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Biomedical subjects

G Klein

Publications and source records attributed to G Klein.

At least 307 records · Page 17Linked to original sources
PubMed1993

Analysis of c-Myc domains involved in stimulating SV40 replication.

We have demonstrated previously that overproduction of c-Myc, N-Myc and, to a lesser extent, L-Myc facilitates the replication of simian virus 40 (SV40)-based vectors in human lymphoid cells. Using a series of c-myc deletion mutants, we investigated which c-Myc regions are important in stimulating SV40 replication. The ability of c-Myc to promote SV40 replication was significantly reduced by deletions in the second exon domain, formerly shown to be crucial for c-Myc's transforming capacity. The c-myc mutants with a disrupted basic region (b) or leucine zipper (Zip) motif were also unable to stimulate SV40 replication. These regions are implicated in protein-DNA and protein-protein interactions, respectively, suggesting that the c-Myc protein might be associated with the DNA-protein replication complex. We present data obtained from gel mobility shift assays and from an immunocomplex-binding assay substantiating this hypothesis.

Base Sequence↗
PubMed1993

EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins.

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment of RB and p53 function.

Amino Acid Sequence↗
PubMed1993

31P-NMR analysis of Entamoeba histolytica. Occurrence of high amounts of two inositol phosphates.

Perchloric-acid extracts of axenic Entamoeba histolytica were investigated by 31P-NMR spectroscopy. All major 31P resonances observed were assigned to specific compounds. The cells contained inorganic phosphate (1039 nmol/g wet cells), pyrophosphate (16 nmol/g wet cells), nucleoside diphosphates (91 nmol/g wet cells), nucleoside triphosphates (275 nmol/g wet cells), NAD(P) (60 nmol/g wet cells), phosphocholine (184 nmol/g wet cells), phosphoethanolamine (214 nmol/g wet cells), cytidine 5'-diphosphocholine (41 nmol/g wet cells) and cytidine 5'-diphosphoethanolamine (55 nmol/g wet cells). The latter four compounds may act as intermediates in the salvage pathway for the synthesis of phosphatidylethanolamine and phosphatidylcholine. E. histolytica trophozoites also contained two inositol phosphates in large quantities, InsP3 (0.26 mumol/g wet cells) and InsP7 (0.11 mumol/g wet cells). These components were identified by 31P-NMR, using homonuclear J-resolved and two-dimensional 1H-31P correlative, analyses as myo-inositol trisphosphate, Ins(2,4,6)P3, and pentakisphospho-myo-inositol diphosphate, Ins(1,2,3,4,6)P5(5)P2.

Animals↗
PubMed1993

Lambda SK diphasmids: phage lambda vectors for genomic, jumping, linking and cDNA libraries.

Fifteen new phage lambda vectors are discussed: lambda SK4, lambda SK6, lambda SK10, lambda SK16, lambda SK17, lambda SK20, lambda SK21, lambda SK22, lambda SK23, lambda SK24, lambda SK25, lambda SK27, lambda SK28, lambda SK40 and lambda SK41. Their structural and functional features facilitate the implementation of a number of new strategies and cloning procedures which simplify, economize and vastly improve the utility and efficiency of library construction in lambda vectors. Such improved strategies and examples of their application, with particular reference to jumping and linking libraries, are presented.

Bacteriophage lambda↗
PubMed1993

Over-expression of C-myc increases the sensitivity of Epstein-Barr virus immortalized lymphoblastoid cells to non-MHC-restricted cytotoxicity.

Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) lines which maintain the phenotypic characteristics of the in vivo tumor cells are more sensitive to natural (NK), interferon-activated (IAK) and IL-2-activated (LAK) cytotoxicity than EBV-immortalized lymphoblastoid cell lines (LCL) of normal B-cell origin. All BL cells carry chromosomal translocations which lead to deregulated expression of the c-myc oncogene. LCLs transfected with constitutively active c-myc alleles display changes in growth properties and surface phenotype. In this study, we have examined the effect of c-myc deregulation on the sensitivity of LCLs to NK, IAK and LAK effectors. C-myc-transfected LCLs showed an increased sensitivity to lysis which correlated with the level of c-myc expression. Expression of HLA class I and sensitivity to allospecific and EBV-specific cytotoxic T-lymphocytes (CTL) remained unchanged. Transfection of a constitutively active v-H-ras gene, which also induces changes in growth properties and cell-surface phenotype, did not alter the sensitivity of LCLs to NK or LAK cytotoxicity.

Cell Transformation, Viral↗
PubMed1993

An HLA-A11-specific motif in nonamer peptides derived from viral and cellular proteins.

T lymphocytes recognize their antigenic targets as peptides associated with major histocompatibility complex molecules. The HLA-A11 allele, a preferred restriction element for Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte responses, presents an immunodominant epitope derived from the EBV nuclear antigen 4. Subpicomolar concentrations of a synthetic nonamer peptide, IVTDFSVIK, corresponding to amino acids 416-424 of the EBV nuclear antigen 4 sequence, can sensitize phytohemagglutinin-stimulated blasts to lysis by EBV-specific HLA-A11-restricted cytotoxic T-lymphocytes. We show that micromolar concentrations of this peptide induce assembly and surface expression of HLA-A11 in an A11-transfected subline of the peptide transporter mutant cell line T2. Using the IVTDFSVIK peptide and a series of synthetic nonamer peptides, differing from the original sequence by single amino acid substitutions, we have defined a motif for HLA-A11-binding peptides. This predicts the presence of a hydrophobic amino acid in position 2, amino acids with small side chains in positions 3 and 6, and a lysine in position 9. Using this motif, we have identified a peptide in the carboxyl-terminal end of wild-type p53, ELNEALELK, which is able to induce HLA-A11 assembly as efficiently as the IVTDFSVIK viral peptide.

Amino Acid Sequence↗
PubMed1993

Antigen processing and presentation by EBV-carrying cell lines: cell-phenotype dependence and influence of the EBV-encoded LMP1.

Burkitt's-lymphoma (BL) lines which have maintained in vitro the tumor-cell phenotype (group-I BLs) are poor antigen-presenting cells (APC), in spite of a relatively high surface expression of MHC class II. In order to investigate the mechanism of this deficiency, we have compared group-I BL lines, their sub-lines which have progressed in vitro towards an LCL-like phenotype (group-III BLs), and EBV-transformed lymphoblastoid cell lines (LCLs), for their ability to bind and process tetanus toxoid (TT). The uptake and internalization of 125I-labelled TT was equivalent in the 3 cell types. Only LCLs and group-III BL lines were able to process the TT, as shown by the identification of discrete proteolytic products after separation of whole-cell extracts in tricine-SDS-polyacrylamide gels, and by the recovery of TCA-soluble radioactivity in the culture supernatant. Processing of TT was induced by expression of the EBV-encoded membrane protein LMP 1 in transfected group-1 BLs. The present findings suggest that the inability of group-1 BLs to act as APC is due to their failure to process exogenous antigens. This function appears to be related to phenotypic properties that can be modulated by the expression of LMP1.

Antigen-Presenting Cells↗
PubMed1993

Position-dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis.

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization.

Animals↗
PubMed1993

Alu-PCR approach to isolating NotI-linking clones from the 3p14-p21 region frequently deleted in renal cell carcinoma.

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating NotI sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.

Animals↗
PubMed1993

Cell phenotype-dependent control of Epstein-Barr virus latent membrane protein 1 gene regulatory sequences.

Growth transformation-associated Epstein-Barr virus-encoded genes are differentially regulated depending on the host cell phenotype. We have previously identified an LMP1 regulatory region (LRS) 5' of the LMP1 gene (+40 to -634) and analyzed its role in transcription initiation in the EBV-negative Burkitt's lymphoma line DG 75 (Fåhraeus et al., 1990a). In order to investigate the cell phenotype dependence of LMP1 gene regulation we have now compared the activity of positive and negative cis-acting LRS elements in cell lines of B lymphoid and epithelial cell origin in the presence and absence of the virus-encoded nuclear antigen EBNA2. Our results show that reporter plasmids that contain only the -54/+40 region of LRS are active in all tested cell lines. Furthermore, the previously identified negative cis-elements in the -144/-54 region were found to suppress promoter activity independent of the cell phenotype. EBNA2 was able to override the effect of the negative elements in all lines of B-cell origin, whereas it had no effect in epithelial lines. The positive effect of EBNA2 was mediated by cis-acting elements in the -214/-144-bp region. In all six tested cell lines of epithelial origin, reporter plasmids that carried the full-length LRS were active, independent of EBNA2. The LMP1 promoter in these constructs was activated by the concerted action of EBNA2-independent, positive elements in the -214/-144 and -324/-214 positions that counteracted the effect of the negative LRS elements.

Animals↗
PubMed1993

Initiation of atrial fibrillation in the Wolff-Parkinson-White syndrome: the importance of the accessory pathway.

Atrial fibrillation in the Wolff-Parkinson-White syndrome may be related to microreentry in the accessory pathway. If such is the case, catheter ablation of the accessory pathway should eliminate atrial fibrillation. Among 95 patients undergoing catheter ablation, 20 had atrial fibrillation during standard electrophysiologic study (atrial vulnerability) before ablation. There were 16 women and four men with a mean age of 32 years. Before ablation six patients required electrical cardioversion. Thirty minutes after ablation, 11 continued to have inducible atrial fibrillation. Atrial fibrillation terminated spontaneously in every patient after ablation. A control group of 20 patients with accessory pathway ablation had no inducible atrial fibrillation before or after ablation. Catheter ablation had no effect on atrial properties including functional refractory period (227 +/- 37 vs 215 +/- 29 msec before versus after ablation, mean +/- SD) or wavelength (7.4 +/- 3.2 vs 7.2 +/- 2.7 before versus after ablation). These data suggest that an intact accessory pathway is not necessary for initiation of atrial fibrillation in most patients with Wolff-Parkinson-White syndrome. A rapid ventricular response over the accessory pathway may facilitate the perpetuation of atrial fibrillation in persons prone to this arrhythmia.

Adult↗
PubMed1993

Restless sleep, illness intrusiveness, and depressive symptoms in three chronic illness conditions: rheumatoid arthritis, end-stage renal disease, and multiple sclerosis.

Restless sleep was compared across 110 out-patients with rheumatoid arthritis (RA), 101 with end-stage renal disease (ESRD), 94 with multiple sclerosis (MS), and an unselected control group of 176 individuals attending their family practitioners (FP). It was also investigated in the three chronic illness groups as a contributor to illness intrusiveness--the extent to which one's illness and/or its treatment interfere with continued involvements in valued activities and interests--a determinant of depression and emotional distress in chronic conditions. Reported frequencies of restless sleep were highest in RA, lower in ESRD, and lowest in MS; FP patients reported frequencies that were similar, overall, to those observed in MS. These differences were evident among nondepressed, (chi 2 9, N = 309, p < 0.0001), but not depressed, individuals. The occurrence of restless sleep correlated significantly with increased illness intrusiveness, r (279) = 0.31, p < 0.001, in RA, ESRD, and MS. Results supported the hypothesis that the relation between restless sleep and emotional distress is mediated by illness intrusiveness. Treatment of restless sleep may offer the added benefit of diminishing illness intrusiveness and may, thereby, enhance quality of life in chronic physical illness.

Activities of Daily Living↗
PubMed1993

Selective induction of allostimulatory capacity after 5-azaC treatment of EBV carrying but not EBV negative Burkitt lymphoma cell lines.

Epstein-Barr virus (EBV) negative and EBV carrying Burkitt lymphoma (BL) lines that remain phenotypically similar to the in vivo tumor cells (operationally defined group I BLs) express high levels of CD10 and CD77, and lack immunoblastic markers such as CD23 and CD39, and the cell adhesion molecules CD11a, CD18, CD54 and CD58. This cell phenotype is associated with poor stimulatory capacity in allogeneic mixed lymphocytes cultures (MLC) [Avila-Carino et al. Int. J. Cancer 40, 691-697 (1987)] EBV carrying BL lines tend to drift spontaneously towards an immunoblastic phenotype in parallel with up-regulation of six EBV-encoded nuclear antigens (EBNA-2 to -6) and two membrane proteins (LMP-1 and -2). These viral antigens are characteristically expressed in all EBV transformed lymphoblastoid cell lines (LCLs) of normal B cell origin and can be induced in group I BL lines by treatment with the DNA demethylating agent 5-azacytidine (5-azaC) [Masucci et al. J. Virol. 65, 1558-1567 (1989)]. We have now studied the effect of 5-azaC on the induction of allogenic T cell proliferation by three EBV negative (Ramos, BL28 and BL41) and four EBV carrying BL lines (Rael, Eli, Chep and Mutu) which stably express a group I phenotype. Pre-treatment with 4-15 microM 5-azaC had no effect on the EBV negative cells but increased the stimulatory capacity of all four EBV carrying lines. LMP-1 was the only viral antigen regularly induced suggesting that its expression may be required for the increase of allostimulation. This was corroborated by the observation that LMP-1 transfection increased 35-70-fold the stimulatory capacity of Rael cells. The cell adhesion molecule CD54 was the only cellular marker selectively up-regulated in all cell lines with increased stimulatory capacity.

Antigens, CD↗
PubMed1993

Light and heavy chains specifying a human IgM kappa autoantibody to a T-cell receptor V beta-antigen.

Humans frequently produce serum IgM autoantibodies reactive with T-cell receptor beta chains at a determinant defined by peptides corresponding to the first complementarity determining region. It is likely that this determinant serves as a public idiotype involved in immunoregulation. Following screening of culture supernatants from over 60 Epstein-Barr virus-carrying B-cell lines of normal and neoplastic origin, we identified a line, IARC307, that secretes an IgM kappa protein showing marked specificity for the V beta 8.1 CDR1 sequence CKPISGHNSLFQWYRQT. We cloned and sequenced the complete variable regions of the V kappa and VH chains used by the autoantibody. The light chain has a V kappa III sequence related to the 'a' subgroup and uses J kappa 2. The heavy chain has a VHIII sequence essentially identical to the germ-line sequence DP54 and uses the JH6C minigene. The CDR3 is unique, differing from those of other autoantibodies. The antibody is rigorously specific in its specificity for the V beta 8 peptide and does not show polyspecificity for protein or DNA antigens.

Amino Acid Sequence↗
PubMed1993

Disease-related differences in antibody patterns against EBV-encoded nuclear antigens EBNA 1, EBNA 2 and EBNA 6.

Antibodies to Epstein-Barr virus (EBV) nuclear antigen family (EBNA) and three of its individual members, EBNA 1, EBNA 2 (A and B) and EBNA 6, were measured by anticomplement immunofluorescence (ACIF) in sera of 75 healthy controls, 13 patients with chronic EBV infection, 38 with non-Hodgkin lymphoma (NHL), 23 with Hodgkin's disease (HD), 105 with nasopharyngeal carcinoma (NPC) and 7 patients with infectious mononucleosis (IM). Their anti-EBV lytic antigens were also measured. We observed that: (1) anti-EBNA 2A and E6 rose in parallel 4-6 weeks after IM, followed by anti-EBNA 1 at 3-6 months, (2) all seropositive individuals had anti-EBNA 1; 74% also had anti-EBNA 2A and E6, (3) anti-EBNA 1 accounted for most of the anti-EBNA reactivity in non-IM sera. Striking disease-associated differences were noted on the humoral responses to the lytic and transformation-associated antigens. Compared to the controls, anti-EBNA 1, -EBNA 2A and -EBNA 6 were simultaneously four to 10 times higher in chronic reactivations, whereas only anti-EBNA 1 was elevated (10 times) in NPC. Individual EBNA titres were normal in NHL or HD patients.

Antibodies, Neoplasm↗
PubMed1993

Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment.

Tenascin is a large extracellular matrix (ECM) glycoprotein found in restricted tissue locations in the adult organism. It is copiously synthesized in regenerative organs or regenerating tissues and by certain tumors. We have analyzed the expression of tenascin in human long term bone marrow cultures as well as in cryostat sections of native bone marrow and found it strongly expressed by the stromal cells of the microenvironment. Two different protein subunits of 280 and 220 kD were detected by immunoblotting. These two forms are derived most likely from two different mRNA splice variants of 6 and 8 kb detected by Northern blotting. The in vivo analysis of cryostat sections showed a codistribution with other ECM molecules such as fibronectin and collagen type III in the microenvironment surrounding the maturing hematopoietic cells. Using two independent cell adhesion assays tenascin could be shown to function as a cytoadhesive molecule for hematopoietic cells. These data suggest a direct involvement of tenascin in the retention of hematopoietic progenitor cells in the stroma.

Blotting, Northern↗