Biomedical subjects

J Papamatheakis

Publications and source records attributed to J Papamatheakis.

At least 19 recordsLinked to original sources

PubMed2003

Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells.

Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour efficacy of scFv(anti-HER-2/neu)/zeta expressing MD.45 cells in severe combined immunodeficiency disease mice/human and murine tumour models. The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu. Our data demonstrate the feasibility of redirecting MD.45 CTL with the scFv(anti-HER-2/neu)/zeta chimeric receptor to respond specifically against HER-2/neu expressing tumour cells in vitro and in vivo. Moreover, they make it likely that T cells transduced with the same chimeric gene might be utilised in the treatment of patients with HER-2/neu(+) tumours.

Amino Acid Sequence↗

PubMed2001

Self-association of class II transactivator correlates with its intracellular localization and transactivation.

Class II transactivator (CIITA) is the master regulator of major histocompatibility complex class II genes that regulates both B lymphocyte-specific and interferon gamma-inducible expression. Here we identify protein regions and examine mechanisms that determine the intracellular distribution of CIITA. We show that two separate regions of CIITA mediate nuclear export: amino acids 1-114 and 408-550. Both regions interact with the export receptor CRM-1. The CIITA region spanning amino acids 408-550 of CIITA also determines its ability for homotypic self-association as well as heterotypic interactions with other regions residing at the amino and carboxyl termini of the protein. These observations are in line with data demonstrating that co-expression of amino- and carboxyl-terminal parts of CIITA promote subcellular relocalization and, remarkably, rescue transcriptional activation by individually inert molecules. CIITA point mutations that impair nuclear import and abolish its activation function show reduced self-association. We propose that the concerted action of homo- and heterotypic interactions of CIITA determine proper protein configuration that in turn controls its nucleocytoplasmic trafficking.

Animals↗

PubMed2000

Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes.

The class II transactivator (CIITA), the master regulator of the tissue-specific and interferon gamma-inducible expression of major histocompatibility complex class II genes, synergizes with the histone acetylase coactivator CBP to activate gene transcription. Here we demonstrate that in addition to CBP, PCAF binds to CIITA both in vivo and in vitro and enhances CIITA-dependent transcriptional activation of class II promoters. Accordingly, E1A mutants defective for PCAF or CBP interaction show reduced ability in suppressing CIITA activity. Interestingly, CBP and PCAF acetylate CIITA at lysine residues within a nuclear localization signal. We show that CIITA is shuttling between the nucleus and cytoplasm. The shuttling behavior and activity of the protein are regulated by acetylation: overexpression of PCAF or inhibition of cellular deacetylases by trichostatin A increases the nuclear accumulation of CIITA in a manner determined by the presence of the acetylation target lysines. Furthermore, mutagenesis of the acetylated residues reduces the transactivation ability of CIITA. These results support a novel function for acetylation, i.e., to regulate gene expression by stimulating the nuclear accumulation of an activator.

Acetylation↗

PubMed1998

Involvement of CREB binding protein in expression of major histocompatibility complex class II genes via interaction with the class II transactivator.

The class II transactivator (CIITA) is a key regulatory factor that controls expression of the major histocompatibility complex (MHC) class II genes that are essential components for antigen presentation and thus regulation of the immune response. We show here that the adenovirus E1A protein interferes with the action of CIITA and inhibits both B-cell-specific and gamma interferon (IFN-gamma)-induced expression of MHC class II promoters. Transfection studies provide evidence for the functional role of the CREB-binding protein (CBP) in IFN-gamma and CIITA-mediated MHC class II promoter activation. We demonstrate that the N-terminally located transcription activation domain of CIITA physically interacts with both the N-terminal and the E1A-binding (C/H3) regions of CBP. These results suggest the involvement of a multisubunit complex, which contains the gene-specific coactivator CIITA and the versatile coactivator CBP, in MHC class II gene regulation, which may be responsible for both high-level expression and modulation by different signaling pathways.

Adenovirus E1A Proteins↗

PubMed1997

cDNA cloning of the translocon associated protein beta-subunit in the chick cerebellum.

The 'translocon associated protein' is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The beta subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the beta subunit from chick nervous tissue, namely cerebellum. Its deduced protein sequence is 91% homologous to both the canine and the human protein sequences, showing that the molecule is highly conserved.

Amino Acid Sequence↗

PubMed1996

The fibronectin domains of the neural adhesion molecule TAX-1 are necessary and sufficient for homophilic binding.

Cell adhesion molecules belonging to the immunoglobulin superfamily promote cell aggregation and neurite outgrowth. These proteins are multidomain molecules comprising a number of distinct modules, notably Ig domains of the C2 class and fibronectin type III repeats. A subgroup of these neural adhesion molecules are linked to the membrane with a glycosylphosphatidylinositol anchor and show a more restricted pattern of expression in the embryo. Among them, the human homologue of the transient axonal glycoprotein, named TAX-1, shares a great degree of similarity at the protein level with rodent TAG-1. In the present study we set out to determine which domains of TAX-1 are involved in promoting the homophilic, adhesive properties of the molecule. We established stable Schneider-2 cell lines expressing the intact molecule, the fibronectin, or the immunoglobulin domains. The fibronectin domains were necessary and sufficient to mediate homophilic binding and induce cell aggregation, a response also observed with cells expressing the intact TAX-1 molecule. Aggregation was inhibited by the secreted form of the TAG-1 protein. On the other hand, the immunoglobulin domains by themselves were not able to induce cell aggregation. In addition, TAX-1 was localized in areas of cell contact among aggregating cells, justifying its role as an adhesion molecule.

Cell Adhesion Molecules, Neuronal↗

PubMed1995

A novel IL-4 responsive element of the E alpha MHC class II promoter that binds to an inducible factor.

Interleukin-4 (IL-4) is a lymphokine with important role in the growth and differentiation of T and B lymphocytes. In the latter, IL-4 induces transcriptionally MHC class II gene expression. Using the M12 mouse lymphoid cell line, we have determined an IL-4 response sequence (ILRS) in the proximal promoter region of the E alpha class II gene. The ILRS extends from -80 to -111 and includes the MHC class II X motif and 19 bp of additional 5' sequence. In mouse lymphoid cells, IL-4 activates a complex (Nuclear Factor-IL-4, NFIL-4), that binds to a novel element within the ILRS. Similar IL-4 inducible complexes bind to the interferon-gamma response element of the Fc gamma receptor (GRR), the acute phase response element (APRE) of the alpha 2 macroglobulin promoters and an INF beta promoter site, overlapping the PRDII/NF kappa B element. The factor contacts all these elements through their common GGAA motif. NFIL-4 is immunologically unrelated to NF kappa B or STAT 1 proteins that also recognize the above elements. Activation of NFIL-4 requires tyrosine phosphorylation, occurs within 2 min and persists as long as IL-4 is present. NFIL-4 has an apparent molecular weight of 75 kDa as determined by sedimentation through glycerol gradients.

Animals↗

PubMed1995

A transient GCN4 mRNA destabilization follows GCN4 translational derepression.

Studies based on experimental strategies that utilized either inhibitors or structural alterations point to the existence of an inverse relationship between translation and stability of a given mRNA. In this study we have investigated the potential link between translation and stability of the yeast GCN4 mRNA whose translational rates change with respect to amino acid availability. We observed that under conditions favoring its translation, the steady state levels of the GCN4 mRNA were decreased, but this was not due to a measurable alternation in its decay rate. We have demonstrated that an extensive destabilization of this message is intimately coupled with its increased access to heavy polysomes, which occurs transiently in the process of translational derepression. This transient change in the stability is what readjusts the steady state levels of the GCN4 mRNA. This study demonstrates in vivo the existence of a mechanism of mRNA degradation that is coupled with the process of translation.

DNA-Binding Proteins↗

PubMed1994

Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene.

Glutamate dehydrogenase, an enzyme central to glutamate metabolism, is deficient in patients with heterogeneous neurological disorders characterized by multiple system atrophy. There is evidence for multiplicity of human glutamate dehydrogenase, which may account for the heterogeneity of the above disorders. However, only one mRNA that is encoded by an intron-containing gene (GLUD1) is presently known. Because blindness due to neuroretinal degeneration can occur in rare forms of multiple system atrophy, we searched for retina-specific GLUD mRNA(s) by screening a lambda gt10 library derived from human retina. A novel cDNA encoded by an X chromosome-linked intronless gene, designated GLUD2, was isolated and characterized. Reverse transcription-polymerase chain reaction analysis of human tissues revealed that the novel cDNA is expressed in human retina, testis, and, at lower levels, brain. In vitro translation of mRNAs derived from GLUD1 and GLUD2 genes generated proteins with distinct electrophoretic characteristics. The retinal cDNA was expressed in the baculovirus heterologous system, producing a protein capable of catalyzing the oxidative deamination of glutamate. The mobility of the expressed protein on SDS-polyacrylamide gel electrophoresis and its catalytic properties were very similar to those of the naturally occurring human brain glutamate dehydrogenases. The novel gene will be useful for understanding the biology of human neural and testicular tissues and in the study of X-linked neurodegenerative disorders.

Amino Acid Sequence↗

PubMed1993

The MHC class II E beta promoter: a complex arrangement of positive and negative elements determines B cell and interferon-gamma (IFN-gamma) regulated expression.

The 5' proximal region of the E beta gene was studied with respect to B lymphoid expression and responsiveness to cytokines, revealing a complex array of general and cell type specific cis-elements and factors. Full lymphoid activity and response to interferon-gamma (IFN-gamma) is generated by the concerted action of the MHC boxes (H, X and Y) and additional elements. Combinatorial interactions between elements and their cognate factors are indicated by several lines of evidence. Thus, mutations within the X box in the promoter context are strongly deleterious to both B lymphoid activity and IFN-gamma regulation. However, the X box alone has minimal lymphoid activity upon heterologous promoters. Data from deletion, insertion and site directed mutagenesis demonstrate that sequences extending approximately 35 bp 5' of the X box (designated as Cytokine Response Sequence--CRS) have a dual role: they are required for cytokine-regulated expression as well as serving as an enhancer element for cell-specific constitutive expression. A region that carries X and CRS permits both lymphoid activity and IFN-gamma response. In contrast, sequences that include X and the downstream Y box are constitutively active in all cell types tested. Combination of the sequences both upstream and downstream of the X box results in a tissue-specific and cytokine-regulated enhancer of full strength. In vivo competition studies show that titratable trans-acting factors, shared by Class I and Class II promoters, mediate the CRS-dependent IFN-gamma response. We report here the identification of novel nuclear complexes that bind to the CRS and recognize sites which correlate with its negative or positive elements. One of these complexes is present in B lymphoid cells only. Three other CRS complexes that are upregulated by either IFN-alpha and IFN-gamma are competed by a non-Class II, IFN-alpha stimulated response element (ISRE), providing evidence for the functional interconnection of these cytokines.

Animals↗

PubMed1993

The human glutamate dehydrogenase gene family: gene organization and structural characterization.

Glutamate dehydrogenase is a mitochondrially located, key metabolic enzyme. In addition to its general metabolic role, GLUD is important in neurotransmission. Significant alterations in GLUD enzymatic activity have been associated with certain neurodegenerative human disorders. Although a single species of human GLUD cDNA molecule has been identified so far, both genomic DNA Southern and cytogenetic analyses have indicated the presence of a GLUD gene family. Screening of a human genomic lambda-phage library with the GLUD cDNA, led us to the isolation of several clones divided into five structurally distinct contigs. We have confirmed the presence of all GLUD-specific sequences in the human genome by detailed genomic Southern analysis. This study allowed the identification of the entire functional GLUD gene, named GLUD1. The GLUD1 gene is about 45 kb long and it is organized into 13 exons. Its nucleotide sequence, exon-intron boundaries, and transcription start sites were determined. Potential binding sites for various regulatory factors such as Sp1, AP-1, and AP-2 were recognized at the promoter region of the gene. The members of the other contigs showed an organization clearly different from GLUD1. Two distinct GLUD-specific gene loci, termed GLUDP2 and GLUDP3, possibly represent truncated pseudogenes. Their high degree of similarity to GLUD1 is limited to the region surrounding exons 2, 3, and 4. Finally, two additional GLUD-specific genomic sequences, termed GLUDP4 and GLUDP5, are structurally similar with the 3' part of the GLUD cDNA sequence. These loci probably represent truncated GLUD pseudogenes generated by retrotransposition. The data presented here suggest that all human GLUD pseudogenes have diverged recently in evolution.

Amino Acid Sequence↗

PubMed1993

Isolation of the cDNA and chromosomal localization of the gene (TAX1) encoding the human axonal glycoprotein TAG-1.

The transient axonal glycoprotein (TAG-1) is a cell adhesion molecule that promotes neurite outgrowth and belongs to the immunoglobulin superfamily. We have isolated cDNAs encoding TAX1, the human homologue of TAG-1. Human TAX1 shows a high degree of homology to rat TAX1 and less to its chick counterpart, axonin-1, with 91 and 75% identity at the amino acid level, respectively. The numbers of immunoglobulin (IgC2) domains and fibronectin repeats present in TAG-1 are conserved among the three species. The highest degree of conservation occurs in the second IgC2 domain (98% with the rat and 82% with the chick). The human homologue also contains a putative N-terminal signal sequence and a C-terminal hydrophobic sequence, suggestive of linkage to the cell membrane via phosphatidylinositol. In addition, the two mammalian TAG-1 proteins share the RGD tripeptide, a motif known to mediate recognition of fibronectin by integrins. In situ hybridization to human metaphase chromosomes maps the TAX1 gene encoding human TAG-1 to a single location on chromosome 1q32.

Amino Acid Sequence↗

PubMed1993

Specific CSF-1 binding on murine placental trophoblasts and macrophages serves as a link to placental growth.

Previous studies have shown that the colony-stimulating factor-1 (CSF-1), stimulates the in vitro proliferation of a fetally-derived adherent, phagocytic and non-specific esterase positive placental cell population which stains positively for cytokeratin and Mac-1. Binding experiments were designed to test whether this is a direct effect of the factor on these cells. Binding/elution as well as autoradiography experiments, show that adherent placental cells specifically bind CSF-1. Based on the expression of the endothelial markers cytokeratin and vimentin three subpopulations of cells were isolated from the murine placenta: labyrinthine-derived trophoblasts (cytokeratin positive, vimentin negative), spongiotrophoblast-derived trophoblasts (cytokeratin positive, vimentin negative) and placental macrophages (cytokeratin negative, vimentin positive). 3H-Thymidine incorporation assays as well as binding experiments, showed that these cells simultaneously respond to and bind the macrophage-specific factor CSF-1. Furthermore, the results indicate that isolated trophoblasts have a low rate of growth and they are very sensitive to mitogenic stimulation, whereas placental macrophages alone have a high rate of growth and therefore are less sensitive to the mitogenic stimulus. These findings are in favour of the existence of an important cytokine regulatory network in the murine placenta, where two major cell populations may collaborate possibly via soluble factors to stimulate placental growth and thus fetal development.

Animals↗

PubMed1992

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.

Animals↗

PubMed1992

The p21ras protein as an intermediate signaling molecule in the IL-4-induced HLA-DR expression on normal and leukemic human myeloid cells.

We have previously demonstrated that the low number of interleukin-4 receptors (IL-4Rc) on HL-60 leukemia cells render this population susceptible to differentiation by IL-4. As it occurs with normal human monocytes, IL-4 induces the expression of HLA-DR surface antigens on HL-60 cells as well. The second messenger pathway(s) involved after the IL-4 stimulation leading to class II up-regulation has not been fully examined. Here we show that IL-4-induced class II antigen expression on the HL-60 cell line or normal human monocytes is calcium/calmodulin-independent since theophylline (TPH, a calmodulin inhibitor) does not block the IL-4 effect. In addition, the pyruvate kinase C (PKC) pathway does not seem to participate in the process either because in our system activation of PKC by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) is insufficient by itself to induce HLA-DR. We found, however, that a second messenger pathway can be mediated by a G protein system since IL-4 concomitantly induces class II and p21ras expression which can be successfully blocked by a highly specific anti-p21ras monoclonal antibody. In addition, using another p21ras inducer, the 5-azacytidine C (5-AzaC), we showed that this agent can also induce the expression of p21ras and class II, both of which can be inhibited by the same antibody. Thus, it appears that IL-4 selects the G protein system as a signaling pathway in order to exert its action for the induction of HLA-DR on human normal monocytes or M2 leukemia target cells. Since monocytes and macrophages participate in virtually all immune reactions, the regulation of class II induction is of obvious importance.

Azacitidine↗

PubMed1992

Common mechanisms govern the expression of p21ras and class II MHC antigens in the murine placenta.

It has been shown that there is an inverse as well as a direct correlation between class II MHC antigen expression and the p21ras protein, depending on the cell type. By using trophoblastic cells derived from the spongiotrophoblast and labyrinthine trophoblast, the two major components of the murine placenta, we have examined the p21ras expression in these populations and how this correlates with the induction of class II MHC antigens. It has been shown that although only a small number of cells express the p21ras and class II proteins, they can be induced to express higher levels of these markers by two different treatments. Thus, gamma-interferon (gamma-IFN) induces p21ras and class II protein expression in spongiotrophoblast-derived cells, whereas 5-Azacytidine (5-AzaC) induces expression of these antigens in labyrinthine trophoblast-derived populations. Furthermore, it has been demonstrated that there is a direct correlation between the two markers, as blocking antibody to p21ras cancels the ability of gamma-IFN and 5-AzaC to induce class II antigens. As previous work from this laboratory has shown that in vivo class II induction in the placenta leads to fetal abortion, the present results suggest that both proteins are involved in a common signal transduction pathway which may lead to disruption of fetal membranes and fetal loss.

Animals↗

PubMed1992

The role of IL-4 in human myeloid leukemia: stimulation of RNA synthesis and transduction of differentiation signals through an IL-4 receptor leads to functional and HLA positive HL-60 cells.

The ectopic expression of lineage markers on irrelevant cell types may be of importance in the differentiation pathway(s) of these cells. One example, that is the subject of this study, is the presence of the interleukin-4 (IL-4) receptor on the surface of the human HL-60 myeloid leukemia cell line. The presence of such a receptor, that at first seems to be a simple genetic misprogramming, has an unusual biological function: It serves as a bridge to link the B cell growth factor IL-4 in order to transduce a number of differentiation signals in this M2 acute myeloid leukemia (AML) population. Signal transduction is followed by stimulation of RNA synthesis and subsequent induction of differentiation. Daily administration of low IL-4 dose yields proliferative senescent cells that exhibit 66% of growth inhibition in a 5-day tritiated thymidine incorporation assay. These cells clearly exit from the standard M2 morphology and show more mature characteristics as assessed by the Giemsa-Wright staining technique, followed by a 2-fold increase of the monocyte-granulocyte-specific Mac-1 surface antigen. Cellular function is also affected positively since phagocytosis of latex beads increases considerably after IL-4 treatment. Finally, as reported for normal human and murine monocytes and macrophages, the receptor-ligand interaction augments the levels of the class I and class II antigenic determinants by approximately 60%. Our results suggest that ectopic expression of markers may be a "distinct" event required during a short period in the differentiation of certain hemopoietic cells leading to mature and normal phenotypes.

Cell Differentiation↗

PubMed1992

Different molecular mechanisms lead to same endpoints with different function: TNF-alpha induces non-functional CSF-1 receptors on HL-60 cells in contrast to interferon-gamma.

Tumour necrosis factor-alpha (TNF-alpha) induces differentiation on human promyelocytic leukemia cells (HL-60) as assessed by growth inhibition accompanied by reduction of c-myc levels, and expression of surface colony-stimulating factor-1 (CSF-1) receptors reported as molecular markers for acute myeloid leukemia (AML). CSF-1 receptors can be readily demonstrated by direct binding of 125I-CSF-1 and expression of v-fms whose gene product retains a complete ligand binding domain. Although the same findings have been previously demonstrated on HL-60 cells after interferon-gamma (IFN-gamma) treatment, the major difference between the two pathways of cellular differentiation is the functional state of the induced receptors: when IFN-gamma is used as differentiation agent, the cells are driven to express a high number of CSF-1 receptors and are able to respond to a CSF-1 stimulus. Such mitogenic response is not obtained when TNF-alpha is applied as inducer indicating that the newly expressed receptors are not functional. This difference may be due to different molecular mechanisms the two factors trigger in order to reach the same endpoint: the maturation of this leukemic population.

Cell Differentiation↗