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Biomedical subjects

G Klein

Publications and source records attributed to G Klein.

At least 379 records · Page 21Linked to original sources
PubMed1991

Clonality and methylation status of the Epstein-Barr virus (EBV) genomes in in vivo-infected EBV-carrying chronic lymphocytic leukemia (CLL) cell lines.

Directly growing Epstein-Barr virus (EBV)-carrying cell lines were established from a chronic lymphocytic leukemia (CLL) patient (PG) on repeated occasions. The lines carried the same ring chromosome 15 as the leukemia cells in vivo and were similarly trisomic for chromosome 12. They all showed the same JH rearrangement, indicating that they had arisen from the same B-cell progenitor. They also had the same single EBV-terminal repeat (TR), indicating that they had been generated by a single EBV infection event. It may be surmised that a single CLL cell had been infected by EBV in vivo and established itself subsequently as a subclone within the CLL population. This subpopulation persists in vivo but does not appear to expand with time. After explantation, it transforms into lymphoblastoid cells and proliferates selectively as immortalized lines. The leukemia-representative CLL lines were phenotypically indistinguishable from the B95-8 virus-transformed normal diploid cells of the patient, established in parallel by in vitro infection. They grew as typical LCL clusters and expressed the same B-cell activation markers. The methylation status of EBV-DNA was different in the CLL lines and the B95-8-virus-transformed LCLs. When Hpall- and Mspl- digested DNA was probed with BamHI C, E, H and W fragments, the CLL lines showed a mixture of methylated and unmethylated restriction fragments as in certain EBV-carrying Burkitt lymphoma (BL) lines. In contrast, the EBV-DNA of B95-8 virus-transformed normal diploid cells was completely unmethylated, as in other LCLs.

Antigens, CD↗
PubMed1991

Sinus node-atrioventricular node isolation: long-term results with the "corridor" operation for atrial fibrillation.

The "corridor" operation is designed to restore sinus rhythm to patients with atrial fibrillation by electrically isolating the sinus node, a band of atrial tissue and the atrioventricular (AV) node from the remaining atrial tissue. Nine patients with drug-refractory atrial fibrillation underwent this operation; four patients had chronic atrial fibrillation and five had paroxysmal atrial fibrillation; the mean duration of symptoms was 12 +/- 8 years. Patient ages ranged from 25 to 68 years (mean 48 +/- 12). At preoperative electrophysiologic study, no patient had evidence of an accessory AV pathway or AV node reentry. Sinus node recovery time could not be determined in five patients because of recurrent atrial fibrillation during or before programmed stimulation. At operation the corridor of atrial tissue connecting the sinus and AV nodes was successfully isolated from the remaining left and right atrial tissue in all patients. One patient required early reoperation for recurrent atrial fibrillation before hospital discharge. At the predischarge electrophysiologic study, the corridor remained isolated in all patients except for one patient who had intermittent conduction between the corridor and excluded right atrium. One patient had nonsustained atrial fibrillation and one had atrial tachycardia evident in the corridor. Atypical AV node reentry of uncertain significance was induced in one other patient. Over a total follow-up of 191 patient months (mean 21 +/- 20), seven patients remained free of atrial fibrillation. Two patients had recurrent atrial fibrillation, which in one patient was effectively controlled by a single antiarrhythmic agent. A permanent pacemaker was implanted in four patients for sinus node dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Arrhythmia, Sinus↗
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↗
PubMed1991

An improved dot immunobinding assay for screening hybridoma supernatants. Non-purified antigen immobilized on nitrocellulose paper discs.

This report describes a modified dot immunobinding assay (DIA) in microplates using a crude mixture of non-purified antigen. Nitrocellulose filter paper discs exposed to the antigen mixture were inserted into the wells and kept in place by a specially constructed device. To test the efficiency of the modification a set of monoclonal antibodies from a mouse immunized with 58 kDa trpE-Bmyc fusion protein were screened. The advantage of this modified method over conventional ELISA is that it permits the use of non-purified antigen for screening large numbers of monoclonal antibodies.

Animals↗
PubMed1991

Expression of Epstein-Barr virus-encoded growth-transformation-associated proteins in lymphoproliferations of bone-marrow transplant recipients.

The expression of Epstein-Barr virus (EBV)-encoded, growth-transformation-associated proteins was studied in lymphoproliferations of 9 allogeneic bone-marrow transplant (BMT) recipients. Immunoblots of cell lysates were probed with polyspecific and monospecific antisera directed against EBNA 1, 2, 3 and 6, and the membrane protein LMP. All tumors expressed EBNA 1 and LMP. EBNA 2 was detected in the tumors of 8 patients, and EBNA 3 and 6 in the tumors of 5 patients. The LMP regulatory sequences, 5' of the LMP gene, were totally unmethylated in all 7 cases, while the coding sequences of LMP and EBNA 2 were more methylated in CpG dinucleotides. EBV-transformed lymphoblastoid cell lines (LCL) express EBNA 1 to 6 and LMP; in contrast, Burkitt lymphomas express only EBNA 1. In vitro experiments have shown that EBNA 2, 3 and LMP can generate targets for cytotoxic T cells (CTL). These combined observations are consistent with the hypothesis that the EBV-associated lymphoproliferative disease of the BMT recipients escapes CTL-mediated rejection due to the failure of host immunosurveillance rather than to the down-regulation of immunogenic EBV-encoded antigens.

Adolescent↗
PubMed1991

Transcriptional deregulation of myc in IgH/myc 6;7 translocation carrying rat immunocytomas.

We have previously shown that the reciprocal translocation t(6;7) associated with the spontaneous immunocytoma of the Louvain rat (RIC) leads to the juxtaposition of myc to the IgH cluster. In 10 of 14 tumors investigated the breakpoints on the myc carrying chromosome were clustered in a 1.5 kb region 5' of the intact gene, proximal to the myc promoters. In this paper we describe the effect of the translocation on myc transcription in the RIC system. Run-on analysis showed transcriptional attenuation in the normal rat myc gene, similar to the situation in mice and humans. The attenuation was almost completely abrogated in the three immunocytomas studied. Sequence analysis of two tumors failed to reveal any structural changes within exon 1, as found by others in Burkitt's lymphoma. We also show that the transcriptional initiation of myc mRNA is changed in the RICs. In an established line of rat fibroblasts (Rat-2), the more distal myc promoter (P2) is the preferred site of initiation. In RIC, however, only 30% of transcripts were initiated from P2. We found that 40% of the transcripts were initiated from P1 and 30% from a novel promoter, designated P1a, located between P1 and P2.

Animals↗
PubMed1991

Involvement of 3p deletions in sporadic and hereditary forms of renal cell carcinoma.

Deletions of the short arm of chromosome 3 and associated allele losses have been reported in the majority of sporadic renal cell carcinomas (RCC). On the basis of the combined cytogenetic and molecular data, it is reasonable to assume that a putative RCC locus, which contributes to tumor development by its loss, is located telomerically of the D3F15S2 site. Using H3E4, a D3F15S2-specific probe, we have isolated a cDNA clone (cl.4-2), and a sequence comparison revealed that the cDNA clone corresponds to the human acyl-peptide hydrolase gene. The gene is fairly universally expressed, but in RCC biopsies its expression is severely reduced, compared to the normal kidney. Cl.4-2 was used for in situ hybridization on metaphase chromosomes prepared from an Epstein-Barr virus (EBV) transformed lymphoblastoid cell line, derived from a t(3;8) (p14.2;q24.1) carrying member of the RCC family described by Cohen et al. in 1979 (N Engl J Med: 301:592-595). Carriers of this translocation regularly develop RCC by middle age. We now report that D3F15S2 is localized on the telomeric side of the constitutional breakpoint, in 3p21. The region of 3p affected by this familial translocation is thus not identical with the region of 3p most frequently deleted in sporadic RCC.

Alleles↗
PubMed1991

Inositol derivatives down-regulate c-myc inducing growth arrest without differentiation.

Cyclitol derivatives that are structurally related to myo-inositol induce growth arrest without differentiation in human promyelocytic leukemia (HL60) cells. An early effect is the rapid down-regulation of c-myc mRNA levels. This was observed also in several mouse and human lines carrying either normal or rearranged myc. The mRNA levels of a constitutive mouse myc construct transfected into HL60 were not affected at the same time. Uridine and thymidine incorporation were significantly decreased by the cyclitol treatment. These effects partly resemble those of certain differentiation inducers and those of hexachlorcyclohexane, another myo-inositol analogue. This new group of agents offers a novel approach to studying control mechanisms involving c-myc.

Blotting, Northern↗
PubMed1991

Co-localization of the retinoblastoma protein and the Epstein-Barr virus-encoded nuclear antigen EBNA-5.

A monoclonal antibody (aRB1C1) raised against an Rb fusion protein detects a limited number (4-10) of relatively large intranuclear foci in an EBV-immortalized cord blood cell line (IB4). These domains also bind an anti-EBNA-5 monoclonal antibody. The Rb antibody reactive sites also co-localize with the SV40 T antigen in transformed monkey cells (COS). The nuclear structures stained by aRB1C1 and EBNA-5 antibodies are distinct from the structures detected with antibodies against centromeric proteins and certain snRNP epitopes. EBNA-5/Rb-positive domains do not selectively react with antibodies against the La antigen known to associate with the small EBV-encoded nuclear RNA species designated as the EBERs.

Antibodies, Monoclonal↗
PubMed1991

Differential c-myc protein expression in Burkitt's lymphomas and EBV-transformed lymphoblastoid lines.

The levels of c-myc protein expression in three types of Epstein-Barr virus (EBV) transformed human B-cell derived lines were examined with an ELISA assay. Six independently maintained sublines of the same EBV-transformed pro-B-cell line (FLEB-14), six B-cell lines (LCL) and six Burkitt's lymphoma lines (BL) were compared. The average amount of c-myc protein, calculated from at least three independent tests on each line, differed between the three groups. Expressed in relative units, the ratio of the means was 1:2:5 for the LCL:FLEB:BL lines. The differences were statistically significant at P less than 0.01.

B-Lymphocytes↗
PubMed1991

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Animals↗
PubMed1991

Immunovirology of transforming viruses.

The paradigmatic shift in cellular immunology, initiated by the realization that cytotoxic T lymphocytes recognize major histocompatibility complex class I antigens that carry endogenous (including virally encoded) protein-derived peptides in a special groove, has made a strong impact on the immunopathology of virus-associated tumors.

Animals↗
PubMed1991

Laminin isoforms and their receptors in the developing kidney.

Epithelial cells have a polarized morphology, with distinct basal, lateral, and apical cell surfaces. It would be of considerable interest to know how the polarized morphology develops during embryogenesis. Both the tubular and glomerular epithelial cells of the kidney develop from mesenchymal stem cells during embryogenesis. A unique conversion of nonpolar cells to polarized epithelial cells thus occurs in the embryonic kidney. This conversion also occurs in vitro if the mesenchymal cells are properly induced. Organ cultures of mesenchymal cells from the mouse embryonic kidney have therefore been much used to study the development of epithelial cell polarity. We have used this model system to study the role of basement membrane glycoproteins in development. The results obtained suggest that laminins are particularly important for epithelial cell development. There are many different types of laminins. Developing kidney tubule cells synthesize a laminin isoform with the chain composition A-B1-B2, and it seems to promote development by interacting with specific integrin receptors on the cell surface. The mesenchymal stem cells also produce laminin B chains, but not the A chain, and they also lack the integrin receptor that interacts with A-B1-B2 laminin.

Animals↗
PubMed1991

[Cardiac adverse effects following an intravenous injection of pirenzepine are an expression of deficient receptor sensitivity].

Under the aspect of life-threatening pneumonias in long-term ventilated patients receiving ant-acids or H2-antagonists the prophylaxis of stress ulcer with pirenzepine gains new importance. This study was aimed at detecting cardiac side effects of pirenzepine due to low receptor specificity. Pirenzepine (10 mg) was given via the peripheral or central venous route at an injection velocity of 5 or 30 seconds as well as a control of 2 ml normal saline solution via the central venous route for 5 seconds, 20 times each. Heart rate and blood pressure were determined before and after the injection. Independent of the injection site or velocity, an increase in the heart rate was found after the injection of pirenzepine. The increase was more marked if the central venous route was used. The percentage rise in heart rate was up to the thirteenth minute highly significant when comparing pirenzepine to normal saline solution. There were no changes in blood pressure. If M1-cholinoceptors are blocked, a reduction of the heart rate is seen. Blocking M2-cholinoceptors causes a rise in the heart rate. Injecting 10 mg of the M1-antagonist pirenzepine results in a significant increase in the heart rate, independent of site or velocity of injection which can be explained by blocking M2-cholinoceptors at the same time. To prevent this probably dose-dependent effect, pirenzepine should be given slowly over a couple of minutes.

Adult↗
PubMed1991

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma.

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

Amino Acid Sequence↗
PubMed1991

B cell phenotype-dependent expression of the Epstein-Barr virus nuclear antigens EBNA-2 to EBNA-6: studies with somatic cell hybrids.

The expression of the transformation-associated Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) 1 to 6 and membrane protein LMP-1 was studied in a series of somatic cell hybrids derived from the fusion of the EBV-transformed lymphoblastoid cell line (LCL) KR-4, and the EBV-carrying Burkitt's lymphoma lines Daudi, P3HR-1 and Raji, with non-B cell lines of fibroblast, erythroid, myeloid and epithelial origin. Expression of EBNAs 2 to 6 was down-regulated in the hybrids in parallel with extinction of the B cell markers CD19, CD20, CD21, CD23, HLA class II, and surface or cytoplasmic immunoglobulin. LMP-1 was expressed independently of EBNA-2 in hybrids derived by the fusion of the LMP-1-positive KR-4 and P3HR-1 cell lines with epithelial and myeloid cells, respectively. LMP-1 was down-regulated in hybrids derived by the fusion of P3HR-1 with an erythroid cell line and in the hybrid between Raji and a mouse fibrosarcoma line. EBNA-1 was the only EBV antigen that was regularly expressed in the hybrids regardless of the dominating cellular phenotype. The autonomous expression of EBNA-1 suggests that its regulatory pathway is independent of phenotype-associated cellular or viral factors. In contrast, the expression of EBNAs 2 to 6 appears to require a B cell environment. EBNA-2 was shown to contribute to the regulation of LMP expression in B cells. We show that in LCL-carcinoma hybrids the dominating epithelial phenotype is permissive for LMP expression in the absence of EBNA-2.

Antigens, Viral↗
PubMed1991

Host cell phenotype-dependent methylation patterns of Epstein-Barr virus DNA.

We have shown previously that the EBNA 1 and latent membrane protein encoding regions of the Epstein-Barr virus (EBV) genome are highly methylated at CCGG sequences in the Burkitt's lymphoma (BL)-derived cell line Rael, but are unmethylated in a lymphoblastoid cell line (LCL) harbouring the same virus. To examine whether this is a regular phenomenon, we compared the methylation patterns of selected regions (BamHI C, W, H, M, E, K and N fragments) of EBV DNA in representative EBV-carrying cell types of normal and neoplastic origin. Analysis of HpaII and MspI cleavage patterns showed that all probed regions were highly methylated in all six BL biopsy samples, but hypomethylated in the four LCLs immortalized by the virus. EBV DNA was also highly methylated in the nude mouse-passaged C15 nasopharyngeal carcinoma strain and partially methylated in the C18 strain. Eight BL lines propagated in vitro, ranging from a typical BL group I to a more LCL-like group III phenotype, showed heterogeneous levels of methylation. Rael, the only stable group I cell line, carried highly methylated viral genomes. The other cell lines, which have drifted to an LCL-like blastic phenotype to various degrees, showed more moderate or low viral DNA methylation. Two sublines of the BL cell line Jijoye, which could be classified as groups II and III, respectively, showed a corresponding difference in EBV DNA methylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Viral↗