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Biomedical subjects

G Klein

Publications and source records attributed to G Klein.

At least 325 records · Page 18Linked to original sources
PubMed1993

Integration of a short Epstein-Barr virus DNA fragment in a B95-8 virus converted Burkitt lymphoma line expressing Epstein-Barr nuclear antigens EBNA2 and EBNA5.

We have analysed the expression of transformation-associated viral antigens, the Epstein-Barr virus (EBV) DNA content and the phenotypic characteristics of two B95-8 virus-converted sublines of the EBV-negative Burkitt's lymphoma (BL) line BL28. The converted lines called E95A-BL28 and E95B-BL28, respectively, differed in their EBV gene expression. The E95B convertant expressed virus-encoded nuclear antigens EBNA1 to -6 and the membrane protein LMP1, but only EBNA2 and EBNA5 were detected by immunofluorescence and immunoblotting in the E95A convertant. Only the entire BamHI W, Y and H regions could be detected in the E95A convertant by hybridization of Southern blots with probes covering the BamHI C, W, Y, H, F, E, K and Nhet regions of the EBV genome. EBV episomes were found to be absent in the E95A convertant as seen by Gardella gels. The E95A convertant retained the phenotypic characteristics of the EBV-negative parental line, and remained highly clonable in agarose. In contrast, expression of EBNA1 to -6 and LMP1 was accompanied by a shift towards a more lymphoblastoid cell line-like phenotype and by loss of agarose clonability in the E95B convertant.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1993

Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy.

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45-60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20-40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.

Animals↗
PubMed1993

Role of histocompatibility antigen gene and protooncogene expressions in intracerebral tumorigenicity of mouse neuroblastoma.

The role of N-myc, c-src, and major histocompatibility complex (MHC, H-2 in the mouse) class I antigen gene expressions in dimethyl sulfoxide (DMSO)-induced differentiation and intracerebral tumorigenicity was examined using a mouse MNB85 neuroblastoma cell line. A fluorescence-activated cell sorter disclosed cell-surface MHC enhancement by DMSO, causing an increase in cytotoxic T-lymphocyte sensitivity. Southern blot analysis verified a single copy of the proto-oncogenes and MHC deoxyribonucleic acids in both untreated and DMSO-treated MNB85 cells. Northern blot analysis indicated that DMSO treatment induced a decrease in N-myc and an increase in c-src and MHC messenger ribonucleic acids. Nuclear run-off transcription assay revealed down-regulation of N-myc at a posttranscriptional level, contrasted with primary up-regulation of c-src at a transcriptional level. Immunoprecipitation after treatment with enzyme endo-beta-N-acetyl-glycoseamidase H proved that the terminal glycosylation of MHC heavy-chain gene products normally occurs in the Golgi apparatus of MNB85 cells. Intracerebral tumorigenicity assay showed that cells highly MHC-expressed by DMSO were less tumorigenic than untreated cells in association with DMSO-augmented cytotoxic T-lymphocyte susceptibility. These results suggest that proto-oncogenes may be linked to cellular differentiation, while cell-surface MHC gene expression influences intracerebral immunosurveillance.

Animals↗
PubMed1993

Endo-lysosomal acidification in Dictyostelium discoideum amoebae. Effects of two endocytosis inhibitors: caffeine and cycloheximide.

Fluid-phase endocytosis (pinocytosis) is highly active in amoebae of the cellular slime mould Dictyostelium discoideum as it provides an efficient entry of nutrients in axenic strains. Detailed kinetic analyses were conducted using fluorescein-labeled dextran (FITC-dextran) as fluid-phase marker and pH probe. Cells were first pulsed with FITC-dextran during a short period then chased by suspension in probe-free medium. Chase kinetics were characterized by a lag phase of about 40 min before pseudo-first order FITC-dextran efflux and thus reflected the progression of the probe cohort through the various endosomal compartments along the endosomal pathway. Temporal evolution of endo-lysosomal pH showed a rapid acidification (T1/2 approximately 10 min) to pH 5.0 followed by an increase up to pH 6.2 to 6.3. The effects of cycloheximide and caffeine, two inhibitors of endocytosis in Dictyostelium amoebae, on the evolution of endosomal pH during fluid-phase endocytosis, have been investigated. Cycloheximide fully blocked the cellular transit of FITC-dextran but acidification of endo-lysosomal compartments still took place. Caffeine increased endo-lysosomal pH, probably as a consequence of an elevation of cytosolic [Ca2+]. Furthermore, it allowed the functional identification of a caffeine-insensitive terminal segment of the endocytic pathway. It corresponded to a recycling, postlysosomal compartment at pH 6.2 to 6.3 with an apparent volume of 160 microns 3/amoebae.

Animals↗
PubMed1993

The long-term antihypertensive efficacy and safety of a new felodipine-metoprolol combination tablet. The Swedish/UK and German Study Groups.

The long-term antihypertensive efficacy and tolerability of a new extended-release tablet containing the vascular selective calcium antagonist felodipine and the beta 1-selective adrenoceptor blocker metoprolol were assessed in two European studies. In a Swedish/UK study, patients were treated with the combination for 12 (n = 122) or 24 (n = 53 of 122) months; in a German study (n = 162), patients were treated for 12 months only. In both studies, the study medication was administered once daily and the antihypertensive effect evaluated 24 h after dose. The dose was individually titrated until blood pressure control was achieved. In the Swedish/UK study, hydrochlorothiazide at 12.5 or 25 mg daily was added if necessary. Results of both studies were similar and the antihypertensive effect was well maintained during long-term treatment with decreases in mean supine systolic/diastolic blood pressure in the order of 25-30/20 mmHg after 12 months of treatment. A similar antihypertensive efficacy was recorded after 24 months of treatment in the Swedish/UK study. In both the Swedish/UK and the German study, the treatment was well tolerated. Overall, the withdrawal rate due to adverse events during the first 12 months of treatment was 6.7%. In both studies, adverse events were generally reported during the first 6 months of treatment and subsequently more rarely. The most commonly reported adverse events were peripheral oedema and headache. No clinically relevant changes in any laboratory variables were observed. The results of these trials confirm that the extended-release formulation combining felodipine and metoprolol maintains a significant antihypertensive effect during long-term treatment and is well tolerated.

Adult↗
PubMed1993

Phosphorylation sites mapping in the N-terminal domain of c-myc modulate its transforming potential.

The nuclear proto-oncoprotein c-Myc is involved in the regulation of cell growth and differentiation. c-Myc is phosphorylated at multiple sites in vivo, two of which we have identified near the amino terminus. In chicken Thr-61/Ser-65 are phosphorylated, as are the comparable positions, Thr-58/Ser-62 in human c-Myc. These residues are located within a domain that is implicated in transactivation and is important for the transforming potential of the protein. Furthermore, these phosphorylation sites or nearby amino acids are frequently mutated in v-myc and in several c-myc genes from Burkitt's lymphoma cells. In vitro these two phosphorylation sites can be modified by glycogen synthase kinase 3 and mitogen activated protein kinase. To address their biological importance we mutated these amino terminal phosphorylation sites separately and together. Stably transfected Rat1A cells expressing the mutated proteins have an increased growth potential in soft agar compared to wt-c-myc transfectants. These altered transformation characteristics indicate that Myc function may be negatively regulated by the amino terminal phosphorylation.

Amino Acid Sequence↗
PubMed1993

Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line.

Using a temperature sensitive p53 construct (ts p53), we have earlier shown that expression of wild-type (wt) p53 triggers apoptosis in a v-myc-induced T-cell lymphoma line that lacks endogenous p53, and in a Burkitt lymphoma line that carries mutant p53. We have suggested that apoptosis is elicited by the contradictory signals emanating from the constitutively activated myc gene and the growth arresting signal of wt p53 (Ramqvist et al., 1993; Wang et al., 1993). Work in other laboratories has shown that constitutive c-myc expression can induce apoptosis when cell proliferation is inhibited due to the lack of growth stimulating factors. Expression of bcl-2 could inhibit apoptosis. In order to test whether p53-induced apoptosis can be prevented by bcl-2, we have introduced a retrovirally driven bcl-2 construct into our v-myc-induced murine T-cell lymphoma line, previously transfected with ts p53. About 90% of the parental ts p53 transfected cells died of apoptosis within 3 days after induction of wt p53 expression at 32 degrees C. Two clones of ts p53/bcl-2 double transfectants that expressed high levels of bcl-2 from the introduced construct were completely protected from apoptosis, following transfer of the cells to 32 degrees C. One clone that expressed the exogenous bcl-2 only at a low level was partially protected from wt p53-induced apoptosis. Clones of the parental ts p53 carrying cells transfected with the puromycin resistance gene vector, without the bcl-2 gene underwent 90% apoptosis. These results suggest that bcl-2 may prevent apoptosis in cells simultaneously exposed to the proliferation-stimulating effect of activated myc and the growth arresting signal of wt p53.

Animals↗
PubMed1993

[Orthotopic arteriovenous fistula in dialysis patients: a special application of fluid dynamic processes].

Our experiments describe the flow patterns in translucent models of AV fistulas. The elasticity of the models is similar to human vessels walls. Dying of particular flow threads with methylene blue visualizes the flow and its disturbances in 4 classical models. The flow dynamics were investigated with parallel inflow. The volume flow was constant at 200 and 400 ml/min, with a volume flow ratio of 80:20. The flow pattern in side to side fistulas and in end to side fistulas with a narrow angle show regions of separation. This may lead to microthrombus formation and early failure. High shear stress causes endothelial lesions and thus induces stenosing intimal reactions. A favourable flow pattern was seen in end to side fistulas with a broad angle, as well as in end to end fistulas.

Anastomosis, Surgical↗
PubMed1993

Reconstitution of wild-type p53 expression triggers apoptosis in a p53-negative v-myc retrovirus-induced T-cell lymphoma line.

Inactivation or mutation of the p53 tumor suppressor gene has been observed in a wide variety of human and murine tumors. We have found that a v-myc retrovirus (J3)-induced T-cell lymphoma line (J3D) has lost one of its p53 alleles, whereas the other has become inactivated due to the insertion of a Moloney murine leukemia provirus in intron 4 with an opposite transcriptional orientation. No p53 protein could be detected by immunoprecipitation with monoclonal anti-p53 antibodies. We have transfected this line with the temperature-sensitive murine Val135 construct that is expressed as mutant p53 at 37 degrees C and largely wild-type p53 at 32 degrees C. There was no difference in the number of viable cells among the p53 transfectants, the parental cells, and neomycin vector-transfected control cells at 37 degrees C. Following a temperature shift to 32 degrees C, the p53 transfectants rapidly lost viability, and 95-100% of the cells were dead by 3 days, whereas the control cells remained unaffected. Examination of DNA isolated from p53-transfected cells grown at 32 degrees C revealed nucleosomal fragmentation, indicating cell death by apoptosis. It is suggested that apoptosis is triggered by contradictory signaling. Constitutively expressed v-myc can stimulate cell proliferation, whereas expression of wild-type p53 in cells that have lost endogenous p53 expression in the course of their neoplastic development may suppress growth.

Alleles↗
PubMed1993

Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1.

Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.

Adenoviridae↗
PubMed1993

Multicenter evaluation of Iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma.

A test kit (Iso-ALP, Boehringer Mannheim) for measuring human bone alkaline phosphatase activity in serum or plasma was evaluated in five laboratories in three countries. The assay is based on the principle described by Rosalki and Foo (Clin Chem 1984;30:1182-6) and uses wheat germ lectin to precipitate bone alkaline phosphatase. Residual ALP in the supernate in comparison with total ALP is used to quantify the bone fraction. The imprecision of residual ALP measurement was low (median between-run CV 4.9%) and comparable with that of total ALP. Linearity of precipitation was demonstrable up to a bone ALP activity (diethanolamine buffer 37 degrees C) of 2000 U/L, though a matrix effect was observed for dilutions of high-activity sera in saline or bovine serum albumin. For assaying patients' samples, different batches of lectin demonstrated excellent comparability. Taking electrophoresis as a basis for standardization, we determined that the lectin precipitated approximately 90% of bone ALP, but < 5% of nonbone ALP. From this we derived serum/plasma upper reference limits for bone ALP activity in adults and children.

Adult↗
PubMed1993

Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53.

All Burkitt lymphoma (BL) biopsies and cell lines carry a c-myc/Ig translocation. The resulting constitutive activation of c-myc is regarded as an essential factor for the progressive growth of the tumor cells. At least 60% of BL cell lines carry a mutated p53 gene as well. It has been shown that the growth of mutant p53 carrying tumor cells could be inhibited by the introduction of wild-type p53. In order to examine whether this also applies to the presumably 'myc-driven' BL cell, we have transfected the Epstein-Barr virus (EBV) negative BL41 cell line with a temperature sensitive p53 mutant (p53-Val135) that expresses p53 with a largely mutant conformation at 37.5 degrees C and mostly wild-type conformation at 32 degrees C. At 37.5 degrees C, the p53-Val135 transfected cells behaved like the parental or neo transfected control cells. However, expression of exogenous wild-type p53 at 32 degrees C resulted in a rapid reduction of the number of viable cells while the parental and neo control cells remained unaffected. Cell death was due to apoptosis as shown by chromatin and nuclear condensation and specific DNA fragmentation. The first signs of apoptosis were evident after 10 h at 32 degrees C and after 3 days 90-100% of the cells had undergone apoptosis. These findings indicate an incompatibility between expression of wild-type p53 and progressive growth of BL cells if their neoplastic development has included a p53 mutation. The question whether apoptosis was induced in by the wild-type protein per se or by the contradictory signals of a constitutively activated c-myc and wild-type p53 needs further investigation.

Apoptosis↗
PubMed1992

Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells.

The EBV-encoded membrane protein LMP is one of 9 viral proteins regularly expressed in virally immortalized B lymphocytes. It is expressed in EBV-carrying lymphoblastoid cell lines of normal origin and in the majority of the poorly differentiated nasopharyngeal carcinomas, but not in Burkitt lymphomas. LMP has been reported to transform rodent fibroblasts, to inhibit epithelial differentiation and to alter morphology and cytokeratin expression in an in vitro immortalized human keratinocyte cell-line, RHEK-I. We now report that an LMP-transfected mouse mammary carcinoma line, SHG, exhibits a similar morphological change to that previously described in the LMP-transfected RHEK-I. In the LMP-transfected RHEK-I and SHG cells, we observed a decreased expression of the calcium-dependent adhesion molecule E-cadherin. The LMP-transfected RHEK-I cells were capable of invading type-I collagen gels while the control cells were not. The LMP-transfected SHG cells showed a significantly higher invasive capacity than the original cell line.

Adenocarcinoma↗
PubMed1992

An anatomically guided approach to atrioventricular node slow pathway ablation.

Radiofrequency ablation of the "slow" pathway of the atrioventricular (AV) node reentrant circuit may be guided by electrophysiologic ("slow pathway potential") or anatomic landmarks. Experience with a systematic, anatomically guided approach in 25 patients (20 women and 5 men, aged 38 +/- 15 years) with typical AV node reentry is described. The slow pathway is assumed to be the posterior input to the AV node, approaching the nodal region in the corridor between the tricuspid annulus and the orifice of the coronary sinus. A series of radiofrequency lesions are given to interrupt this corridor at its entrance to Koch's triangle. If this is unsuccessful, the series of lesions are repeated progressively at higher levels approaching the AV node. The major end point for success is elimination of the slow pathway as determined by extrastimulus testing. A mean of 1.2 +/- 0.2 ablative sessions (20 +/- 12 applications of energy) achieved clinical success in 24 of 25 patients (96%) at a follow-up of 8.6 +/- 2.2 months. Anterograde Wenckebach cycle length increased from 361 +/- 67 ms to 398 +/- 70 ms (p = 0.01), yet the atrio-Hisian interval in sinus rhythm did not change (69 +/- 17 ms before vs 65 +/- 15 ms after ablation), p = 0.22. Retrograde Wenckebach cycle length was not affected (348 +/- 78 ms before vs 366 +/- 82 ms after ablation). During ablation, transient third-degree AV block occurred in 6 patients with no permanent sequelae. This approach provides a systematic, expedient technique to eliminate slow pathway conduction based on anatomic landmarks.

Adult↗
PubMed1992

Expression of the Epstein-Barr virus (EBV)-encoded membrane protein LMP1 impairs the in vitro growth, clonability and tumorigenicity of an EBV-negative Burkitt lymphoma line.

In a previous study on several independently established Epstein-Barr virus (EBV)-converted sublines of the EBV-negative Burkitt lymphoma (BL) line BL41, we found that expression of the virally encoded membrane protein LMP1 was accompanied by reduced agarose clonability and tumorigenicity. In order to investigate whether LMP1 can induce these phenotypic changes by itself, we have now studied the growth in suspension culture, the clonability in agarose and the tumorigenicity in immunosuppressed and SCID mice of 4 LMP1-transfected sublines of BL41 that carry the gene under the control of the ZnSO4-inducible metallothionein promoter. Expression of LMP1 at levels comparable to those detected in EBV-transformed lymphoblastoid cell lines (LCL) correlated with impairment of growth in suspension and reduction of clonability and tumorigenicity. Only minor changes were observed in transfectants expressing low LMP1 levels. Up-regulation of LMP1 by ZnSO4 treatment of the low LMP1 clone MTLM5 was accompanied by a slowing down of proliferation, increased cell clumping and decreased clonability. The results suggest that expression of LMP1 at levels which are compatible with immortalization of normal B-cells antagonizes the ability of BL cells to grow in vitro and in vivo, and illustrate a possible mechanism by which down-regulation of this viral antigen may favor tumorigenicity in EBV-carrying BLs.

Animals↗