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Biomedical subjects

G Klein

Publications and source records attributed to G Klein.

At least 253 records · Page 14Linked to original sources
PubMed1995

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.

DNA, Single-Stranded↗
PubMed1995

Intra-species characterization of clinical isolates and biotechnologically used strains of Lactobacillus rhamnosus by analysis of the total soluble cytoplasmatic proteins with silver staining.

Lactobacillus rhamnosus is often used in milk products because of its technological properties, however it is supposed to be related to human infections as well. Therefore, 23 L. rhamnosus strains, including the type strain, were investigated. Nine strains resulted from biotechnological sources, 14 isolates had a clinical background, 13 came from a swedish culture collection. Biochemical and physiological properties were proved by classical tests. Analysis of the total soluble cytoplasmatic protein patterns was performed with diamine silver staining, a technique not previously applied to lactobacilli. Classical tests were able to confirm all strains as L. rhamnosus. Analysis of protein patterns allowed to differentiate between three clusters. Cluster I contained the type strain and biotechnologically used strains and one clinical strain isolated in Berlin. The second consisted of both, technological and clinical strains. The third cluster contained clinical isolates alone. SDS-PAGE of proteins together with diamine silver staining seems to be helpful to detect intra-species differences. It was stated, that technologically used strains of L. rhamnosus could clearly be differentiated from each other and also from clinical isolates.

Bacterial Proteins↗
PubMed1995

Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells.

Human perlecan is a heparan sulfate proteoglycan with a large core protein of 467 kDa to which three glycosaminoglycan side chains are attached. It belongs to the heparan sulfate proteoglycan family which has been implicated in strong interactions between developing hematopoietic cells and their microenvironment in the bone marrow. Here we report that perlecan is highly expressed in the human bone marrow, as well as in long-term bone marrow cultures which are thought to mimic hematopoiesis in vitro. Expression of perlecan in this tissue was shown by Northern blotting of the 14-kb mRNA of the core protein and by immunofluorescence stainings. Functionally, perlecan shows a strong anti-adhesive effect on unfractionated bone marrow cells and on various hematopoietic cell lines, repelling the cells from the perlecan-coated area. In contrast, perlecan is adhesive for fibroblasts and endothelial cells. It is suggested that the anti-adhesive site is located within the core protein of perlecan since heparitinase-treated perlecan still shows the repellent effect. Although anti-adhesive for hematopoietic cells, perlecan is able to bind growth factors like granulocyte/macrophage-colony stimulating factor and present them to hematopoietic progenitor cells in a semi-solid colony assay. The functional role of a growth-factor-binding extracellular matrix component in the bone marrow microenvironment with anti-adhesive properties is uncertain but may be related to compartmentalization.

Adult↗
PubMed1995

Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas.

All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.

Carcinoma↗
PubMed1995

Antibodies to LMP2A/2B in EBV-carrying malignancies.

Antibodies to the Epstein-Barr virus (EBV)-encoded membrane proteins, LMP2A and LMP2B, were assayed in 540 individuals, including 154 patients with nasopharyngeal carcinoma, 16 with African Burkitt's lymphoma, 113 with Hodgkin's disease, 14 with EBV-carrying gastric carcinoma, 14 with oral hairy leucoplakia (HIV+ patients), 37 with non-Hodgkin's lymphoma, 49 with tumours of the head/neck, 19 with infectious mononucleosis, 62 with chronic illnesses with EBV titres consistent with re-activations, and 62 healthy controls. A novel assay, mouse monoclonal enhanced indirect immunofluorescence assay (MIFA) was designed and used to test the sera for antibodies to the LMP2A and 2B proteins, expressed in human keratinocytes. Antibody to both LMP2A and LMP2B was strikingly specific to NPC. Virtually all (99 of 101) of the LMP2 antibody positive individuals were NPC patients, 95% of whom had antibodies that reacted both with the LMP2A- and LMP2B-transfected indicator cells, while the remaining 5% reacted only with the LMP2B expressing cells.

Antibodies, Viral↗
PubMed1995

The amino-terminal phosphorylation sites of C-MYC are frequently mutated in Burkitt's lymphoma lines but not in mouse plasmacytomas and rat immunocytomas.

We sequenced the region encoding the amino-terminal phosphorylation sites of C-MYC in the Ig/MYC translocation-carrying Burkitt lymphomas (BL), mouse plasmacytomas (MPC) and rat immunocytomas (RIC). Mutations affecting the Thr-58 codon or the immediate flanking region were found in seven of the 10 in vitro propagated BL lines. No mutations were found in any of the eight BL biopsies analysed. Germ-line sequences were also found in six in vivo and five in vitro passaged MPCs and in four in vivo transplanted RICs. These findings indicate that mutations in this region do not represent a general phenomena in Ig/MYC translocation-carrying tumours, but may confer growth advantage on BL cells under continuous in vitro propagation.

Animals↗
PubMed1995

Infrequent MDM2 gene amplification and absence of gross WAF1 gene alterations in nasopharyngeal carcinoma.

We have investigated the possible involvement of MDM2 and WAF1 gene alterations in the development of nasopharyngeal carcinoma (NPC). MDM2 and WAF1 were analysed in 46 primary NPCs by Southern blot analysis. Forty-five tumours showed a normal EcoRI hybridisation pattern and hybridisation intensity with a human MDM2 cDNA probe. One tumour had more intense normal size MDM2 hybridising bands. Densitometric scanning revealed a 10-12-fold MDM2 gene amplification, as compared with human placenta DNA. All 46 tumours showed normal size WAF1 EcoRI bands that hybridised with normal intensity. This is the first demonstration of MDM2 gene amplification in NPC. Nonetheless, our analysis indicates that gross structural alterations of the MDM2 and WAF1 genes are infrequent events in the genesis of NPC.

Blotting, Southern↗
PubMed1995

E-cadherin is functionally involved in the maturation of the erythroid lineage.

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.

Base Sequence↗
PubMed1995

HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA-1, EBNA-2 and BZLF-1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1.

B lymphocytes immortalized with EBV in vitro, lymphoblastoid cell lines (LCL), express eight EBV-encoded proteins, EBV nuclear antigens -1 to -6 (EBNA-1 to -6), and latent membrane proteins 1 and 2 (LMP 1 and 2). After appropriate stimulations of blood lymphocytes from seropositive individuals, MHC-restricted cytotoxic T cells (CTL), which lyse LCL cells, can be generated in vitro. Such CTLs can recognize EBNA-2 to -6, and LMP 1 and 2, but not EBNA-1-derived peptides presented on the cell surfaces. We posed the question whether this exceptional feature of EBNA-1 is due to lack of MHC class I binding peptides. A computer search for 11 human leukocyte antigen (HLA) alleles showed that EBNA-1 has a lower number and lower proportion of relevant binding motifs to several alleles than EBNA-2 to -6 and LMP 1 and 2. The low motif numbers in EBNA-1 is in line with its apparent failure to generate a CTL response, and it may be the consequence of immunoselection allowing the existence of EBV genome-carrying B cells in the immunocompetent hosts. The binding capacities of synthetic peptides of EBNA-1 and -2 and of the immediate early lytic cycle protein BZLF-1 to HLA-A0201 (A2) and HLA-B7 molecules were tested in an MHC stabilization assay. The peptide transporter-deficient T2 line, which expresses a low level of HLA-A2 and its HLA-B7 transfectant subline, were used for this purpose because specifically bound peptides elevate the surface expression of these MHC molecules. Of five synthetic nonamer EBNA-1 peptides which include the relevant binding motif, four bound to A2. In a series of 20-amino acid-long overlapping EBNA-1 peptides none showed binding to A2, while eight peptides bound to B7. Two 20-amino acid-long EBNA-2 and seven BZLF-1 peptides were identified as A2 binders, and four EBNA-2 and eight BZLF-1 peptides bound to B7. Thus, we have exclude the possibility that the inability of the EBNA-1 protein to generate HLA-restricted CTLs could be due to the lack of HLA class I binding peptides in its sequence. The finding that several EBNA-1 peptides could bind to these two HLa molecules does not, however, necessarily reflect the natural situation because the peptides may not be processed and/or transported to the cell surfaces. We have stimulated lymphocytes of healthy donors with relevant HLA types with the autologous LCL.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1995

Propofol has no direct effect on sinoatrial node function or on normal atrioventricular and accessory pathway conduction in Wolff-Parkinson-White syndrome during alfentanil/midazolam anesthesia.

BACKGROUND: Propofol has been implicated as causing intraoperative bradyarrhythmias. Furthermore, the effects of propofol on the electrophysiologic properties of the sinoatrial (SA) node and on normal atrioventricular (AV) and accessory pathways in patients with Wolff-Parkinson-White syndrome are unknown. Therefore, this study examined the effects of propofol on the cardiac electrophysiologic properties in humans to determine whether propofol promotes bradyarrhythmias and its suitability as an anesthetic agent in patients undergoing ablative procedures. METHODS: Twelve patients with Wolff-Parkinson-White syndrome undergoing radiofrequency catheter ablation were studied. Anesthesia was induced with alfentanil (50 micrograms/kg), midazolam (0.15 mg/kg), and vecuronium (20 mg) and maintained with alfentanil (2 micrograms.kg-1.min-1) and midazolam (1-2 mg, every 15 min, as needed). A electrophysiologic study was performed consisting of measurement of the effective refractory period of the right atrium, AV node, and accessory pathway and the shortest cycle length of the AV node and accessory pathway during antegrade stimulation plus the effective refractory period of the right ventricle and accessory pathway and the shortest cycle length of the accessory pathway during retrograde stimulation. Determinants of SA node function including sinus node recovery time, corrected sinus node recovery time, and SA conduction time; intraatrial conduction time and atrial-His interval also were measured. Reciprocating tachycardia was induced by rapid right atrial or ventricular pacing, and the cycle length and atrial-His, His-ventricular, and ventriculoatrial intervals were measured. Alfentanil/midazolam was then discontinued. Propofol was administered (bolus 2 mg/kg + 120 micrograms.kg-1.min-1), and the electrophysiologic measurements were repeated. RESULTS: Propofol caused a statistically significant but clinically unimportant prolongation of the right atrial refractory period. The effective refractory periods of the AV node, right ventricle, and accessory pathway, as well as the shortest cycle length, were not affected. Parameters of SA node function and intraatrial conduction also were not affected. Sustained reciprocating tachycardia was inducible in 8 of 12 patients, and propofol had no effect on its electrophysiologic properties. All accessory pathways were successfully identified and ablated. CONCLUSIONS: Propofol has no clinically significant effect on the electrophysiologic expression of the accessory pathway and the refractoriness of the normal AV conduction system. In addition, propofol has no direct effect on SA node activity or intraatrial conduction; therefore, it does not directly induce bradyarrhythmias. It is thus a suitable agent for use in patients undergoing ablative procedures who require either a neuroleptic or general anesthetic.

Adolescent↗
PubMed1995

Coupled transcription of Epstein-Barr virus latent membrane protein (LMP)-1 and LMP-2B genes in nasopharyngeal carcinomas.

The presence of transcripts of the Epstein-Barr virus genes for Epstein-Barr nuclear antigen (EBNA)-1 and EBNA-2 and for latent membrane protein (LMP)-1, LMP-2A and LMP-2B was investigated in 24 nasopharyngeal carcinoma (NPC) biopsies of Chinese origin and two NPC-derived solid tumour lines, CAO and C15, of Chinese and north African origin respectively, propagated by serial transplantation in nude mice. Transcripts were detected by PCR amplification of cDNA. EBNA-1 transcripts were present in all biopsies tested and originated exclusively from the FQ promoter while the C and W promoters were inactive. Using nested primers, LMP-1 and LMP-2B RNAs were found to be co-ordinately expressed in 22 of the 24 biopsies, while the two remaining tumours were negative for both. LMP-2A transcription was detected in 17 of the 22 LMP-1-positive biopsies. In summary, the following patterns of viral gene expression were observed in the tumour biopsies: (i) LMP-1-LMP-2B and LMP-2A positive (17 biopsies); (ii) LMP-1-LMP-2B positive but LMP-2A negative (five biopsies); (iii) no viral gene other than EBNA-1 expressed (two biopsies).

Animals↗
PubMed1995

Reversible nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion.

The Epstein-Barr virus (EBV)-encoded, nuclear matrix-associated EBNA-5 protein is preferentially localized within distinct nuclear blobs in EBV-immortalized lymphoblastoid cell lines. We have previously found that the same blobs also contain retinoblastoma (Rb) protein. We now show that they contain hsp70 protein as well. Both EBNA-5 and hsp70 translocate to the nucleolus under cell density congestion or after heat shock. Both proteins relocate to their original position upon the re-establishment of normal physiological conditions. EBNA-5 is tightly bound to the nuclear matrix. The translocated EBNA-5 is also tightly associated with matrix structures, as shown by sequential elution-based cell fractionation. The Rb protein does not translocate to the nucleolus. The virally encoded EBNA-1, -2, -3 and -6, and cellular PCNA, snRNP and cyclin E are not affected either. The translocation of EBNA-5 to the nucleolus is not species- or cell type-specific since stress conditions induced the same phenomenon in EBNA-5-transfected human, mouse and rat cells of different tissue origins.

Animals↗
PubMed1995

The vascular cell adhesion molecule (VCAM-1) is expressed on a subset of lymphoid and myeloid leukaemias.

The expression of the vascular cell adhesion molecule (VCAM-1), recently identified as cytokine-inducible ligand of the beta 1-integrin VLA-4, was investigated on normal and malignant haemopoietic precursors as well as on haemopoietic cell lines. VCAM-1 was demonstrated on > 20% blasts in 4/22 acute myeloid leukaemia (AML) and 6/10 acute lymphoblastic leukaemia (ALL) specimens but was absent from CD34+ normal bone marrow precursor cells. Interestingly, the VCAM-1+ AMLs classified as M1 and M5 simultaneously expressed N-CAM (CD56), a member of the immunoglobulin family. In ALL, VCAM-1 expression was restricted to Calla+ CD19+ precursors of the c-ALL subtype. VCAM-1 was also found on some cell lines, mainly of the B-lymphocytic type. Furthermore, in 13/20 chronic lymphocytic leukaemia (CLL) samples > 20% of the CD19+ B-lymphocytic precursors carried VCAM-1, which seemed to correlate with more advanced disease. Therefore VCAM-1 expression appeared to characterize leukaemic cells of the B-cell lineage as well as a CD56+ subset of AML. Since its expression was clinically correlated with dermal infiltrates of leukaemic cells in AML and with advanced Rai stages in CLL, VCAM-1 may play a role in enhanced adhesion of the malignant cells to tissues and/or to each other.

Acute Disease↗
PubMed1995

Characterization of heat-shock response of the marine bacterium Vibrio harveyi.

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 degrees C was the highest temperature at which V. harveyi was able to grow steadily. A shift from 30 degrees C to 39 degrees C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15kDa protein. We showed that a protein of approximately 43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (sigma 32) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.

Bacterial Proteins↗
PubMed1995

A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1.

Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.

Animals↗
PubMed1995

Localization of human ARF2 and NCK genes and 13 other NotI-linking clones to chromosome 3 by fluorescence in situ hybridization.

Two human genes containing NotI sites, ADP-ribosylation factor (ARF2) and melanoma NCK protein, were mapped by fluorescence in situ hybridization to 3p21.2-->p21.1 and 3q21, respectively. Thirteen other NotI-linking clones, representing sequence tagged sites, were also mapped to different regions of human chromosome 3. Two of these clones that contain sequences 80% homologous to the rat tropoelastin gene and brain Cl- channel protein CLC-2 gene probably represent new human genes closely related to the known rat genes.

ADP-Ribosylation Factors↗
PubMed1995

Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles.

Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested 'in-gel' with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.

Amino Acid Sequence↗