic epithelial cells.

Immunoassay for bovine serum thymopoietin: Discrimination from splenin by monoclonal antibodies

A polyclonal rabbit anti-bovine thymopoietin antiserum was used to develop a radioimmunoassay and sandwich enzyme-linked immunosorbent (ELISA) assay for the thymic hormone thymopoietin. Both assays showed slightly less sensitivity for the closely related splenic hormone splenin (SP) than thymopoietin (TP) and markedly less sensitivity for the human as compared with the bovine polypeptides. A number of murine monoclonal antibodies specific for bovine thymopoietin were generated; they were unreactive with bovine splenin and were also unreactive with human thymopoietin and splenin. A sandwich ELISA using these monoclonal anti-TP antibodies together with polyclonal rabbit anti-TP was specific for bovine thymopoietin and measured 300-500 ng/ml thymopoietin in bovine serum. Similar approaches are being pursued to develop an immunoassay for thymopoietin in human serum.