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- Zhang, Qian-Jin
(författare)
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Influence of MHC class I peptide interaction on antigen presentation in normal and malignant cells
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The presentation of MHC class I peptide complexes in normal and malignant cells was studied. An HLA A11 binding motif was defined by comparing synthetic peptide analogues of a known cytotoxic T Iymphocyte (CTL) target epitope for induction of surface A11 expression and triggering of cytotoxic activity. The motif predicts the presence of hydrophobic aminoacids in position 2 (P2), small amino acids in P3 and P6 and Lys in P9. The off-rates of P2 analogues discriminated between "apparent" and "true" binders that formed complexes with a half life longer than 72 hours. The persistence of MHC-peptide complexes the surface of antigen presenting cells was shown to correlate with the immunogenicity two A11-restricted epitopes of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4 (designated IVT and AVF, respectively). Molecular modeling and alanine scanning mutagenesis of the IVT and AVF peptides demonstrated that solvent exposed peptide side chains affect CTL recognition as well as antibody binding suggesting that allospecific antibodies recognize the complexes in a fashion similar to T cell receptors. Class I molecules are important for target recognition by T cells as well as natural killer (NK) cells. Overexpression of the c-myc oncogene increased the sensitivity of EBV transformed Iymphoblastoid cell lines (LCLs) to NK lysis. The reactivity with allospecific antibodies was significantly reduced suggesting that a different set of antigenic peptides may occupy the class I groove, resulting in inability to deliver a negative regulatory signal to NK cells. Defective presentation of endogenously expressed EBNA4 was demonstrated in A11 positive Burkitt's Iymphoma (BL) lines. The defect was not overcome by cytosolic expression of the preformed epitope. The tumor cell expressed low levels of the transporter associated with antigen processing (TAP) and behaved poorly in a streptolysin-O mediated peptide translocation assay but presentation was not restored by upregulation TAP activity following treatment with IFN-y. Efficient maturation of class I molecules to Endo H resistant species was demonstrated in pulse-chase experiments. The results localize the defect to functions downstream of the generation of antigenic peptides but upstream of TAP-dependent peptide transport and class I assembly and maturation.
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- Zhang, Shi-Jin, 1957-
(författare)
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Regulation of Intracellular Calcium in Brown Adipocytes
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intracellular Ca2+ is considered a primary regulator of cell function. In the present study, the control and the effects of intracellular Ca2+ in brown adipocytes have been investigated. Cytosolic Ca2+ levels ([Ca2+]i) are the resultant of the activity of Ca2+ transport systems. Results concerning Ca2+transport systems in the plasma membrane, endoplasmic reticulum and mitochondria are presented.[Ca2+]i, monitored with Fura-2/AM, is increased when brown adipocytes are stimulated with norepinephrine (NE). The NE effect is mediated via a1-adrenoceptors and involves both release from intracellular Ca2+ stores and influx of extracellular Ca2+. The NE-induced [Ca2+]i response could be desensitized by pretreatment with NE. The desensitization is also mediated by a1-receptors and intracellularly by increased [Ca2+]i and calmodulin but not by protein kinase C. The kinetics of the desensitization are similar to those of inhibition of protein synthesis or transcription and the desensitization is associated with a comparable decrease in the number of a1-receptors.Mitochondrial Ca2+ levels ([Ca2+]m) were monitored within brown adipocytes with mitochondrially targeted aequorin. [Ca2+]m was not a simple reflection of [Ca2+]i; rather, evidence is presented for the existence of a b-adrenergic, cAMP-mediated signal that augments the [Ca2+]m/[Ca2+]i ratio. This signal causes the mitochondria to sequester Ca2+ even in the absence of increased cytosolic levels. Inhibition of mitochondrial Ca2+ uptake augments the cytosolic responses. Mitochondria may thus play an important role even in cytosolic Ca2+homeostasis in brown adipocytes.Chronic treatment of brown adipocytes with NE resulted in marked alterations of cytosolic Ca2+ handling, but the mitochondria retained their ability to sequester Ca2+during adrenergic stimulation, i.e. under conditions when UCP1 should be active.The effects of an increase in [Ca2+]i involve activation of a cAMP phosphodiesterase, and the presence of this component explains the unusual kinetic characteristics of norepinephrine-induced cAMP accumulation. [Ca2+]i is also involved in the regulation of gene expression: increased [Ca2+]i interacts synergistically with cAMP in the control of c-fos expression which may be of significance for regulation of cell proliferation and differentiation.It was concluded that Ca2+ is a primary regulator of physiological functions in brown adipocytes. The Ca2+ transport systems in brown adipocytes are involved in the regulation of intracellular and intraorganellar Ca2+. Changes of the free cytosolic Ca2+ concentration by hormone stimulation induces the activation of many physiological processes.
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