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Biomedical subjects

V Ling

Publications and source records attributed to V Ling.

At least 19 recordsLinked to original sources
PubMed2001

Differential expression of inducible costimulator-ligand splice variants: lymphoid regulation of mouse GL50-B and human GL50 molecules.

The process of immunological costimulation between APC and T cells is mediated by protein ligand:receptor interactions. To date, costimulatory receptors known to be expressed by T cells include the structurally related proteins CD28 and the inducible costimulator (ICOS). The ligands to human and mouse ICOS, human GL50 (hGL50), and mouse GL50 (mGL50) were recently cloned and demonstrated to have sequence similarity to the CD28 ligands B7-1 and B7-2. Examination of mGL50 cDNA transcripts by 3'RACE revealed an alternatively spliced form, mGL50-B, that encoded a protein product with a divergent 27-aa intracellular domain. Both mGL50- and mGL50-B-transfected cells exhibited binding to human and mouse ICOS-Ig fusion protein, indicating that the alternate cytoplasmic domain of mGL50-B does not interfere with extracellular interactions with ICOS receptor. Flow cytometric and RT-PCR analysis of BALB/c and RAG1(-/-) mice splenocytes demonstrate that freshly isolated B cells, T cells, macrophages, and dendritic cells express both splice variant forms of ICOS ligand. Comparative analyses with the human ICOS ligand splice variants hGL50 and B7-H2 indicate that differential splicing at the junction of cytoplasmic exon 6 and exon 7 may be a common method by which GL50-ICOS immunological costimulatory processes are regulated in vivo.

Alternative Splicing↗
PubMed2001

Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.

Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp(-/-) mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp(-/-) mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp(-/-) mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

ICOS is critical for CD40-mediated antibody class switching.

The inducible co-stimulatory molecule (ICOS) is a CD28 homologue implicated in regulating T-cell differentiation. Because co-stimulatory signals are critical for regulating T-cell activation, an understanding of co-stimulatory signals may enable the design of rational therapies for immune-mediated diseases. According to the two-signal model for T-cell activation, T cells require an antigen-specific signal and a second, co-stimulatory, signal for optimal T-cell activation. The co-stimulatory signal promotes T-cell proliferation, lymphokine secretion and effector function. The B7-CD28 pathway provides essential signals for T-cell activation, but does not account for all co-stimulation. We have generated mice lacking ICOS (ICOS-/- ) to determine the essential functions of ICOS. Here we report that ICOS-/- mice exhibit profound deficits in immunoglobulin isotype class switching, accompanied by impaired germinal centre formation. Class switching was restored in ICOS-/- mice by CD40 stimulation, showing that ICOS promotes T-cell/B-cell collaboration through the CD40/CD40L pathway.

Animals↗
PubMed2000

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Adjuvants, Immunologic↗
PubMed2000

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Amino Acid Sequence↗
PubMed2000

CTLA-4 (CD152) can inhibit T cell activation by two different mechanisms depending on its level of cell surface expression.

CTLA-4 (CD152) engagement results in down-regulation of T cell activation. Two mechanisms have been postulated to explain CTLA-4 inhibition of T cell activation: negative signaling and competitive antagonism of CD28:B7-mediated costimulation. We assessed the contributions of these two mechanisms using a panel of T cell lines expressing human CTLA-4 with mutations in the cytoplasmic region. Under conditions of B7-independent costimulation, inhibition of IL-2 production following CTLA-4 engagement required the CTLA-4 cytoplasmic region. In contrast, under B7-dependent costimulation, inhibition of IL-2 production by CTLA-4 engagement was directly proportional to CTLA-4 cell surface levels and did not require its cytoplasmic region. Thus, CTLA-4 down-regulates T cell activation by two different mechanisms-delivery of a negative signal or B7 sequestration-that are operational depending on the levels of CTLA-4 surface expression. These two mechanisms may have distinct functional outcomes: rapid inhibition of T cell activation or induction of T cell anergy.

Abatacept↗
PubMed2000

M-ABC2, a new human mitochondrial ATP-binding cassette membrane protein.

We have isolated a human cDNA encoding a novel ATP-binding cassette (ABC) protein whose gene was previously localized to chromosome 1q42 [Allikmets et al. (1995) Mamm. Genome 6, 111-117]. The gene transcript is expressed in all human tissues examined, with the highest levels in bone marrow. A non-expressed pseudogene also exists at chromosome 15q13-14. The new protein, which is most similar to the mitochondrial (M)-ABC1 protein, was also localized to mitochondria and therefore designated 'M-ABC2'. The N-terminus of M-ABC2 was shown to contain a mitochondrial-targeting signal sequence.

ATP-Binding Cassette Transporters↗
PubMed2000

Characterization of ABCB9, an ATP binding cassette protein associated with lysosomes.

We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a "half" ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two "half" ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.

ATP-Binding Cassette Transporters↗
PubMed2000

Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor.

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.

Amino Acid Sequence↗
PubMed2000

Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence.

We have investigated the substrate specificity of the Escherichia coli hemolysin transporter system. Translocation of hemolysin is dependent on a C-terminal signal sequence located within the last 60 amino acids of this protein. Previous comparative studies of the signal sequence have revealed a conserved helix(alpha1)-linker-helix(alpha2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the alpha1, linker, and alpha2 regions, as well as an alpha1-amphiphilic helical library to identify features buried within the structural motif that contribute to transport. Combinatorial variants were generated by altering the primary sequence of specific regions, and correlation between the genotype and phenotype of the mutant populations allowed us to objectively identify any functional features involved. It was found that the alpha1-amphiphilic helix and the linker are both important for function. To our surprise, the second helix of the conserved structural motif was not essential for transport. The finding that a predicted amphiphilic helix and hydrophobicity, rather than primary sequence, contribute to transport in the alpha1 region allows us to speculate on the mechanism of multiple substrate recognition. This may have implications for understanding the broad substrate specificity common among other ATP-binding cassette transporters.

Amino Acid Motifs↗
PubMed2000

The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation.

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules.

Abatacept↗
PubMed2000

Cell-cycle-dependent turnover of P-glycoprotein in multidrug-resistant cells.

Regulation of P-glycoprotein (Pgp) expression occurs not only at the DNA and mRNA level but also at the protein level. We showed previously that Pgp was stabilized when multidrug-resistant CH(R)C5 and SKVCR 2.0 ovarian cell lines were subjected to serum-starved or high-cell-density growth conditions, whereas Pgp turnover in a leukemic multidrug-resistant cell line, CEMVLB0.1, was not affected by serum starvation (Muller et al., 1995). On further analysis, we have observed that the majority of the CH(R)C5 and SKVCR 2.0 cells under these conditions were in the G1/G0 phase of the cell cycle, whereas the cell cycle of CEMVLB0.1 cells was not affected. Pgp in CEMVLB0.1 cells was stabilized only when the cell cycle was delayed in the G1/G0 phase by using amino acid-deficient growth medium. In CH(R)C5 cells, Pgp half-life was also considerably increased when the cell cycle of these ovary-derived cells was delayed in the G1/G0 phase by using high concentrations of progesterone under normal serum growth conditions. In contrast, Pgp stability was not greatly affected if these cells were delayed in the S or G2/M phase of the cell cycle with Ara-C, cisplatin, or colchicine under the same conditions. Insulin-like growth factors could release the serum-starved CH(R)C5 and SKVCR2.0 cells from the G1/G0 phase and destabilized Pgp. These results indicate that Pgp turnover is a cell-cycle-related process in MDR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Complete sequence determination of the mouse and human CTLA4 gene loci: cross-species DNA sequence similarity beyond exon borders.

CTLA4 (CD152), a receptor for the B7 costimulatory molecules (CD80 and CD86), is considered a fundamental regulator of T-cell activation. In this paper, we present the complete primary structure of the mouse and human CTLA4 gene loci. Sequence comparison between the mouse and the human CTLA4 gene loci revealed a high degree of sequence conservation both for homologous noncoding regions (65-78% identity) and for coding regions (72-98% identity), with an overall score of 71% over the entire length of the two genes. Of the CTLA4 genomic regions aligned, five simple repetitive elements were found in the mouse locus, whereas two simple repetitive sequences were localized on the human locus. RNA blot analysis of mouse and human primary tissues indicated that both CTLA4 and T-cell receptor transcripts were found in most organs with generally higher levels in lymphoid tissues. The conservation of CTLA4 gene patterning raises the possibility that constrained gene evolution of CTLA4 may be linked to conserved transcriptional control of this locus.

Abatacept↗
PubMed1999

A human nucleobase transporter-like cDNA (SLC23A1): member of a transporter family conserved from bacteria to mammals.

A family of related polytopic membrane proteins that mediate the transport of nucleobases has been extended to Homo sapiens by the cloning of a full-length human cDNA that encodes a nucleobase transporter-like protein. The protein is predicted to contain 11-14 transmembrane-spanning regions, exhibits 20-28% overall sequence identity to fungal and bacterial transporters, and contains a conserved signature motif found in this family. Fluorescence in situ hybridization localized the gene (HGMW-approved symbol SLC23A1) to human chromosome 20p13. Human nucleobase transporter-like mRNA was present in all tissues examined, with lower levels found in heart, skeletal muscle, and ovary. Expression of the 60-kDa cDNA-encoded protein was demonstrated by an in vitro transcription-translation approach. The identification of this nucleobase transporter-like protein will allow the further elucidation of the interaction of human cells with physiological nucleobases and pharmacologically important drugs such as 5-F-uracil, dideoxynucleosides, and acyclic nucleosides.

Amino Acid Sequence↗
PubMed1999

Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis.

Ligand-dependent changes in accessibility of purified P-glycoprotein, functionally reconstituted in liposomes, were investigated by fluorescence measurements. Trp quenching experiments provided evidence that P-glycoprotein adopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATPgammaS. Five anthracycline derivatives were tested as drug substrates: daunorubicin, 4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among them, daunorubicin and 4'-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, whereas the three others have been shown to accumulate in multidrug-resistant cells overexpressing P-glycoprotein and therefore retain their cytotoxic activity. A small conformational change was associated with nucleotide binding and amplified after nucleotide hydrolysis. Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgATPgammaS. These conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells. These data also suggest that the cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related to the fact they are not transported by P-glycoprotein. On the contrary, methoxy-morpholino-doxorubicin cytotoxicity may be explained in terms of its rapid reincorporation into the plasma membrane after being transported by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

A mammalian lysosomal membrane protein confers multidrug resistance upon expression in Saccharomyces cerevisiae.

Mouse transporter protein (MTP) is a highly conserved polytopic membrane protein present in mammalian lysosomes and endosomes. The role of MTP in regulating the in vivo subcellular distribution of numerous structurally distinct small molecules has been examined in this study by its expression in a drug-sensitive strain of the yeast Saccharomyces cerevisiae. Surprisingly, the expression of MTP in membranes of an intracellular compartment resulted in a cellular resistance or hypersensitivity to a range of drugs that included nucleoside and nucleobase analogs, antibiotics, anthracyclines, ionophores, and steroid hormones. The intracellular bioavailability of steroid hormones was altered by MTP, as determined using an in vivo glucocorticoid receptor-driven reporter assay in yeast, suggesting that the MTP-regulated drug sensitivity arose due to a change in the subcellular compartmentalization of steroid hormones and other drugs. MTP-regulated drug sensitivity in yeast was blocked to varying degrees by compounds that inhibit lysosomal function, interfere with intracellular cholesterol transport, or modulate the multidrug resistance phenotype of mammalian cells. These results indicate that MTP is involved in the subcellular compartmentalization of diverse hydrophobic small molecules and contributes to the inherent drug sensitivity or resistance of the mammalian cell.

Base Sequence↗
PubMed1999

Identification and characterization of a mammalian mitochondrial ATP-binding cassette membrane protein.

Membrane proteins of the ATP-binding cassette (ABC) superfamily are involved in the transport of diverse substrates across organellar and plasma membranes of the mammalian cell. Most human ABC proteins identified to date are associated with genetically linked diseases or clinically relevant phenotypes. We describe a new human half-molecule ABC protein, designated M-ABC1, that contains a predicted single membrane and ATP-binding cassette domain. M-ABC1 is localized to membranes of the mitochondria and its transcript is expressed in all tissues. The N-terminal region of the M-ABC1 protein was shown to function independently as a mitochondrial signal sequence by its ability to target the green fluorescent protein to the mitochondria. The monomeric 60 kDa M-ABC1 protein was chemically crosslinked in vivo into a major protein species of 120-130 kDa, thereby confirming that M-ABC1 exists within a higher ordered ABC protein complex. A dominant negative repression approach using M-ABC1 protein with site-directed mutations in its Walker A motif revealed that the mutant protein was rapidly degraded and indicated that the intact Walker A motif of M-ABC1 was required for its stability. The identification of M-ABC1 extends the known distribution of members of the ABC protein family into the mammalian mitochondrion.

ATP Binding Cassette Transporter 1↗