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Effect of negatively charged linear phosphates on MHC-restricted and non-MHC-restricted cytolytic activity mediated by lymphocytes
10
Abstracts
#3 4.1
#4 7.3
ORIGINS OF HLA-DRB GENETIC DIVERSITY. MT Povce-Jacino 1, P Santa.maria 1,2, SS Rich3, AJ Faras 1. Institute of Human Genetics, division of Endocrinology and Metabolism, Department of Medicine and Department of Laboratory Medicine and Pathology, Unviersity of Minnesota, Minneapolis, MN. The genetic mutations which comprise the more than 40 alleles of the DRB loci in humans may be hypothesized to have arisen by one of two mutation mechanisms, point mutation and gene conversion. Others have studied DRB allele evolution by sequencing a few individuals from representative populations, which has helped the basic understanding of the origins of HLA but not in the understanding of the selective pressures which regulate the generation of HLA diversity. We wanted to study DRB diveristy in a larger non-human primate population to determine if point mutation or gent conversions, or a combination of both, have been the driving mechanism of diversity in DRB. We combined sequence data from previously published DRB alleles with new human DRB alleles we have identified using sequence-based typing and with DRB sequence data from a series of six unrelated chimpanzees from the Yerxes primate center and 15 unrelated Rhesus monkeys from the Wisconsin prim~:te center. The DRB genes were amplified by PCR from RNA (chimps) or DNA (Rhesus) and cloned for sequencing. Preliminary results indicate that variations in DRB sequences in non-human primates follow the same distribution pattern, with unique point mutations within the beta pleated sheet region and with variation within the alpha helix region consisting of the same variable amino acids as in human DRB alleles. These results suggest that generation of diversity in the alpha helix of primate DRBs occurs primarily via gene conversions involving "casettes" of alpha helix-encoding sequences which predate human speciatlon.
EFFECT OF NEGATIVELY CHARGED LINEAR PHOSPHATES ON MHCR E S T R I C T E D AND N O N - M H C - R E S T R I C T E D C Y T O L Y T I C A C T I V I T Y MEDIATED BY LYMPHOCYTES. Bajpai, A. and ~ Z. Indiana University School of Medicine, Indianapolis. Extracellular ATP affect a variety of cell functions via ATP receptors on the plasma membrane. In this study, we examined the mechanism of inhibition of human lymphocyte mediated eytotoxicity (CMC) by 6 nuoleotides: As effector cells (EC) we used highly purified, freshly isolated NK cells and an MHC-restricted cell llne, YTINDY. ATP, GTP, ADP and GDP all strongly inhibited CMC in a d o s e - d e p e n d e n t f a s h i o n w h e r e a s AM P and GMP inhibited the least. Preincubation of either EC or TC with the diphosphates or triphosphates did not lead to inhibition, suggesting that these compounds inhibited a post-binding event. Furthermore, the inhibition was observed only when the nucleotides were present during the initial 30 min incubation of EC with TC. Next we wanted to ascertain whether the phosphates themselves or the negative charges on these molecules were responsible for the inhibition. We therefore tested the effect of p e n t a s o d i u m t r i p h o s p h a t e (PST) and g l y c o s a m i n o g l y o a n sulfate (heparin) on CMC: Both inhibited CMC in a dosedependent manner. The inhibition mediated by all the c o m p o u n d s t e s t e d was r e v e r s i b l e and s e r i n e e s t e r a s e levels as well as g r a n u l e c o n t e n t of EC w e r e not affected. This phenomenon, therefore, is not restriuted to ATP as previously reported.
Abstracts
#3 4.1
#4 7.3
ORIGINS OF HLA-DRB GENETIC DIVERSITY. MT Povce-Jacino 1, P Santa.maria 1,2, SS Rich3, AJ Faras 1. Institute of Human Genetics, division of Endocrinology and Metabolism, Department of Medicine and Department of Laboratory Medicine and Pathology, Unviersity of Minnesota, Minneapolis, MN. The genetic mutations which comprise the more than 40 alleles of the DRB loci in humans may be hypothesized to have arisen by one of two mutation mechanisms, point mutation and gene conversion. Others have studied DRB allele evolution by sequencing a few individuals from representative populations, which has helped the basic understanding of the origins of HLA but not in the understanding of the selective pressures which regulate the generation of HLA diversity. We wanted to study DRB diveristy in a larger non-human primate population to determine if point mutation or gent conversions, or a combination of both, have been the driving mechanism of diversity in DRB. We combined sequence data from previously published DRB alleles with new human DRB alleles we have identified using sequence-based typing and with DRB sequence data from a series of six unrelated chimpanzees from the Yerxes primate center and 15 unrelated Rhesus monkeys from the Wisconsin prim~:te center. The DRB genes were amplified by PCR from RNA (chimps) or DNA (Rhesus) and cloned for sequencing. Preliminary results indicate that variations in DRB sequences in non-human primates follow the same distribution pattern, with unique point mutations within the beta pleated sheet region and with variation within the alpha helix region consisting of the same variable amino acids as in human DRB alleles. These results suggest that generation of diversity in the alpha helix of primate DRBs occurs primarily via gene conversions involving "casettes" of alpha helix-encoding sequences which predate human speciatlon.
EFFECT OF NEGATIVELY CHARGED LINEAR PHOSPHATES ON MHCR E S T R I C T E D AND N O N - M H C - R E S T R I C T E D C Y T O L Y T I C A C T I V I T Y MEDIATED BY LYMPHOCYTES. Bajpai, A. and ~ Z. Indiana University School of Medicine, Indianapolis. Extracellular ATP affect a variety of cell functions via ATP receptors on the plasma membrane. In this study, we examined the mechanism of inhibition of human lymphocyte mediated eytotoxicity (CMC) by 6 nuoleotides: As effector cells (EC) we used highly purified, freshly isolated NK cells and an MHC-restricted cell llne, YTINDY. ATP, GTP, ADP and GDP all strongly inhibited CMC in a d o s e - d e p e n d e n t f a s h i o n w h e r e a s AM P and GMP inhibited the least. Preincubation of either EC or TC with the diphosphates or triphosphates did not lead to inhibition, suggesting that these compounds inhibited a post-binding event. Furthermore, the inhibition was observed only when the nucleotides were present during the initial 30 min incubation of EC with TC. Next we wanted to ascertain whether the phosphates themselves or the negative charges on these molecules were responsible for the inhibition. We therefore tested the effect of p e n t a s o d i u m t r i p h o s p h a t e (PST) and g l y c o s a m i n o g l y o a n sulfate (heparin) on CMC: Both inhibited CMC in a dosedependent manner. The inhibition mediated by all the c o m p o u n d s t e s t e d was r e v e r s i b l e and s e r i n e e s t e r a s e levels as well as g r a n u l e c o n t e n t of EC w e r e not affected. This phenomenon, therefore, is not restriuted to ATP as previously reported.