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.

     

Muscarinic Agonists Evoke Neurotransmitter Release: Possible Roles for Phosphatidyl Inositol Bisphosphate Breakdown Products in Neuromodulation

Carbachol (CCh), a muscarinic agonist that elicits the formation of inositol trisphosphate (IP3) and diacylglycerol (DG), induces a calcium-dependent [3H]norepinephrine ([3H]NE) release [IC50 = (2.7 +/- 0.5) X 10(-4) M] in rat brain slices. Similarly, other muscarinic agonists evoke [3H]NE release which is specifically inhibited by muscarinic antagonists such as 3-quinuclidinyl benzilate, atropine, and N-methyl-4-piperidyl benzilate. The atropine-sensitive evoked release is effectively inhibited by neomycin (IC50 = 50 microM), a phospholipase C inhibitor that interferes with IP3-dependent cellular processes. In addition, polymyxin B, a rather selective inhibitor of protein kinase C (PK-C), abolishes the agonist-mediated release with a half-maximal effective concentration of 0.53 microM (750 ng/ml). These results have a significant implication for the mechanism by which agonists generating IP3 and DG act as inducers of neurotransmitter release in the CNS. However, since both neomycin and polymyxin B act also as N-calcium-channel blockers, other possible mechanisms are discussed. The CCh-induced release suggests that in the CNS an agonist-receptor interaction leads to a calcium-dependent neurotransmitter release, most likely via promoting the IP3/DG as second messengers followed by activation of PK-C.     

The phylogenic expression of plasmolipin in the vertebrate nervous system

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.Abbreviations EDTA Ethylene diamine N.,NN tetracetic acid - EGTA Ethylene glycol bis-(B-Aminoethyl Ether) N,,NN tetracetic acid - MES ([N-Morpholino] ethane sulfonic acid) DCCD, N, Dicyclohexyl carbodiimide     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin

Prosolin is a major cytosolic protein (Mr 18400, isoelectric point 5.9) first reported in HL-60 promyelocytic leukemia cells. It is rapidly phosphorylated (15 to 30 min) in response to TPA treatment as an early event in a sequence that leads to cessation of cell proliferation and to differentiation of promyelocytes into monocytes. In our study we examined the expression of prosolin in human peripheral lymphocytes and investigated the effects of TPA treatment on prosolin phosphorylation and on lymphocyte proliferation. Prosolin was not expressed in resting PBL but was induced after 24 to 36 h of PHA stimulation, simultaneously with induction of DNA synthesis. In rapidly proliferating (IL-2 dependent) PBL prosolin was a major cytosolic component, comprising 0.5% of total cytosolic protein, of which approximately 28% was phosphorylated. Expression of prosolin decreased again when either mitogen-induced or IL-2-dependent proliferation diminished during extended periods in culture. Thus, expression of prosolin is correlated with periods when PBL are cycling through S-phase. TPA treatment of IL-2-dependent PBL at the peak of their growth caused phosphorylation of about two-thirds of preexisting unphosphorylated prosolin within 1 h. This was accompanied by cessation of cell proliferation, as indicated by measurements of TdR incorporation. Although TPA has well known mitogenic effects in lymphocytes during initial activation, this result shows that it exerts an antiproliferative effect in rapidly dividing PBL. It is suggested that increased phosphorylation of prosolin may be an initiating event in the antiproliferative response to TPA, which would occur only in proliferating lymphocytes expressing prosolin.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.