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N J Stam

Publications and source records attributed to N J Stam.

At least 19 recordsLinked to original sources
PubMed1997

Human serotonin 5-HT7 receptor: cloning and pharmacological characterisation of two receptor variants.

Two splice variants of the 5-HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5-HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH-3T3 cells, both variants encode functional 5-HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5-HT7 receptor genes may exist in human.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Molecular cloning and characterization of a novel orphan receptor (P2P) expressed in human pancreas that shows high structural homology to the P2U purinoceptor.

Here we report the cloning of a gene encoding a new member of the superfamily of G protein-coupled receptors. The gene encodes a protein of 365 amino acids closely resembling two recently cloned nucleotide binding receptors, called P2U and P2Y purinoceptors (71% and 49% sequence identity within the transmembrane domains, respectively). Our studies show that this new putative purinoceptor (designated P2P) is encoded by an intronless single copy gene that is exclusively expressed in pancreas, in contrast to the P2U and the P2Y purinoceptors which are widely distributed throughout the periphery. The identification of a pancreas-specific human putative P2 purinoceptor makes it attractive to speculate that the reported actions of ADP/ATP analogues in pancreas on insulin secretion are mediated through this receptor.

Amino Acid Sequence↗
PubMed1996

Radiolabelling of the human 5-HT2A receptor with an agonist, a partial agonist and an antagonist: effects on apparent agonist affinities.

Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed1994

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor.

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

3T3 Cells↗
PubMed1993

Agonist-induced down-regulation of human 5-HT1A and 5-HT2 receptors in Swiss 3T3 cells.

We have used single cell clones of Swiss 3T3 cells transfected with genes for the human 5-HT1A or 5-HT2 receptor to study down-regulation and desensitization. After pre-incubation of the cells with serotonin agonists, a time-dependent decrease in [3H]8-hydroxy-2-(di-n-propylamino) tetralin or [3H]ketanserin binding was observed. The pertussis toxin sensitive, 5-HT mediated inhibition of forskolin-stimulated cAMP accumulation in 5-HT1A receptor transfected cells was diminished by 68% after a 2 h pre-incubation of the cells with 10 microM 5-HT. The pertussis toxin insensitive, 5-HT mediated PI turnover in 5-HT2 receptor transfected cells was decreased by 65% after pre-treatment. While this decrease was paralleled by a decreased potency of 5-HT to stimulate PI turnover, in 5-HT1A cells the potency of 5-HT to inhibit cAMP formation was comparable to control values. The down-regulation and desensitization of the 5-HT2 receptor can be explained by phosphorylation via activated PKC. In contrast, the attenuation of the 5-HT1A receptor-coupled inhibition of cAMP accumulation has to occur by an alternative, as yet unknown, molecular mechanism.

3T3 Cells↗
PubMed1992

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes.

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.

3T3 Cells↗
PubMed1991

Aberrant expression of HLA class-I antigens in Burkitt lymphoma cells.

HLA class-I expression has been investigated by biochemical methods in 14 Burkitt lymphoma (BL) cell lines and the corresponding Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the same individuals. Selective down-regulation of one or more HLA class-I specificities was demonstrated in 9 out of 14 BL lines. The defect was restricted to a single HLA-A allele in 3 of the lines (BL29, BL72, WW-I-BL). Four lines (BL28, BL37, BL41 and Jijoye M13) showed down-regulation of both HLA-A and -C alleles, and one (BL36) failed to express one HLA-C allele. Only one BL line (WW-2-BL) had lost one HLA-A and one HLA-B allele. The allele-specific defects were mainly detected in cell lines that had maintained the phenotypic characteristics of the original tumor. Expression of B-cell activation markers and the EBV-encoded nuclear antigen (EBNA)-2 correlated with up-regulation of the Cw4 allele in the P79 subline of the BL line Jijoye. Treatment with gamma-interferon (IFN) resulted in full or partial reversion of the HLA class-I defects in some of the cases but had no significant effect in others. This was not due to a cell-line-related unresponsiveness to IFN, nor did it reflect an allele-specific mode of regulation because the same allele could respond differently in different cell lines. The data suggest that defective expression of HLA class-I antigens, which appears to be more prevalent for alleles within the HLA-A and -C loci, is a common feature of BL cell lines. Different regulatory mechanisms appear to be involved.

Alleles↗
PubMed1990

Empty MHC class I molecules come out in the cold.

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.

Animals↗
PubMed1990

HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy.

The mAb HCA2 reacts preferentially with HLA-A locus heavy chains. Its reactivity contrasts with that of HC10, a mAb with more pronounced specificity for HLA-B and -C heavy chains. Both HCA2 and HC10 were raised against free class I heavy chains of HLA-A and -B antigens respectively, to obtain mAbs that would still react with denatured class I antigens, as they occur in Western blotting, conventional light microscopical analysis of formalin-fixed, paraffin-embedded sections, and cryo-immuno-electron microscopy. HCA2 and HC10 indeed retain strong reactivity with free class I heavy chains in Western blots. The HCA2 mAb in particular reacts in a locus-specific manner by biochemical criteria. Conditions are described for use of HCA2 and HC10 in immunohistochemical staining of formalin-fixed, paraffin-embedded sections. HCA2 and HC10 also produce strong reactivity in immuno-electron microscopy. Their use allows the determination of tissue and subcellular distribution of class I antigens.

Antibodies, Monoclonal↗
PubMed1989

Suppression of class I human histocompatibility leukocyte antigen by c-myc is locus specific.

The c-myc oncogene downregulates class I HLA expression in human melanoma. The major class I HLA antigens are encoded by three loci, A, B, and C, and we investigated whether these loci are suppressed equally by c-myc. In three melanoma cell lines with high c-myc expression, we analyzed mRNA, protein, and cell surface expression of the class I HLA antigens. Whereas the HLA-B locus expression was found to be strongly reduced, the HLA-A locus was expressed normally. Analysis of c-myc-transfected clones of two melanoma cell lines confirmed that c-myc preferentially suppresses the class I HLA-B locus. Immunohistochemical analysis of fresh melanoma lesions also showed that in the tumors the HLA-A loci are expressed normally, while on the majority of tumor cells no HLA-B antigen expression was found. This downregulation may have consequences for the recognition of malignant cells by tumor-infiltrating lymphocytes. Our results predict that HLA-B-restricted cytotoxic T cells will be unable to kill high c-myc-expressing melanoma cells.

Alleles↗
PubMed1989

Lack of correlation between levels of MHC class I antigen and susceptibility to lysis of small cellular lung carcinoma (SCLC) by natural killer cells.

Small cellular lung carcinoma (SCLC) cell lines are susceptible to lysis by NK cells. SCLC, normally negative for MHC class I Ag, were rendered positive for HLA-A and -B Ag by two methods: treatment with IFN-gamma or transfection with HLA class I genes. Exposure to IFN-gamma induced high levels of class I Ag and reduced susceptibility to NK-mediated lysis. However, transfection with either HLA-A2, HLA-B27, or HLA-B27 with beta 2m did not result in reduced susceptibility to NK cells. These transfectants expressed amounts of HLA class I Ag comparable to those in IFN-gamma-treated, untransfected cells. Transfection with the beta 2m gene or plasmid alone neither influenced levels of surface class I Ag nor resulted in reduced susceptibility to lysis by NK cells. Thus, the effects of IFN-gamma on NK susceptibility can be dissociated from the induction of class I Ag.

Carcinoma, Small Cell↗
PubMed1989

Molecules relevant for T cell-target cell interaction are present in cytolytic granules of human T lymphocytes.

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

Clone Cells↗
PubMed1989

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines.

We have reported that Burkitt lymphomas (BL) that arise in HLA-A11 positive individuals are resistant to lysis by HLA-A11-specific and HLA-A11-restricted CTLs(10,11). Here we show that this phenomenon can be explained by a selective loss of the HLA-A11 polypeptide. The HLA-A11 negative phenotype is due to a regulatory phenomenon, rather than a structural defect, as proven by the ability to rescue expression of HLA-A11 in in vitro Epstein-Barr virus (EBV)-converted sublines of EBV negative BLs.

Alleles↗
PubMed1988

Biochemical complexity of serum HLA class I molecules.

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around Mr 44,000, 40,000, and 35,000-37,000. HLA-A24-positive individuals are distinguished by higher serum levels of Mr 44,000 and 40,000 class I-like molecules than those found in HLA-A24-negative individuals. The Mr 44,000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The Mr 35,000 and 37,000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum Mr 44,000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The Mr 40,000 molecules do not contain a TM region. Mr 40,000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.

Animals↗
PubMed1987

Association with beta 2-microglobulin controls the expression of transfected human class I genes.

The genes encoding HLA-B27K and HLA-B27W were transfected into murine recipient cells. A monoclonal antibody HC-10, directed against free B-locus heavy chain, was the only reagent capable of efficiently detecting the HLA-B27 heavy chains in detergent lysates. These heavy chains were devoid of sialic acid. Trace amounts of HLA-B27 could be isolated with the anti-HLA-A,-B antibody W6/32, which reacts with the heavy chain beta 2-microglobulin complex. In marked contrast, HLA-A2 and -B7 genes, when transfected, yielded easily detectable amounts of antigen precipitable with W6/32, which carried the usual complement of sialic acids. Because the alpha 3 domains of HLA-B27 and HLA-B7 and the more COOH-terminal portions are identical in amino acid sequence, structural elements in the polymorphic alpha 1 and alpha 2 domains must control association of heavy chain with beta 2-microglobulin. Introduction of a human beta 2-microglobulin gene into L cells transfected with the HLA-B27 gene rescued the expression of HLA-B27 at the cell surface, as evidenced by reactivity with W6/32, surface staining, and the presence of sialic acid on the heavy chain.

Animals↗
PubMed1986

Monoclonal antibodies raised against denatured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C locus products.

Twelve monoclonal antibodies (HC-MoAb) were prepared against a mixture of denatured HLA-B7 and -B40 heavy chains and were characterized by biochemical methods. The patterns of reactivity of 11 out of 12 of these MoAb were very similar and showed marked preference for the HLA-B locus heavy chains. A single antibody, HC-3, reacted with a subset of the HLA class I heavy chains recognized by the other HC-MoAb, which react with all HLA-B locus heavy chains tested. In pulse-chase experiments, only biosynthetic intermediates consisting of free class I heavy chains were precipitated by the HC MoAb. In addition to free HLA-B and minor quantities of some HLA-A heavy chains, additional class I polypeptides were recognized by the HC-MoAb. A number of these additional class I polypeptides showed a striking correlation with HLA-C as determined serologically. None of these polypeptides could be clearly identified in immunoprecipitations prepared from continuously labeled cells with the MoAb W6/32, previously thought to recognize all HLA-A, B, and C specificities. These findings suggest that in human cells, HLA-C locus products may be associated only weakly with B2m, explaining some of the difficulties encountered in biochemical studies of HLA-C antigens.

Antibodies, Monoclonal↗
PubMed1986

An analysis of class I antigens of man and other species by one-dimensional IEF and immunoblotting.

A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.

Animals↗