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M Ritter

Publications and source records attributed to M Ritter.

At least 55 records · Page 3Linked to original sources
PubMed2000

The promoter for constitutive expression of the human ICln gene CLNS1A.

The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.

Animals↗
PubMed2000

Cardiac unloading alters contractility and calcium homeostasis in ventricular myocytes.

Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca(2+)](i)transients, SR Ca(2+)content); and L-type Ca(2+)and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca(2+)](i)transient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal I(Ca,L)and I(Na/Ca)densities, and SR Ca(2+)content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca(2+)buffers, may contribute to the increase in the [Ca(2+)](i)transient in this model.

Animals↗
PubMed2000

Eradication of Helicobacter pylori and stability of remissions in low-grade gastric B-cell lymphomas of the mucosa-associated lymphoid tissue: results of an ongoing multicenter trial.

The normal human stomach is devoid of any organized lymphatic tissue. Acquisition of mucosa-associated lymphatoid tissue (MALT) in the stomach is considered to be a direct consequence of chronic infection with Helicobacter pylori. Thus, MALT appears to be part of the host defense against the pathogen H. pylori. Consequently, lymphomas arising from gastric MALT may be seen as an end point of a clonal evolution starting from the infection. Cumulative data from several studies show that eradication of H. pylori induces complete histologic remissions in about 70%-80% of the patients. Here we present data of an extended analysis of an ongoing multicenter trial. Eighty-four patients with low-grade gastric MALT lymphoma in stage EI were treated using a dual regimen to eradicate H. pylori. Complete remission was observed in 68 (81%) patients; a partial remission was found in seven (8%) patients. In contrast, nine (11%) patients revealed "no change" and were referred for alternative treatment strategies. The majority of these cases were found to harbor high-grade lymphomas in deeper mucosal areas. Polymerase chain reaction (PCR) performed on the VDJ rearrangements of the immunoglobulin heavy chain yielded monoclonal bands in 50 of 65 analyzed patients (77%) at diagnosis. Interestingly, in patients analyzed during follow up after achieving complete histologic remission, ongoing PCR monoclonality was found in 19 of 39 eligible patients (49%). Several patients who developed local relapse of the lymphoma were found in the group with ongoing PCR monoclonality. Together with data from the literature, these results suggest that the majority of low-grade gastric MALT lymphomas in stage EI respond to eradication of H. pylori. Longer follow-up investigations are necessary to determine whether remissions really indicate a cure from the disease and to elucidate whether PCR monoclonality after complete histological remission is predictive of increased relapse rate.

Anti-Bacterial Agents↗
PubMed2000

Functional reconstitution of ICln in lipid bilayers.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

3T3 Cells↗
PubMed2000

The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels.

1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2000

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes.

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.

Animals↗
PubMed2000

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must - at some time point - lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na(+)/H(+) exchange and Na(+),K(+),2Cl(-) cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na(+)/H(+) exchanger and by activation of Cl(-) channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K(+) channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca(++) entry through I(CRAC) and the generation of cytosolic Ca(++) oscillations in proliferating cells. As a matter of fact I(CRAC) is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K(+) channels and Na(+)/H(+) exchanger as well as Ca(++) oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl(-) channels and organic osmolyte release. Inhibition of K(+) channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K(+) channels. Moreover, cell proliferation may at some point require activation of Cl(-) channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Apoptosis↗
PubMed2000

Structure and function of the ion channel ICln.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water - a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a Madin Darby canine kidney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell.

Amino Acid Sequence↗
PubMed2000

Cytokine-driven differentiation of blasts from patients with acute myelogenous and lymphoblastic leukemia into dendritic cells.

We investigated the ability of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts to differentiate into dendritic cells (DC) in vitro. Cytokine-supplemented suspension cultures of leukemic blasts in 98 patients with AML and five patients with ALL (normal karyotype, n = 2; BCR/ABL, n = 3) were performed. Mononuclear cells out of peripheral blood or bone marrow containing between 60% and 90% leukemic blasts were cultured for eight days using different growth factor combinations. The highest yield of CD1a(+)/CD14(-) cells could be obtained with stem cell factor, transforming growth factor-beta, tumor necrosis factor-alpha, GM-CSF, and FLT-3-ligand. In the AML samples the median content of CD1a(+)/CD14(-) cells after eight days of culture was 3.5% (r = 0%-82%). In five informed patients CD1a(+)/CD14(-) cells were sorted by fluorescence-activated cell sorting or immunomagnetic separation. Cytogenetic and polymerase chain reaction analyses showed known primary chromosomal aberrations (monosomy 7 and inversion 16) in the sorted fractions, respectively. Dendritic cells (DC) could be generated out of leukemic blasts in 68% of AML patients. Leukemic DC showed no phagocytosis of latex beads, but stimulated allogeneic naive cord blood-derived T cells more efficiently than did uncultured blasts. In ALL patients the median percentage of CD1a(+)/CD14(-) cells was 1.2% (r = 0.7%-3.8%) after culture. The sorted CD1(+)/CD14(-) fractions were BCR/ABL-negative when analyzed with fluorescence in situ hybridization, indicating their nonleukemic origin. Leukemic DC can be generated out of leukemic progenitors in patients with AML. These cells might become relevant for autologous and allogeneic immunotherapy in selected patients. BCR/ABL-positive lymphoblasts could not be transformed into cells with an early dendritic phenotype with the cytokines used in our experiments.

Adolescent↗
PubMed2000

Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli.

CD163, also referred to as M130, a member of the scavenger receptor cysteine-rich family (SRCR) is exclusively expressed on cells of the monocyte lineage. In freshly isolated monocytes the CD14bright CD16+ monocyte subset revealed the highest expression of CD163 among all monocyte subsets. CD163 mRNA and protein expression is up-regulated during macrophage colony-stimulating factor (M-CSF)-dependent phagocytic differentiation of human blood monocytes. In contrast, monocytic cells treated with GM-CSF and interleukin-4 (IL-4) for dendritic differentiation down-regulate this antigen. CD163 expression is also suppressed by proinflammatory mediators like lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha, whereas IL-6 and the antiinflammatory cytokine interleukin-10 (IL-10) strongly up-regulate CD163 mRNA in monocytes and macrophages. The effects of the immunosuppressants dexamethasone, cyclosporin A (CA), and cortisol differ in their capacity to influence CD163 mRNA levels. Our results demonstrate that CD163 expression in monocytes/macrophages is regulated by proinflammatory and antiinflammatory mediators. This expression pattern implies a functional role of CD 163 in the antiinflammatory response of monocytes.

Antigens, CD↗
PubMed1999

Lipopolysaccharide inhibits the expression of the scavenger receptor Cla-1 in human monocytes and macrophages.

Human Cla-1 is the likely homologue of the murine scavenger receptor class B type I (SR-BI). SR-BI mediates selective transfer of cholesterol to high-density lipoprotein (HDL) and the efflux of endogenously synthesized and plasma membrane sterols to HDL. HDL protects against atherosclerosis but also reduces endotoxic activity by complexation and neutralization of LPS. We found that Cla-1 is upregulated during phagocytic as well as dendritic differentiation of monocytes, indicating a function of this receptor for cholesterol homeostasis in phagocytes and antigen-presenting cells. Cla-1 expression is suppressed by the proinflammatory stimuli lipopolysaccharide, interferon-gamma, and tumor necrosis factor alpha in monocytes and macrophages. Downregulation of Cla-1 mRNA by LPS is likely due to a modification and subsequent destabilization of the mRNA. We propose that suppression of Cla-1 expression may help to stabilize the lipoprotein status in the blood compartment important for host defense.

CD36 Antigens↗
PubMed1999

Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily.

The human protein CD163 (M130) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, which is exclusively expressed by monocytes and macrophages. Here, we investigated the genomic organization and the chromosomal localization of the human CD163 gene. The CD163 gene is composed of 17 exons and 16 introns and spans over 35 kb. Each of its nine SRCR domains is encoded by a separate exon, which is similar to other members of the group B SRCR subfamily. Two cytoplasmic variants of CD163 arise from alternative splicing of intron 15, while a truncated and an extracellular variant results from alternative splicing of intron 5 or intron 7, respectively. Using fluorescence in situ hybridization we mapped this gene to the human chromosome 12p13. The transcription initiation sites of the CD163 gene were determined and the 5'-flanking region was sequenced. The nucleotide analysis revealed several putative binding sites for transcription factors, which have been shown to play an important role in myeloid specific gene expression. In addition, we identified a L1 element located 1.4 kb upstream of the major transcription initiation site.

Alternative Splicing↗
PubMed1999

Lack of polymorphism of the conversion of losartan to its active metabolite E-3174 in extensive and poor metabolizers of debrisoquine (cytochrome P450 2D6) and mephenytoin (cytochrome P450 2C19).

OBJECTIVE: Losartan was given to subjects with known phenotypes of the polymorphic enzymes CYP2D6 and CYP2C19 to study any possible influence on the metabolism of the drug. METHODS: Plasma concentrations of losartan and E-3174 were studied after oral intake of 50 mg losartan in 24 healthy, male, Swedish Caucasian subjects who were extensive or poor metabolizers (EM/PM) of debrisoquine [cytochrome P450 2D6 (CYP2D6)] or mephenytoin [cytochrome P450 2C19 (CYP2C19)]. RESULTS: The areas under the curve (AUCinfinity) of losartan and E-3174 did not differ between poor and extensive metabolizers of debrisoquine or mephenytoin, respectively. CONCLUSION: About 14% of the antihypertensive drug losartan is metabolized to the active carboxylic acid metabolite E-3174, which contributes to the effect of losartan. The present study suggests that CYP2D6 and CYP2C19 are not involved to any major extent in the in vivo conversion of losartan to E-3174.

Adult↗
PubMed1999

Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection.

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells.

Amelogenin↗
PubMed1999

Detection of cytogenetic aberrations both in CD90 (Thy-1)-positive and (Thy-1)-negative stem cell (CD34) subfractions of patients with acute and chronic myeloid leukemias.

Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are thought to arise from malignant hematopoietic progenitor cells representing early and undifferentiated stem cell clones. In CML there is evidence for a progenitor cell subset free of leukemic clones, depending on the course of the disease. Additionally, it has been suggested that in AML, the early stem cell compartment (CD34+/90+) does not harbor the malignant clone. We analyzed white blood cells from leukemia patients for the presence of aberrant cells in stem cell subfractions. Sixteen patients with CML, six patients with AML, two patients with acute lymphatic leukemia (ALL) and one with chronic myelomonocytic leukemia (CMMOL), all with known cytogenetic abnormalities, were evaluated according to their CD90 (Thy-1)-positive or -negative phenotype. Subsets were sorted on to slides and further characterized by FISH and/or standard cytogenetic testing. The bcr-abl translocation or gross chromosomal abnormalities could be detected in equally high amounts of 92.2% and 89.2% in both stem cell subsets. We conclude, that in progressed AML and CML cells characterized by specific genetic aberrations implicated in the malignant state can be found in the CD34+/CD90+ and CD90- population, thus making CD90 an inappropriate marker to distinguish benign from malignant cells in these leukemias.

Acute Disease↗