Biomedical subjects

M S Sy

Publications and source records attributed to M S Sy.

At least 37 records · Page 2Linked to original sources

PubMed2000

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.

Antioxidants↗

PubMed2000

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.

Amino Acid Sequence↗

PubMed2000

Differential contribution of superoxide dismutase activity by prion protein in vivo.

Normal prion protein (PrP(C)) is a copper binding protein and may play a role in cellular resistance to oxidative stress. Recently, copper-bound recombinant PrP(C) has been shown to exhibit superoxide dismutase (SOD)-like activity. However, as PrP(C) affinity for copper is low in comparison to other cupro-proteins, the question remains as to whether PrP(C) could contribute SOD activity in vivo. To unravel this enigma, we compared the SOD activity in lysates extracted from different regions of the brain from wild-type mice before and after the depletion of PrP(C). We found that removal of PrP(C) from the brain lysates reduced the levels of total SOD activity. The level of contribution to the total SOD activity was correlated to the level of PrP expressed and to the predominant form of PrP present in the specific brain region. Collectively, these results provide strong evidence that PrP(C) differentially contributes to the total SOD activity in vivo.

Animals↗

PubMed2000

Copper has differential effect on prion protein with polymorphism of position 129.

The pathology of human prion diseases is affected by polymorphism at amino acid residue 129 of the prion protein gene. Recombinant mouse prion proteins mimicking either form of the polymorphism were prepared to examine their effect on the conformation and the level of superoxide dismutase (SOD) activity of the prion protein. Following the binding of copper atoms to prion protein, antibody mapping and CD analysis detected conformational differences between the two forms of protein. However, neither the level of copper binding nor the level of SOD activity associated with this form of prion protein altered with the identity of codon 129. These results suggest that in the holo-metal binding form of the protein, prion structure but not its SOD activity is affected by polymorphism at codon 129.

Animals↗

PubMed2000

Regulation of no synthesis induced by inflammatory mediators in RAW264.7 cells: collagen prevents inhibition by osteopontin.

Osteopontin has been shown to inhibit the induction of inducible nitric oxide synthase (iNOS, or NOS2) by lipopolysaccharide and interferon-gamma in the RAW264.7 mouse monocyte/macrophage line and in primary mouse proximal tubule epithelial cells. However, the RAW264.7 cells become refractory to the action of OPN after several subcultures or under dilute culture conditions, possibly because of changes in the composition of the extracellular matrix. We make this suggestion because if the cells are plated on a collagen type I or collagen type IV substrate the inhibitory action of OPN is completely suppressed; this is not the case on substrates consisting of laminin, fibronectin, poly-D-lysine, or poly-(2-hydroxyethylmethylacrylate). These observations imply that macrophages are sensitive to regulation by OPN only in certain physiological contexts. Both hyaluronate, which binds CD44, and rat IgGs are also able to inhibit the induction of NO synthesis by the inflammatory mediators. The similar actions of HA and OPN are consistent with the possibility that CD44 may be a receptor for OPN.

Animals↗

PubMed2000

Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain.

The hallmark of prion diseases is the cerebral accumulation of a conformationally altered isoform (PrP(Sc)) of a normal cellular protein, the prion protein (PrP(C)). In the inherited form, mutations in the prion protein gene are thought to cause the disease by altering the metabolism of the mutant PrP (PrP(M)) engendering its conversion into PrP(Sc). We used a cell model to study biosynthesis and processing of PrP(M) carrying the glutamic acid to lysine substitution at residue 200 (E200K), which is linked to the most common inherited human prion disease. PrP(M) contained an aberrant glycan at residue 197 and generated an increased quantity of truncated fragments. In addition, PrP(M) showed impaired transport of the unglycosylated isoform to the cell surface. Similar changes were found in the PrP isolated from brains of patients affected by the E200K variant of Creutzfeldt-Jakob disease. Although the cellular PrP(M) displayed some characteristics of PrP(Sc), the PrP(Sc) found in the E200K brains was quantitatively and qualitatively different. We propose that the E200K mutation cause the same metabolic changes of PrP(M) in the cell model and in the brain. However, in the brain, PrP(M) undergoes additional modifications, by an age-dependent mechanism that leads to the formation of PrP(Sc) and the development of the disease.

Amino Acid Substitution↗

PubMed2000

Functional and structural differences between the prion protein from two alleles prnp(a) and prnp(b) of mouse.

The prion protein is a glycoprotein expressed by neurones and other cells. In its holo-form it binds copper and exhibits superoxide dismutase activity. Studies in mice have led to the description of two distinct alleles. Differences in these alleles are linked to long and short incubation times following infection with scrapie. We studied recombinant mouse protein corresponding to the products of either allele and two intermediates carrying single amino-acid residue substitutions. The different forms of the prion protein exhibited differences in superoxide dismutase (SOD) activity and conformation. Intermediates with single substitutions were less stable than either allelic product. The findings provide insight into the differences between the two alleles and might have consequences for understanding differences in susceptibility to prion disease.

Alleles↗

PubMed1999

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy.

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann-Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild-type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild-type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease-associated PrP.

Alleles↗

PubMed1999

Prion replication-once again blaming the dendritic cell.

The lymphoid system is known to be involved in the propagation and spread of scrapie. However, the identity of the cell type responsible for scrapie replication remains controversial. A new study provides evidence that the follicular dendritic cells in the spleen are the targets of this infectious form of prion (pages 1308-1312).

Animals↗

PubMed1998

Identification of two regions in the cytoplasmic domain of CD44 through which PMA, calcium, and foskolin differentially regulate the binding of CD44 to hyaluronic acid.

CD44 contains two clustered basic residues of three arginines and three lysines in the membrane-proximal region of its cytoplasmic domain. These two clusters are conserved among different species and different splicing forms. The function of these two motifs is not known. We substituted either the three-arginine or the three-lysine motif with alanine (CD44.3R3A and CD44.3K3A) and established stable CD44 transfectants. The effects of these mutations on the binding of CD44 to one of its ligands, hyaluronic acid (HA), were studied. When stimulated with PMA, transfectants bearing CD44.3K3A and CD44.3R3A proteins have reduced HA-binding capacity. When stimulated with forskolin, an activator of cAMP-dependent PKC, CD44.3R3A transfectants were able to bind low but detectable level of fluorescent-conjugated HA (F-HA). In contrast, CD44.3K3A transfectants were unable to bind any F-HA. Elevation of intracellular calcium concentrations either by ionomycin or thapsigargin also induced binding of HA in CD44.3R3A but not in CD44. 3K3A transfectants. These results provide evidence that both the arginine and the lysine motifs are important in the binding of CD44 to high levels of HA when stimulated with PMA. In contrast, when transfectants were stimulated with either forskolin or a Ca2+ mobilizer to bind a low level of F-HA, the lysine cluster, but not the arginine cluster, is required. These two closely located basic clusters are, therefore, differentially involved in the binding of CD44 to HA, depending on the level of ligand binding and the nature of the stimulatory signals.

Arginine↗

PubMed1998

Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines.

Tumor cells may alter the expression of proteins involved in antigen processing and presentation, allowing them to avoid recognition and elimination by cytotoxic T cells. In this study, reverse transcription-PCR was used to assess the expression in human tumor cell lines of mRNA for multiple components of the class I MHC antigen-processing pathway, including several proteasome subunits that have been implicated in antigen processing but have not been previously examined in this context (e.g., low molecular weight polypeptide proteasome subunit (LMP) 10, proteasome activator (PA) 28alpha, and PA28beta). Deficiencies in the expression of antigen-processing genes were demonstrated in 9 of 27 cell lines, representing a variety of histological types. In some cases, virtually complete deficiencies were observed in the expression of the four genes encoded within the MHC (TAP1, TAP2, LMP2, and LMP7), as well as LMP10, which is encoded outside the MHC. Combined deficiencies of these gene products were common, and marked deficiency of LMP10 was found in five of the nine cell lines with deficits. The existence of deficiencies in the expression of genes at dispersed loci suggested that the basis for the deficiencies was a regulatory mechanism, as opposed to mutation or deletion of these genes. Furthermore, most of the deficiencies were reversed by treatment with IFN-gamma. In contrast to such extreme deficiencies, we found unaltered or only partially decreased expression of PA28alpha and PA28beta in tumor cell lines. Thus, tumors may evade immune surveillance by simultaneously down-regulating multiple components of the MHC-I antigen-processing pathway, thereby altering the processing and presentation of tumor antigens. Expression of essential proteasome subunits, however, may still be maintained.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Prion protein expression in different species: analysis with a panel of new mAbs.

By immunizing prion knockout mice (Prnp-/-) with recombinant murine prion protein (PrPc), we obtained a panel of mAbs specific for murine PrPc. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrPc) and protease-resistant (PrPres) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrPc. These newly generated PrPc antibodies will help to explore the biology of PrPc and to establish the diagnosis of prion diseases in both humans and animals.

Animals↗

PubMed1998

Mechanisms regulating the binding activity of CD44 to hyaluronic acid.

CD44 is a cell surface glycoprotein present on many cell types. Many CD44 isoforms have been identified. All CD44 isoforms utilize identical transmembrane and cytoplasmic domains. The hematopoietic form of CD44 (CD44H) is the major CD44 protein present on normal human lymphocytes and monocytes. One of the ligands for CD44 is hyaluronic acid (HA), a polymer consisting of repeat units of disaccharide; N-acetyl-D-glucosamine and N-acetyl-D-glucuronic acid. Since HA is present ubiquitously in extracellular matrix and in circulation, promiscuous binding of HA to CD44 may have undesirable affect. Similar to other adhesion molecules, binding of HA to cell surface CD44 requires regulation. In this review, we summarized our studies using a human lymphoma cell line, Jurkat. We found that binding of CD44+ Jurkat transfectants to HA requires cellular activation. Cellular activation induces the reorganization of the cytoskeleton proteins. Reorganization of cytoskeletal proteins results in clustering of CD44 on the cell surface. Clustering of CD44 on the cell surface is a prerequisite for the homodimerization of CD44. Our studies on Jurkat transfectants and results from other investigators suggest that interactions between CD44 and HA is a dynamic process and requires the participation of different cellular components; depending of the nature of the cell type and/or the nature of the activation signals.

Amino Acid Sequence↗

PubMed1998

Over-expression of human CD44s in murine 3T3 cells: selection against during primary tumorigenesis and selection for during micrometastasis.

Human CD44 standard isoform (hCD44s) cDNA regulated by a high-expressing promoter was transfected into Balb/c 3T3 cells and the tumorigenic and metastatic capacities of the transfectants were investigated in nude mice at the subcutaneous site. One of three transfectants was tumorigenic. hCD44s expression was lost in the cells of large primary tumors using this tumorigenic clone. These tumors were extremely aggressive giving overt metastases and micrometastases to several sites including mesentery, stomach, liver, diaphragm, pancreas and lung. Micrometastatic cells re-expressed hCD44s, consistent with its importance for early steps in the metastatic cascade. hCD44s was not expressed in overt metastases; most probably the expression was lost during the outgrowth of micrometastases into overt metastatic tumors. Thus hCD44s expression in murine 3T3 cells does induce tumorigenicity in select cases, is not compatible with aggressive outgrowth of primary or secondary tumors, and is advantageous for early steps in metastatic spread. These results suggest that CD44s is an example of a novel type of 'metastasis' molecule that is disadvantageous for tumor growth and is only transiently advantageous during metastatic spreading of tumor cells to distant organs.

3T3 Cells↗

PubMed1997

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: facilitated retention in the lung vasculature.

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell-cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell-cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.

3T3 Cells↗

PubMed1997

Counter-selection for over-expressed human CD44s in primary tumors versus lung metastases in a mouse fibrosarcoma model.

Human CD44 standard isoform cDNA (hCD44s) was transfected into sis-transformed Balb/c 3T3 cells and into ras-revertant IIIA4 cells (both tumorigenic but nonmetastatic). Transfectants were injected subcutaneously into athymic nude mice to elucidate the functional role of hCD44s over-expression in progression and metastasis. The transfectants (but not parental cells) were capable of lung micrometastasis and of binding exogenously-added hyaluronan. hCD44s protein expression was conserved in lung micrometastases suggesting that it may have been necessary for their formation. In contrast, no hCD44s protein was detected in large subcutaneous (s.c.) tumors but normal levels of murine CD44 were detected. A second round of tumor development, using these two tumor cell classes, demonstrated that hCD44s-nonexpressing s.c. tumor cells re-expressed it in lung micrometastases. Conversely, hCD44s-expressing lung micrometastatic cells, when injected into a second group of mice, downregulated hCD44s expression in order to grow sizable s.c. tumors. S.c. tumor cells still contained the hCD44s gene but its expression was inhibited by epigenetic mechanisms, one of which was shown to be methylation of the hCD44s gene. These studies demonstrate (a) opposing selective pressures on CD44s over-expression for s.c. tumor growth and for metastatic spread to the lung and (b) further credence for the significance of CD44 for metastatic spread of fibrosarcomas. Therefore, CD44s may be a critical component of the metastatic phenotype induced by specific oncogenes.

3T3 Cells↗

PubMed1997

Phorbol myristate acetate stimulates the dimerization of CD44 involving a cysteine in the transmembrane domain.

Receptor oligomerization is important for ligand binding and signal transduction. CD44 is a transmembrane protein present on many cell types. One of the ligands for CD44 is hyaluronic acid (HA). HA binding activity of CD44 is linked to cellular activation in some cell types. Clustering of CD44 has been speculated to be important for binding of HA. However, the molecular mechanisms for converting CD44 from an inactive receptor to an active receptor are not known. Here we report that PMA stimulates the binding of CD44 to HA by inducing clustering of CD44 followed by covalent homodimerization of CD44 on the cell surface. Covalent dimerization involves a cysteine (Cys286) in the transmembrane domain of CD44 and is essential for binding of high levels of fluorescein-conjugated HA. Activation-induced clustering followed by disulfide bond-mediated dimerization of CD44 represents an additional signal transduction mechanism for regulating receptor-ligand interactions.

Carcinogens↗

PubMed1997

CD44 as a marker in human cancers.

Tumor metastasis is one of the most life-threatening aspects of tumor progression in patients with cancer. One of the cell surface molecules that has been implicated to play an important role in tumor metastasis is CD44. Earlier results provide the initial optimism that CD44 isoform expression may be a marker for human cancers. However, more recent studies revealed that regulation of CD44 isoform expression is a complex and not well-understood phenomenon. Expression of CD44 in tumor cells can be regulated quantitatively by increasing the expression of one particular CD44 isoform or quantitatively by altering the expression of CD44 isoforms. Downregulation of CD44 is important in the metastasis of some tumor cells. We summarize some of the recent results on the potential of CD44 as a diagnostic or prognostic marker for patients with cancers.

Antigens, Neoplasm↗