Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

α-MELANOCYTE-STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α-melanocyte-stimulating hormone (α-MSH) in the entire hypothalamus as well as the amount of α-MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1-24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso-osmotic sucrose solution, and free granules and synaptosomes containing α-MSH were fractionated by means of continuous sucrose density gradient centrifugation. α-MSH was quantified by radioimmunoassay. The total amount of α-MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α-MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5-24-month-old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α-MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.     

Estrogen hydroxylase activity in the human placenta at term

Placental estrogen hydroxylase (EH) enzyme activity was measured at term using the catechol-O-methyl transferase coupled method in normal and high risk conditions. The identity and ratio of products formed during incubation of microsomes as analysed by high performance liquid chromatography in chronic hypertension, toxemia and diabetes mellitus was not different from controls. The mean enzymatic activity was also not different among the conditions studied as expressed mean +/- SE pmol/min/mg, protein: chronic hypertension (7.8 +/- 1), toxemia (8 +/- 1.6), diabetes mellitus (6.1 +/- 0.9) and controls (8.3 +/- 1.5). The cofactor dependence of EH was studied showing that NADPH is a better substrate for the enzyme than NADH.     

Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution.

In Swiss 3T3 cells, depletion of protein kinase C (PKC) by prolonged incubation with phorbol esters potentiates the formation of total inositol phosphates in response to bombesin or vasopressin [Blakeley, Corps & Brown (1989) Biochem. J. 258, 177-185]. The characteristics of the accumulation of inositol phosphates in control and PKC-depleted cells stimulated by bombesin, vasopressin or prostaglandin F2 alpha (PGF2 alpha) have now been compared. The potentiation of the PGF2 alpha response was greater than that of the vasopressin response which was, in turn, greater than that of the bombesin response. The time courses of the responses to all three agonists were biphasic, and both phases of the response were amplified in the PKC-depleted cells. These results provide further evidence for the involvement of a PKC-mediated negative-feedback loop regulating phosphoinositide hydrolysis in response to several 3T3 cell mitogens. The differential potentiation of the response to these agonists suggests that PKC might act at multiple sites within the signal transduction pathway.     

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and α-melanocyte-stimulating hormone (α-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of α-MSH in the homogenate, 36% was recovered in the 11,500 g pellet and 31% sedimented between 11,500 and 105,000 g. α-MSH was not detected in the 105,000 g supernatant fluid. When the 900 g supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing α-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain α-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing α-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing α-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing α-MSH are synaptosomes.     

A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KC1 resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.