Search PubMed⌕ Search

Biomedical subjects

G Meinhardt

Publications and source records attributed to G Meinhardt.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of trovafloxacin on the IL-1-dependent activation of E-selectin in human endothelial cells in vitro.

E-selectin is an endothelial-specific surface protein, which is transiently expressed in response to inflammatory cytokines and plays an important role in the recruitment of leukocytes to the site of infection. The effect of two fluoroquinolones, ciprofloxacin (cipro) and trovafloxacin (trova), on the interleukin-1 (IL-1)-dependent activation of E-Selectin was studied on human umbilical vein endothelial cells (HUVEC) in vitro. Trova, at 80 microg/ml, affected the transient expression of E-selectin mRNA after pro-inflammatory stimulation with IL-1 leading to a sustained expression over 24 h. Surface expression of E-selectin remained upregulated after 24 h in a higher percentage of cells when they were activated in the presence of trova, as determined by flow cytometry analysis. Moreover, the concentration of shedded soluble E-selectin (sE-selectin) in the cell supernatant increased by 3.5 fold compared to those stimulated in the presence of cipro or without fluoroquinolones. Analogously, the antiproliferative effect of trova on endothelial cells was found to be more pronounced compared to cipro leading to an accumulation of cells arrested in G1-phase. These data provide evidence that accumulation of high concentration of trova in vivo in inflamed tissue might alter inflammatory responses.

Anti-Infective Agents↗
PubMed2000

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis.

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.

Annexin A5↗
PubMed2000

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways.

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.

Antineoplastic Agents↗
PubMed1999

Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis.

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.

Apoptosis↗
PubMed1999

Molecular pathogenesis of chronic lymphocytic leukemia: factors and signaling pathways regulating cell growth and survival.

Chronic lymphocytic leukemia is a malignant disease characterized by clonal expansion of relatively mature B-lymphocytes with a high percentage of cells arrested in the nonproliferative G0/G1 cell cycle phase. Possibly reflecting the clinical heterogeneity observed in patients, various signaling pathways may become affected during the initiation and course of this disease. This review discusses frequent alterations concerning proliferative, differentiation-inducing, and apoptotic pathways elucidated in the recent years that have improved our understanding of this disease.

Animals↗
PubMed1999

Evidence for different nonlinear summation schemes for lines and gratings at threshold.

Within a wide class of multichannel models of the visual system it is suggested that spatial distributions of luminance are processed by the independent activation of grating detectors, or spatial frequency channels. Probability summation is often described in terms of Quick's nonlinear pooling model [Quick RF (1974) Kybernetik 16:65-67]. Using this model, we find evidence for the existence of different kinds of nonlinear summation at threshold; for compound gratings with well-separated spatial frequency components, the threshold functions indicate nonlinear summation which is not compatible with probability summation, while for line patterns well separated in the spatial domain the probability summation rule proves compatible with the data.

Animals↗
PubMed1998

Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.

The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.

Amino Acid Sequence↗
PubMed1998

Detection of aperiodic test patterns by pattern specific detectors revealed by subthreshold summation.

The hypothesis that the visual system detects, under certain conditions, stimulus patterns by means of filters matched to these patterns (Hauske et al. 1976) may be challenged by the argument that other coding mechanisms like spatial frequency channels, Gabor or Hermite filters mimic the behaviour of matched filters, a view supported by the finding of non-linear contrast-interrelationship functions (CIFs), as determined in superposition experiments. In this paper we argue that an overall non-linear CIF does not contradict the hypothesis of detection by a single matched filter: we find that the sensitivity functions determined in our experiments can be separated into two components reflecting (i) a bandpass filter and (ii) a filter characterized by the spectrum of the test-pattern.

Animals↗
PubMed1996

Specific determination of tyrosine-phosphorylated proteins and peptides by differential iodination.

A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.

Alkaline Phosphatase↗
PubMed1995

Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease.

Amino Acid Sequence↗
PubMed1995

Differential expression of c-myc, max and mxi1 in human myeloid leukemia cells during retrodifferentiation and cell death.

Previous studies in human myeloid leukemia cells (HL-60, U-937, THP-1) suggested an involvement of the c-myc gene in the control of mutually exclusive pathways, such as retrodifferentiation and cell death. Treatment of U-937 cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) which is associated with the induction of a monocytic differentiation program and growth arrest, revealed an initial up-regulation of c-myc, c-max, and mxi1 mRNAs after 1-6 h. Thereafter expression of these genes significantly declined to barely detectable levels when the cells ceased to grow after 12-24 h of TPA treatment. Between 7 and 11 days of TPA-induced G0/G1 cell cycle arrest, expression of the c-max and mxi1 genes continuously increased up to 8-fold until 32 days and declined to control levels when the cells regained proliferative capacity by 36 days. In contrast, c-myc mRNAs remained down-regulated during periods of growth arrest and increased only during re-entry into the cell cycle after 36 days. This effect is consistent with a retrodifferentiation process, whereby previously differentiated cells revert back to the undifferentiated phenotype and re-enter the cell cycle. Different results were obtained during serum starvation-induced cell death of U-937 cells. After 48-72 h of serum-starvation, expression of the c-myc and c-max genes were significantly down-regulated by 4-fold and 3-fold, respectively, while there was little, if any, change in mxi1 mRNA levels. Analysis of cell death in serum-starved U-937 cells demonstrated progressively increasing DNA fragmentation reaching 45.4% +/- 0.9% after 72 h. Synchronization of proliferating U-937 cells throughout distinct phases of the cell cycle exhibited little, if any, change in c-myc, c-max and mxi1 mRNAs. Furthermore, like c-myc, c-max and mxi1 mRNA transcripts appeared to be regulated primarily by post-transcriptional mechanisms, and c-max and mxi1 half-lives exceeded 4 h in contrast to < 60 min for the c-myc gene. Taken together, these findings suggested differential regulation and inverse expression levels of c-myc compared to c-max and mxi1 during differentiation, retrodifferentiation and cell death.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1994

Characterization of human TUR leukemia cells: continued cell cycle progression in the presence of phorbol ester is associated with resistance to apoptosis.

Human TUR leukemia cells were generated as a subclone of U937 monoblastoid leukemia cells. There was no obvious difference in the ultrastructure of both cell lines. Like in U937 cells, the expression of monocyte-specific surface markers such as CD14 was negligible in TUR cells. U937 cells and other human myeloid leukemia cell lines (HL-60, THP-1) can be induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the monocytic pathway. In contrast, exposure to TPA had no effect on the induction of the differentiation program in TUR cells. Thus, the presence of leukocyte integrins including CD11 and CD18, which are significantly induced during TPA-induced differentiation of HL-60, U937 and THP-1 cells, remained nearly unchanged at low levels in both TUR and TPA-treated TUR cells. Furthermore, while expression of major histocompatibility complex (MHC) class II antigens on U937 and TPA-treated U937 cells is barely detectable, there was a significantly constitutive expression of MHC class II, particularly human lymphocyte antigen (HLA-DR) on the surface of TUR and TPA-treated TUR cells. Exposure of human myeloid leukemia cells to TPA is also associated with growth arrest resulting either in a retrodifferentiation process or in programmed cell death. In contrast, TUR cells continued to proliferate in the presence of TPA although the proliferative capacity was continuously reduced by increasing concentrations of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗
PubMed1993

Resistance to phorbol ester-induced differentiation of a U-937 myeloid leukemia cell variant with a signaling defect upstream to Raf-1 kinase.

Previous studies have demonstrated that treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with growth arrest and induction of monocytic differentiation. The present work describes the isolation of a U-937 cell variant, designated TUR, which is unresponsive to the growth-inhibitory effects of this agent. The results demonstrate that, in contrast to U-937 cells, the TUR line fails to respond to TPA with induction of the c-jun, junB, c-fos, and EGR-1 early response genes. The finding that these cells also fail to exhibit adherence or induction of the tumor necrosis factor and c-fms genes further supports their resistance to TPA-induced differentiation. In contrast, TUR cells responded to 1,25-dihydroxyvitamin D3, another inducer of monocytic differentiation, with growth arrest and induction of early response gene and c-fms transcripts. TUR cells also responded to okadaic acid, an inhibitor of type 1 and 2A protein phosphatases, with similar changes in gene expression. Further characterization of TUR cells has demonstrated decreased expression of protein kinase C as compared to wild-type U-937 cells. We also demonstrate that although treatment of U-937 cells with TPA is associated with activation of the Raf-1 serine/threonine kinase, there was no detectable decrease in electrophoretic mobility of this protein in TPA-treated TUR cells. Taken together, these findings indicate that the TUR variant is defective in TPA-induced signaling events upstream to activation of Raf-1 kinase.

Cell Differentiation↗
PubMed1992

Repeated evaluation of human papillomavirus 16 status in cervical swabs of young women with a history of normal Papanicolaou smears.

This study was designed to compare single-point prevalence estimates with a cumulative prevalence estimate of human papillomavirus (HPV) type 16 in cervical smears. The influence of the menstrual cycle and the long-term effect of HPV 16 positivity on the development of cervical intraepithelial neoplasia (CIN) were monitored. We examined 21 women (median age 23.6 years) every 5 weeks for 1 year. All women had a history of negative Papanicolaou smears for at least 5 years before enrollment. Cervical swabs were collected at each visit for Papanicolaou smears and HPV 16 detection by the polymerase chain reaction. Five years after completion of the last visit, self-reported information regarding cervical neoplasia was obtained. Human papillomavirus type 16 point-prevalence estimates per visit varied between 14.3-33.3%. The cumulative prevalence was 66.7%; 14 women were positive at least once and seven women were continuously negative for HPV 16. Detection of HPV 16 was significantly higher in the luteal phase. Repeated positivity for HPV 16 by Southern blot and polymerase chain reaction preceded and accompanied CIN in one patient, whereas in the remaining patients, positivity for HPV 16 by polymerase chain reaction alone was not associated with CIN during a 5-year follow-up. Single-point measurements of HPV 16 in cervical smears by polymerase chain reaction are of limited value for assessment of an individual's HPV status. This should be kept in mind when HPV testing for screening programs is considered.

Adolescent↗
PubMed1991

Prevalence of human papillomavirus genomes in tissues from the lower genital tract as detected by molecular in situ hybridization.

Incidence cases of human papillomavirus (HPV)-related disease of the lower anogenital tract were prospectively collected and submitted to RNA-DNA in situ hybridization, which allows a direct correlation between morphology and the presence of viral DNA. The overall HPV detection rate in condylomata and intraepithelial or invasive neoplasia varied for the different genital areas: cervix (64%), vagina (56%), vestibule (59%), vulva (50%), and penis (42%). HPVs 6/11 were associated with condylomata, whereas HPV 16 was associated with intraepithelial and invasive neoplasia. Not a single case of intraepithelial or invasive neoplasia contained HPV 6/11, which supports the notion of the oncogenic potential of HPV 16. Condylomata were positive for HPV 16 only when adjacent to HPV 16-positive cervical intraepithelial neoplasias (CINs). The amount of viral DNA (copy number) was low in invasive cancers, intermediate in intraepithelial neoplasia, and conspicuously high in condylomata. In biopsies (n = 142) taken from lesions suggestive of subclinical papillomavirus infection on the basis of minor colposcopic and histologic abnormalities, HPV DNA was not detectable. Histologically normal tissue (n = 210) adjacent to condyloma, CIN, or cancer, and thus potential carrier of latent HPV infection, also proved negative for HPV DNA by in situ hybridization. The inability to detect viral DNA by in situ hybridization in tissues diagnosed as subclinically or latently infected may have been due to limited sensitivity of the hybridization method applied in this study.

Carcinoma↗
PubMed1991

Platelet-targeted fibrinolysis enhances clot lysis and inhibits platelet aggregation.

BACKGROUND: Although plasminogen activator therapy has been shown to reduce mortality in patients with severe myocardial infarction, several problems fuel the search for more potent and specific thrombolytic agents. METHODS AND RESULTS: To explore the effect of plasminogen activator targeting to platelets, we covalently linked urokinase that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate to the Fab' of a monoclonal antibody (7E3) that selectively binds to platelet membrane glycoprotein (GP) IIb/IIIa. In an assay measuring (as reflected by plasmin generation) a plasminogen activator's ability to bind GP IIb/IIIa immobilized on plastic, urokinase-7E3 Fab' produced 31-fold more plasmin than did urokinase (p = 0.0001). The addition of solubilized GP IIb/IIIa blocked this enhancement of plasmin generation, indicating that binding was impaired. Plasmin generation reflecting binding to immobilized intact platelets was 2.4-fold greater for urokinase-7E3 Fab' than for unconjugated urokinase (p = 0.002). In a plasma clot lysis assay, urokinase-7E3 Fab' was at least 25-fold more potent than either urokinase alone or a mixture of urokinase and 7E3 (Fab')2 (p less than 0.009), and potency could be related to platelet concentration in the clot. Ex vivo, ADP-induced platelet aggregation was inhibited by a urokinase-7E3 IgG conjugate at a concentration of 8 nM, whereas a mixture of urokinase and 7E3 (Fab')2 in equimolar amounts required 60 nM and urokinase alone required 1 microM to achieve the same effect. CONCLUSIONS: Therefore, the targeting of urokinase to the GP IIb/IIIa platelet receptor both accelerates clot lysis (when platelets are associated with a fibrin clot) and inhibits platelet aggregation.

Antigens↗
PubMed1987

Sensitivity of the cytologic diagnosis of cervical condyloma in comparison with HPV-DNA hybridization studies.

The cytologic diagnosis of cervical condyloma is based on criteria developed over the last 10 years. It has now become possible to document the presence of human papilloma virus (HPV) DNA directly in cervical swabs by the highly sensitive technique of DNA filter hybridization in situ. The purpose of this article is to evaluate critically the empirically established cytologic criteria of condyloma by comparing them with HPV-DNA hybridization studies in the same material. The results of this study indicate that "classic" koilocytosis and dyskeratocytosis are not highly sensitive criteria for the presence of HPV infection, identifying only 15% of the HPV-DNA-positive cases correctly. In an attempt to improve the sensitivity of the cytologic diagnosis of HPV infections, a panel of nine "nonclassic" criteria was evaluated. The five most valuable signs were "mild koilocytosis," mild dyskeratocytosis," hyperchromatic nuclei, bi- and multinucleation, and cleared cytoplasm. Using these criteria in combination, statistically discriminant analysis could correctly identify 84% of the HPV-positive group.

Condylomata Acuminata↗