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1.
Chem Commun (Camb) ; 53(8): 1393-1396, 2017 Jan 24.
Article in English | MEDLINE | ID: mdl-28074973

ABSTRACT

A new fluorescence turn-on type of PCR monitoring system (Hpro-PCR) using a hairpin probe and a primer having a tag sequence at the 5' end with the fluorescent molecule 2,7-diamino-1,8-naphthyridine derivative (DANP) has been developed. The Hpro-PCR exploited the modulation of the equilibrium states between the DANP-bound hairpin structure and probe-tag duplex, and the PCR progress alternated the equilibrium state, resulting in the change of fluorescent intensity.


Subject(s)
Fluorescence , Fluorescent Dyes/chemistry , Naphthyridines/chemistry , Polymerase Chain Reaction
2.
Chem Commun (Camb) ; 50(96): 15195-8, 2014 Dec 14.
Article in English | MEDLINE | ID: mdl-25338232

ABSTRACT

The progress of a polymerase chain reaction (PCR) was sensitively monitored based on the increase in fluorescence of N,N'-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine, which was covalently anchored on the cytosine bulge directly neighbouring the 5'-T_G-3'/5'-CCA-3' sequence in the hairpin tag at the 5' end of the PCR primer.


Subject(s)
Cytosine/chemistry , DNA Primers/metabolism , Fluorescent Dyes/chemistry , DNA Primers/chemistry , Naphthyridines/chemistry , Plasmids/metabolism , Real-Time Polymerase Chain Reaction
3.
J Immunol ; 167(8): 4180-6, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11591738

ABSTRACT

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.


Subject(s)
Antigens, Ly , Cell Line , Killer Cells, Natural/immunology , Membrane Glycoproteins/metabolism , T-Lymphocyte Subsets/immunology , Animals , Antigen-Presenting Cells , Bone Marrow Cells/immunology , Cell Culture Techniques/methods , Galactosylceramides/immunology , Genes, MHC Class I , Histocompatibility Antigens Class I/immunology , Lectins, C-Type , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , NK Cell Lectin-Like Receptor Subfamily A , Receptors, NK Cell Lectin-Like , Spleen/cytology , Spleen/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Tissue Distribution
4.
Immunol Rev ; 181: 90-103, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11513155

ABSTRACT

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.


Subject(s)
Antigens, CD/genetics , Antigens, Ly , Lectins, C-Type , Membrane Glycoproteins/genetics , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Evolution, Molecular , Gene Expression Regulation, Developmental , Humans , Killer Cells, Natural/immunology , Mice , Molecular Sequence Data , Multigene Family , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Papio/genetics , Papio/immunology , Phylogeny , Receptors, NK Cell Lectin-Like , Receptors, Natural Killer Cell , Sequence Homology, Nucleic Acid
5.
Immunogenetics ; 53(3): 215-24, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11398966

ABSTRACT

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.


Subject(s)
Antigens, Ly , Membrane Glycoproteins/genetics , Promoter Regions, Genetic , Receptors, Immunologic/genetics , Animals , Binding Sites , DNA-Binding Proteins/metabolism , Gene Expression , Humans , Lectins, C-Type , Lymphoid Enhancer-Binding Factor 1 , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A , Receptors, NK Cell Lectin-Like , Regulatory Sequences, Nucleic Acid , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , TATA Box , Transcription Factors/metabolism , Transcription, Genetic
6.
Int Immunol ; 13(3): 321-7, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11222501

ABSTRACT

The CD94/NKG2A heterodimer is an inhibitory receptor expressed on a subset of mouse NK cells. CD94/NKG2A recognizes the non-classical MHC class I (class Ib) molecule Qa-1(b) and inhibits NK cytotoxicity. Qa-1(b) presents a peptide derived from the leader sequence of classical MHC class I molecules. Here, we examined the role of CD94/NKG2A in T cell-mediated cytotoxicity. Soluble tetrameric Qa-1(b) bound to almost all CD8(+), but not CD4(+), T cells. This binding seems to be mediated by CD8, because COS cells transfected with CD8 also bound Qa-1(b) tetramer. Therefore, the expression of CD94/NKG2 in T cells was further examined by single-cell RT-PCR. Most murine CD8(+) T cells constitutively expressed CD94 and NKG2A transcripts, whereas they were not detected in CD4(+) T cells. Co-expression of Qa-1(b) and D(k) on target cells significantly inhibited cytotoxicity of D(k)-specific cytotoxic T lymphocytes generated by mixed lymphocyte reaction, indicating that Qa-1(b) on antigen-presenting cells interacts with CD94/NKG2A on CD8 T cells and regulates classical MHC class I-restricted cytotoxic T cells. These results suggest a significant role of CD94/NKG2A as an inhibitory receptor on CD8(+) T cells.


Subject(s)
Cytotoxicity, Immunologic/immunology , H-2 Antigens/immunology , Histocompatibility Antigens Class I/immunology , Lectins, C-Type , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COS Cells , Chlorocebus aethiops , Dimerization , Genes, MHC Class I , H-2 Antigens/genetics , Histocompatibility Antigen H-2D , Lymphocyte Culture Test, Mixed , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , RNA, Messenger/biosynthesis , Rats , Receptors, Immunologic/immunology , Receptors, Natural Killer Cell , Recombinant Fusion Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction , Transfection
7.
Immunogenetics ; 52(3-4): 212-23, 2001.
Article in English | MEDLINE | ID: mdl-11220623

ABSTRACT

The Ly49 multigene family consists of at least 14 closely related genes located in the natural killer (NK) gene complex on mouse Chromosome 6. Reverse transcriptase (RT)-PCR on single NK cells has shown that Ly49c is expressed on approximately 50% of NK cells, whereas the closely related Ly49j gene is expressed on 5-8% of NK cells. In this study, we examined three regions to determine whether they contain cis-acting elements involved in regulating the expression of these two closely related Ly49 genes within NK cells. Luciferase reporter assays in EL-4 cells suggested that the 5' regions of Ly49c and j contain promoter elements and repressor sequences. In addition, luciferase assays suggest that Ly49j also contains an active promoter in the first intron, although the transcripts produced from this promoter appear to be severely truncated. Finally, comparisons of the 3' noncoding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c 130 bp downstream of the termination codon. The polyadenylation signal for Ly49j is located downstream of the Ly49c poly(A) site, which results in a much longer 3' untranslated region (UTR). When the Ly49j 3'UTR was used to provide the polyadenylation signal for the green fluorescent protein (GFP) reporter gene, GFP expression was reduced twofold. These results suggest that both internal promoters, repressors, and 3' regions play a role in regulating Ly49 gene expression.


Subject(s)
Antigens, Ly , Membrane Glycoproteins/genetics , Promoter Regions, Genetic/genetics , 3' Untranslated Regions/genetics , Animals , Base Sequence , COS Cells , Exons/genetics , Gene Expression Regulation , Genes, Reporter/genetics , Introns/genetics , Killer Cells, Natural/metabolism , Lectins, C-Type , Mice , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily A , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, NK Cell Lectin-Like , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
8.
J Neurosurg ; 93(2): 245-53, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10930010

ABSTRACT

OBJECT: This prospective study is based on a consecutive series of 20 patients with pineal region tumors who underwent minimally invasive preferential management. The purpose of this report is to discuss the role of neuroendoscopic procedures in the management of pineal region tumors. METHODS: If the tumor markers alpha-fetoprotein and human chorionic gonadotropin were not detected in serum and there was significant ventricular dilation visualized on neuroimages, neuroendoscopic surgery was first applied for tumor debulking with tissue diagnosis and gross morphological analysis of the tumor and the intraventricular structures, followed by third ventriculostomy. Subsequent procedures were determined on the basis of verified individual tumors. For treatment of germinomas and pineoblastomas, if no tumor dissemination was confirmed by pre-, intra-, or postoperative findings, stereotactic radiotherapy or radiosurgery was performed after one course of chemotherapy with the ICE regimen (isofomid, cisplatin, and etoposide) and followed by two additional courses of chemotherapy. For treatment of malignant germ cell tumors, after extensive surgery, adjuvant chemotherapy with the ICE regimen was performed in three courses in all cases. Then radiotherapy was started using various methods, depending on the evidence of tumor dissemination. For treatment of teratomatous and neuroectodermal tumors other than pineoblastomas, extensive surgical removal was performed. As for adjuvant therapy, if the tumor was a low-grade glioma or if the patient was younger than 5 years of age, postoperative treatment did not include radiotherapy. If the tumor was a malignant teratoma or high-grade glioma, conventional focal radiotherapy was performed, followed by chemotherapy with ICE for 1 year. All but two treated patients had ventriculomegaly. Neuroendoscopic procedures were performed in six of 15 treated patients. Neuroendoscopic biopsy with tumor debulking offered enough material for tissue diagnosis, including immunohistochemical analysis and, in one case, revealed evidence of tumor dissemination undetectable on neuroimaging. With one exception, no shunt was required in any patient undergoing endoscopic third ventriculostomy. Stereotactic radiotherapy was performed in indicated cases. Favorable therapeutic outcomes were obtained in all cases of germinoma and pineoblastoma, with follow-up periods ranging from 24 months to 6.5 years. CONCLUSIONS: Our minimally invasive preferential regimen clarified the precise indication for neuroendoscopic procedures, and the majority of our patients with dilated ventricles and no evidence of tumor markers were treated satisfactorily with effective neuroendoscopic procedures as the initial procedure, avoiding unnecessary craniotomy and radiotherapy and promising excellent therapeutic outcomes. The treatment for malignant pineal region tumors remains a subject for further study.


Subject(s)
Brain Neoplasms/surgery , Endoscopy , Minimally Invasive Surgical Procedures , Pineal Gland/surgery , Pinealoma/surgery , Radiosurgery , Adolescent , Adult , Aged , Biomarkers, Tumor/analysis , Brain Neoplasms/pathology , Chemotherapy, Adjuvant , Child , Female , Glioma/pathology , Glioma/surgery , Humans , Male , Middle Aged , Neoplasm Staging , Pineal Gland/pathology , Pinealoma/pathology , Prospective Studies , Radiotherapy, Adjuvant , Risk Factors , Treatment Outcome
9.
Eur J Immunogenet ; 27(3): 149-51, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10940084

ABSTRACT

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.


Subject(s)
Antigens, CD/genetics , Membrane Glycoproteins/genetics , Alternative Splicing/genetics , Animals , Cloning, Molecular , Exons/genetics , Humans , Killer Cells, Natural/immunology , Lectins/genetics , Lectins, C-Type , Mice , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily D , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid/genetics , Restriction Mapping , Sequence Alignment , Sequence Analysis, DNA
10.
Eur J Immunol ; 30(7): 2074-82, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10940897

ABSTRACT

In the mouse, two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, have been identified on NK cells. Individual NK cells can express several Ly49 molecules as well as members of the CD94/NKG2 family. The expression of multiple receptors with different specificities for MHC class I is thus thought to generate NK cells with diverse recognition patterns. To delineate the mechanism by which NK cells begin to express different patterns of Ly49 and CD94/NKG2 molecules, we developed a clonal assay in which NK1.1(-), IL-2/ IL-15 receptor beta+ NK precursors generated by culture of multipotential Lin(-), c-kit+ progenitors in IL-7, stem cell factor and flt3 ligand are induced to differentiate into NK1.1+ , Ly49+ NK cells. Examination of the clonal populations thus generated revealed heterogeneity in the pattern of Ly49 and CD94/NKG2 gene expression. In addition, a distinct kinetic pattern of expression was observed. CD94, NKG2A, NKG2C and Ly49B were expressed first followed by Ly49G, then Ly49C and I and finally, Ly49A, D, E and F. The data suggest a stochastic but ordered acquisition of class I receptors on NK cells in which developing NK cells become capable of expressing distinct receptors at different times but show no absolute prerequisite to express the receptors that are acquired early in NK development for the expression of those that are acquired later.


Subject(s)
Antigens, CD/genetics , Antigens, Ly , Hematopoietic Stem Cells/metabolism , Killer Cells, Natural/metabolism , Lectins, C-Type , Membrane Glycoproteins/genetics , Receptors, Immunologic/genetics , Animals , Antigens , Antigens, Surface , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Line , Gene Expression , Hematopoietic Stem Cells/cytology , Killer Cells, Natural/cytology , Leukopoiesis , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A , NK Cell Lectin-Like Receptor Subfamily B , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Proteins , Receptors, Interleukin-2 , Receptors, NK Cell Lectin-Like , Receptors, Natural Killer Cell
11.
Chemistry ; 6(6): 983-93, 2000 Mar 17.
Article in English | MEDLINE | ID: mdl-10785818

ABSTRACT

Living polymerization of chiral aryl isocyanides, such as m- and p-menthoxycarbonylphenyl isocyanides 2 and 5, initiated by the Pd-Pt mu-ethynediyl dinuclear complex 1, proceeds with a high screw-sense selectivity to give the poly(isocyanide)s 3 and 6, which exhibit a large specific rotation and an intense CD band at lambda = 364 nm as a consequence of a helical chirality. The molar optical rotation and molar circular dichroism of the resulting polymers 3 and 6 reach a constant value at a degree of polymerization (Pn) of more than 30. Screw-sense-selective polymerization of achiral aryl isocyanides that bear very bulky substituents, such as 3,5-di(propoxycarbonyl)phenyl isocyanide (11), 3,5-di(butoxycarbonyl)phenyl isocyanide (13), and 3,5-di(cyclohexyloxycarbonyl)phenyl isocyanide (15), is achieved by the use of chiral oligomer complexes 3(30) and 6(30), prepared from the reaction of 1 with 30 equivalents of 2 or 5, as an initiator to give predominantly single-handed helical polymers. In contrast, smaller aryl isocyanides are also polymerized by 3(30) and 6(30) with screw-sense selectivity in the initial stage of the reaction, but the single-handed helix is not preserved up to high molecular weight. Kinetic studies of the polymerization of (L)- and (D)-2, or (L)- and (D)-5 with chiral oligomer complexes (L)-3(50) or (L)-6(100) suggests that the screw sense of the polymer backbone is not controlled kinetically, but rather that the thermodynamically stable screw sense is produced.

12.
J Immunol ; 163(12): 6488-93, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10586040

ABSTRACT

Activation of NK cells by target cells leads to cytotoxicity as well as production of various cytokines including IFN-gamma. MHC class I molecules on target cells regulate NK cytotoxicity. However, little is known about the regulation of IFN-gamma production by NK cells. We examined the production of IFN-gamma in individual murine NK cells stimulated with tumor cell lines by flow cytometric analysis of intracellular IFN-gamma. Among several tumor lines tested, the rat basophilic leukemia line RBL-1 induced particularly high level of IFN-gamma production in IL-2-activated NK cells, whereas other lines, including the prototypic NK target YAC-1, induced very low or no IFN-gamma production. Transfection of murine classical MHC class I molecules into RBL-1 cells substantially inhibited IFN-gamma production. This inhibition of IFN-gamma production by MHC class I was independent of Ly-49 or CD94/NKG2A expression on NK cells. These results indicate that some target cells directly stimulate IL-2-activated NK cells and induce IFN-gamma production, but the requirements for the induction of IFN-gamma production seem different from those for NK cytotoxicity. Furthermore, similar to NK cytotoxicity, induction of IFN-gamma production is inhibited by MHC class I on stimulating cells. However, the MHC class I-specific receptors inhibiting IFN-gamma production are different from those for NK cytotoxicity.


Subject(s)
Antigens, Ly , Cytotoxicity, Immunologic , Histocompatibility Antigens Class I/physiology , Interferon-gamma/biosynthesis , Interleukin-2/pharmacology , Killer Cells, Natural/immunology , Lymphocyte Activation , Animals , Binding Sites/immunology , Histocompatibility Antigens Class I/metabolism , Interferon-gamma/antagonists & inhibitors , Killer Cells, Natural/metabolism , Lectins, C-Type , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Rats , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/metabolism , Receptors, KIR , Receptors, NK Cell Lectin-Like , Tumor Cells, Cultured
13.
J Immunol ; 163(1): 212-6, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10384118

ABSTRACT

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.


Subject(s)
Aging/immunology , Animals, Newborn/immunology , Antigens, Ly , Killer Cells, Natural/metabolism , Lectins, C-Type , Receptors, Immunologic/biosynthesis , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Blotting, Southern , Lymphocyte Count , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Receptors, Immunologic/genetics , Receptors, NK Cell Lectin-Like , Receptors, Natural Killer Cell , Reverse Transcriptase Polymerase Chain Reaction , Stochastic Processes
14.
Immunogenetics ; 49(7-8): 685-91, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10369927

ABSTRACT

Five new Ly49 genes, named Ly49j-n, have recently been identified in C57BL/6 mice. This study examined the expression of three of these new genes, Ly49j, k, and n. To determine whether the Ly49j, k, and n genes were transcribed, gene-specific primers were used to amplify cDNA clones for each gene from C57BL/6 interleukin-2-activated natural killer (NK) cell cDNA. A full-length cDNA for Ly49j was detected which encodes a 267 amino acid protein and shares approximately 96% nucleotide identity with Ly49c and i. COS cells transfected with the Ly49j cDNA were shown to react with the monoclonal antibody 8H7, suggesting that the gene likely encodes a functional protein. Many different sized Ly49k and n transcripts were observed, although it is likely that they do not encode functional proteins due to missing exons or severe truncations in the open reading frames. Interestingly, the most abundant Ly49j transcript detected was shown to lack exon 3, which encodes the transmembrane domain. Similar studies performed on the same source of NK cell cDNA using Ly49c- and i-specific primers revealed the presence of transmembrane-less Ly49i transcripts, although at a much lower frequency than observed for Ly49j. We also detected Ly49g and h transcripts lacking the transmembrane domain. Despite the absence of the transmembrane region, the resulting Ly49 transcripts maintain their open reading frames, and therefore could potentially encode cytoplasmic proteins with a role in NK cell function.


Subject(s)
Antigens, Ly , Antigens, Surface/genetics , Membrane Glycoproteins/genetics , Receptors, Immunologic/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Membrane/metabolism , DNA, Complementary , Gene Expression , Lectins/genetics , Lectins, C-Type , Mice , Mice, Inbred C57BL , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily A , Protein Isoforms , RNA, Messenger , Receptors, NK Cell Lectin-Like
15.
J Immunol ; 162(12): 7271-6, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10358175

ABSTRACT

The Ly-49 family of inhibitory receptors plays a major role in regulating mouse NK cell cytotoxicity. Two of its members, Ly-49C and I, are recognized by the mAb 5E6, which also defines a subset of NK cells involved in the hybrid resistance phenomenon. Previous studies have shown that Ly-49C binds to a broad spectrum of class I MHC molecules, while Ly-49I apparently does not bind to any class I MHC molecules tested. In the present investigation we have defined the amino acid residues of Ly-49C that are critical for determining its ligand specificities. First, using quantitative COS cell adhesion assays, we demonstrated that Ly-49CB6 bound to Dd, Db, Kb, or Kk as well as to murine leukemic cell lines GM979 (H-2s) and IC-21 (H-2b). In contrast, COS cells expressing Ly-49IB6 did not significantly bind to any of the class I MHC tested. To determine which amino acid residues of Ly-49C are critical for their specific binding to class I MHC, a series of chimeric and mutant Ly-49C and I were generated and tested. Exchanging the critical residues between Ly-49C and I significantly affected their binding specificities. Finally, we identified the epitopes on Ly-49C recognized by mAbs 5E6 and 4LO3311 that functionally inhibit Ly-49C recognition of its ligands. These results further define the class I specificities of Ly-49C and provide insight into the structural basis for how class I MHC is recognized by the Ly-49 family of NK receptors.


Subject(s)
Antigens, Ly/metabolism , H-2 Antigens/metabolism , Killer Cells, Natural/metabolism , Membrane Glycoproteins/metabolism , Receptors, Immunologic/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antigens, Ly/chemistry , Antigens, Ly/genetics , Antigens, Ly/immunology , Binding Sites, Antibody/genetics , COS Cells , H-2 Antigens/genetics , Hybridomas , Killer Cells, Natural/immunology , Lectins, C-Type , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Point Mutation , Protein Binding/genetics , Protein Binding/immunology , Rats , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, NK Cell Lectin-Like , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Transfection , Tumor Cells, Cultured
16.
J Immunol ; 162(10): 5792-9, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10229812

ABSTRACT

The inside-out signaling involved in the activation of LFA-1-mediated cell adhesion is still poorly understood. Here we examined the role of the SH2-containing inositol phosphatase (SHIP), a major negative regulator of intracellular signaling, in this process. Wild-type SHIP and a phosphatase-deficient mutant SHIP were overexpressed in the murine myeloid cell line, DA-ER, and the effects on LFA-1-mediated cell adhesion to ICAM-1 (CD54) were tested. Overexpression of wild-type SHIP significantly enhanced cell adhesion to immobilized ICAM-1, and PMA, IL-3, or erythropoietin further augmented this adhesion. In contrast, phosphatase dead SHIP had no enhancing effects. Furthermore, PMA-induced activation of LFA-1 on DA-ER cells overexpressing wild-type SHIP was dependent on protein kinase C but independent of mitogen-activated protein kinase activation, whereas cytokine-induced activation was independent of protein kinase C and mitogen-activated protein kinase activation but required phosphatidylinositol-3 kinase activation. These results suggest that SHIP may regulate two distinct inside-out signaling pathways and that the phosphatase activity of SHIP is essential for both of them.


Subject(s)
Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Phosphoric Monoester Hydrolases/metabolism , src Homology Domains , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Adhesion , Erythropoietin/pharmacology , Hematopoietic Stem Cells/physiology , Interleukin-3/pharmacology , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/genetics , Protein Binding , Protein Kinase C/metabolism , Rats , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology
17.
Chest ; 115(5): 1402-6, 1999 May.
Article in English | MEDLINE | ID: mdl-10334160

ABSTRACT

STUDY OBJECTIVES: To investigate intracranial pressure (ICP) changes and their mechanisms in chronic hypercapnia. DESIGN: After 12 male Wistar rats were maintained in CO2-mixed air (mean PaCO2, 71.0 mm Hg) for 21 weeks, their ICP levels were measured during the breathing of 0, 5, 10, 12, and 14% CO2-mixed air before and after the i.v. administration of nitro-L-arginine methyl ester (L-NAME). The ICP responses to i.v. norepinephrine and i.v. adenosine were also tested. Ten rats that were maintained in room air served as the control group. RESULTS: The mean ICP in the study group (5.9 mm Hg) was not significantly different from the mean ICP in the control group. In the study group, the ICP response to changes in PaCO2 was significantly blunted when compared to the response seen in the control group. In both groups, i.v. norepinephrine significantly increased the ICP. In the control group but not in the study group, i.v. L-NAME suppressed the ICP response to changes in PaCO2. In both groups, i.v. adenosine significantly increased the ICP. CONCLUSIONS: The ICP response to PaCO2 was blunted in rats with chronic hypercapnia, and the mechanism of this reduced response may involve nitric oxide.


Subject(s)
Carbon Dioxide/pharmacology , Hypercapnia/physiopathology , Intracranial Pressure , Adenosine/pharmacology , Animals , Blood Pressure/drug effects , Cerebrovascular Circulation/drug effects , Intracranial Pressure/drug effects , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/physiology , Nitric Oxide Synthase/antagonists & inhibitors , Norepinephrine/pharmacology , Rats , Rats, Wistar , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacology
18.
Eur J Immunol ; 29(3): 755-61, 1999 03.
Article in English | MEDLINE | ID: mdl-10092077

ABSTRACT

Multiple NK cell receptors for MHC class I have been identified. They include killer inhibitory receptors and CD94/NKG2 heterodimers in humans and the Ly49 family in mice. Here we report the cloning of murine NKG2A, B and C. The deduced amino acid sequence of mouse NKG2A contains only one consensus cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). NKG2A from B6 and BALB/c mice differ by six amino acid residues in the extracellular domain. Murine NKG2B, like its human conterpart, appears to be a splice variant of NKG2A and lacks a large portion of the stalk region. Murine NKG2C lacks an ITIM in its cytoplasmic domain, a feature shared by human and rat NKG2C. However, unlike the human counterpart, the transmembrane domain of mouse NKG2C does not contain a charged amino acid residue. Mouse NKG2A mRNA was detected in IL-2-activated NK cells and spleen cells but not in other tissues. The NKG2A gene was localized on the distal portion of chromosome 6 where the NK complex has been located. These results further extend the repertoire of C-type lectin receptors on murine NK cells.


Subject(s)
Antigens, CD/genetics , Killer Cells, Natural/metabolism , Lectins, C-Type , Lectins/metabolism , Membrane Glycoproteins/genetics , Receptors, Immunologic/genetics , Receptors, Mitogen/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Gene Expression , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Rats , Receptors, Natural Killer Cell , Sequence Analysis, DNA
19.
Cell Immunol ; 191(2): 145-51, 1999 Feb 01.
Article in English | MEDLINE | ID: mdl-9973537

ABSTRACT

Murine cytomegalovirus (CMV)-encoded protein m144 is homologous to class I MHC heavy-chain and is thought to regulate NK-cell-mediated immune responses in vivo. To examine the effects of m144 on NK cytotoxicity in vitro, various cell lines were transfected with wild-type m144 or a chimeric construct in which the cytoplasmic domain of m144 was replaced with green fluorescence protein. Burkitt lymphoma line Raji expressed a significant level of m144 as determined by anti-m144 mAb binding or the green fluorescence of the fusion protein. The level of m144 expression was relatively low compared with that of transfected murine class I MHC Dd. However, m144 on Raji cells partially inhibited antibody-dependent cell-mediated cytotoxicity of IL-2-activated NK cells. NK cells from the CMV-susceptible BALB/c as well as those from the resistant C57BL/6 mice were inhibited by m144. Antibodies against the known murine NK inhibitory receptors Ly-49A, C, G, and I did not affect the inhibitory effect of m144. These results suggest that the murine CMV class I MHC homologue m144 partially inhibits NK cells by interacting with a novel inhibitory receptor.


Subject(s)
Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, Ly , Cytomegalovirus/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , Viral Proteins/immunology , Animals , Cytomegalovirus/pathogenicity , Histocompatibility Antigens Class I/chemistry , Immunity, Innate , Lectins, C-Type , Major Histocompatibility Complex , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Receptors, NK Cell Lectin-Like , Recombinant Proteins/immunology , Sequence Homology, Amino Acid , Tetrahydronaphthalenes , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism , beta 2-Microglobulin/metabolism
20.
Leukemia ; 12(10): 1565-72, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9766501

ABSTRACT

SR-91 is a natural killer (NK)-resistant leukemic cell line expressing a low level of ICAM-1. Pre-treatment of SR-91 cells with TNF-alpha or IFN-gamma, increased both ICAM-1 (CD54) expression on SR-91 cells and binding to the human NK cell line NK-92. However, only TNF-alpha-treated SR-91 cells became sensitive to killing by NK-92 cells. The increased binding induced by both cytokines and the TNF-alpha-induced sensitivity of SR-91 cells to NK-92 cell killing were abrogated by anti-LFA-1 mAb as well as by a combination of antibodies against the three ligands of LFA-1 (CD11a/CD18), ICAM-1 (CD54), ICAM-2 (CD102) and ICAM-3 (CD50). This indicated that LFA-1 interaction with the three ICAMs on SR-91 cells is essential for effector-target cell binding (which is a prerequisite for subsequent target cell lysis), but is insufficient to render the SR-91 cells sensitive to killing by NK-92 cells. TNF-alpha, but not IFN-gamma also induced the activation of LFA-1, CD44 and beta1 integrins on SR-91 cells. Based on these observations we propose that the differential effect of TNF-alpha and IFN-gamma could be related to the activation of certain adhesion molecules on the surface of SR-91 cells by TNF-alpha that, upon interaction with their counter-receptors on NK-92 cells, lead to the activation of the NK-92 cells.


Subject(s)
Antigens, Differentiation , Cell Adhesion Molecules/physiology , Cytotoxicity, Immunologic , Hyaluronan Receptors/physiology , Killer Cells, Natural/immunology , Tumor Necrosis Factor-alpha/physiology , Antigens, CD/physiology , Burkitt Lymphoma , Cell Adhesion , Cell Adhesion Molecules/genetics , Cytotoxicity, Immunologic/drug effects , DNA Fragmentation , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , K562 Cells , Leukemia , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology
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