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Biomedical subjects

J Reinhardt

Publications and source records attributed to J Reinhardt.

At least 19 recordsLinked to original sources
PubMed2003

FLT3 mutations in acute myeloid leukemia cell lines.

Internal tandem duplications (ITD) and D835 point mutations of the receptor tyrosine kinase (RTK) FLT3 are found in a high proportion of cases with acute myeloid leukemia (AML). These genetic aberrations may lead to the constitutive activation of the receptor, thus providing the molecular basis for a persisting growth stimulus. We have screened 69 AML-derived cell lines for FLT3 mutations. Four of these cell lines showed ITD of the FLT3 gene, none carried a D835 point mutation. Two cell lines (MUTZ-11 and MV4-11) expressed exclusively the mutated allele, the other two cell lines (MOLM-13 and PL-21) displayed a mutated and the wild-type version of the gene. Although mutationally activated FLT3 is supposed to substitute for the stimulatory signal of a growth factor, one of these cell lines (MUTZ-11) was strictly cytokine-dependent. FLT3 transcripts were found in all four cell lines, but the constitutively phosphorylated receptor protein was clearly detectable only in cell line MV4-11, possibly explaining why MUTZ-11 cells were growth-factor dependent. Thus, not all FLT3 ITD-positive cells express high levels of the active receptor protein, a finding that might be of relevance for a possible future application of a kinase inhibitor as therapeutic agent. It had been described that STAT-5 phosphorylation was part of the FLT3 signalling chain and that STAT-5 molecules were constitutively phosphorylated in FLT3 ITD-positive cells. Although we observed the constitutive phosphorylation of STAT-5 molecules in FLT3-mutant cells, FLT3 ligand (FL) did not induce STAT-5 phosphorylation in FLT3 wild-type cells. These results suggest that the signalling mechanisms of the mutated FL receptor differ at least to some extent from those conferred by wild-type FLT3. In conclusion, (1) not all cells with FLT3 ITD express significant amounts of the mutated receptor protein; (2) signals downstream from wild-type and mutant FLT3 receptors are not 100% identical; and (3) MV4-11 represents a model cell line for FLT3 ITD signalling.

Acute Disease↗
PubMed2002

Aldosterone signaling pathway across the nuclear envelope.

We describe the route by which aldosterone-triggered macromolecules enter and exit the cell nucleus of Xenopus laevis oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2-30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5-15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small ( congruent with 100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large ( congruent with 800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (i) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (ii) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (iii) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.

Aldosterone↗
PubMed2002

Laser fluorescence bronchoscopy for detection of fluorescent reporter genes in airway epithelia.

Current methods for detecting successful gene transfer to airway epithelia involve obtaining a sample of the target tissue. This may affect the longevity of expression of the transgene under evaluation. We describe a laser fluorescence bronchoscopic system that can detect the expression of the fluorescent protein, green fluorescence protein (GFP), in the airway of monkeys that have been transfected with adenovirus, without the need for obtaining tissue. This technique will have applications in pre-clinical and clinical studies of gene transfer to airway epithelia and other surface epithelia accessible by endoscopy.

Adenoviridae↗
PubMed2002

Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1.

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.

Animals↗
PubMed2001

Expression of the ATP-binding cassette transporter gene ABCG1 (ABC8) in Tangier disease.

Several members of the ATP-binding cassette (ABC) transporter family are involved in cholesterol efflux from cells. A defect in one member, ABCA1, results in Tangier disease, a condition characterized by cholesterol accumulation in macrophages and virtual absence of mature circulating high-density lipoproteins. Expression of a second member, ABCG1, is increased by cholesterol-loading in human macrophages. We now show that ABCG1, which we identified by differential display RT-PCR in foamy macrophages, is overexpressed in macrophages from patients with Tangier disease compared to control macrophages. On examination by confocal laser scanning microscopy, ABCG1 was present in perinuclear structures within the cell. In addition, a combination of in situ hybridization and indirect immunofluorescence microscopy revealed that ABCG1 is expressed in foamy macrophages within the atherosclerotic plaque. These data indicate that not only ABCA1 but also ABCG1 may play a role in the cholesterol metabolism of macrophages in vitro and in the atherosclerotic plaque.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2001

ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages.

Apolipoprotein E (apoE) produced by macrophages in the arterial wall protects against atherosclerosis, but the regulation of its secretion by these cells is poorly understood. Here we investigated the contribution of the adenosine triphosphate binding cassette transporters ABCA1 and ABC8 to the secretion of apoE from either primary human monocyte-derived macrophages (HMDM) or human THP1 macrophages. During incubations of up to 6 h, apoE secretion from both THP1 macrophages and HMDM was stimulated by 8-Br-cAMP, which activates ABCA1 expression. The putative ABCA1 inhibitor glyburide and antisense oligonucleotides directed against ABCA1 mRNA significantly reduced apoE secretion from THP1 macrophages and HMDM. Antisense oligonucleotides directed against ABC8 mRNA also inhibited apoE secretion, although this inhibition was less pronounced and consistent than in the case of ABCA1. ApoE secretion from HMDM of ABCA1-deficient patients with Tangier disease was also decreased. ApoE mRNA expression was not affected by inhibition of ABCA1 or ABC8 in normal HMDM or the lack of functional ABCA1 in HMDM from Tangier disease patients. Inhibition of ABCA1 in HMDM prevented the occurrence of anti-apoE-immunoreactive granular structures in the plasma membrane. We conclude that ABCA1 and, to a lesser extent, ABC8 both promote secretion of apoE from human macrophages.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Immunocytochemical analysis of cell lines derived from solid tumors.

Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.

Antibodies, Monoclonal↗
PubMed2000

The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C.

The M1 protein of influenza A virus has multiple regulatory functions during the infectious cycle, which include mediation of nuclear export of viral ribonucleoproteins, inhibition of viral transcription and a crucial role in virus assembly and budding. The only known modification of the M1 protein is by phosphorylation through yet-to-be-identified kinases. We postulated that at least some of the M1 functions are exerted or regulated through interactions with cellular components. In a screen for such cellular mediators, the protein receptor of the activated C-kinase (RACK 1) was identified by its interaction with the viral M1 protein in the yeast two hybrid system. The physical M1-RACK 1 interaction was confirmed in glutathione-S-transferase-based coprecipitation assays for the diverged M1 proteins of avian, swine and human influenza A virus strains. This conservation suggests that the M1-RACK 1 interaction is of general importance during influenza A virus infections. RACK 1 has previously been identified to specifically bind the activated form of protein kinase C (PKC) and is assumed to anchor the kinase at membranes in the vicinity of its substrates. Since the M1 protein becomes phosphorylated during influenza virus infection, we examined if PKC could catalyze the phosphate transfer. We demonstrate that virion-derived and recombinant M1 protein can indeed be efficiently phosphorylated by purified PKC. Moreover, in cell extracts, we detected M1 phosphorylation activity that was strongly reduced in the presence of the PKC-specific inhibitor compound GF109203X. These data suggest that PKC is the main M1-phosphorylating activity in the cell. Since both, the M1 protein and PKC have been shown to interact with RACK 1, we suggest that the M1-RACK 1 interaction is involved in M1 phosphorylation.

Animals↗
PubMed2000

A short leucine-rich sequence in the Borna disease virus p10 protein mediates association with the viral phospho- and nucleoproteins.

Borna disease virus (BDV) is unique among the non-segmented negative-strand RNA viruses of animals and man because it transcribes and replicates its genome in the nucleus of the infected cell. It has recently been discovered that BDV expresses a gene product of 87 amino acids, the p10 protein, from an open reading frame that overlaps with the gene encoding the viral p24 phosphoprotein. In addition, the p10 protein has been localized to intranuclear BDV-specific clusters containing viral antigens. Here, characterization of p10 interactions with the viral nucleoprotein p38/p39 and the p24 phosphoprotein is reported. Immunoaffinity chromatography demonstrated the presence of high-salt stable complexes of p10 containing the p24 and p38/p39 proteins in extracts of BDV-infected cells. Analyses in the yeast two-hybrid system and biochemical co-precipitation experiments suggested that the p10 protein binds directly to the p24 phosphoprotein and indirectly to the viral nucleoprotein. Mutational analysis demonstrated that a leucine-rich stretch of amino acids at positions 8-15 within the p10 protein is critical for interaction with p24. Furthermore, binding of p10 to the viral phosphoprotein was shown to be important for association with the BDV-specific intranuclear clusters that may represent the sites of virus replication and transcription in infected cells. These findings are discussed with respect to possible roles for the p10 protein in viral RNA synthesis or ribonucleoprotein transport.

Binding Sites↗
PubMed2000

Differential localization of human nongastric H(+)-K(+)-ATPase ATP1AL1 in polarized renal epithelial cells.

The human H(+)-K(+)-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K(+)-transporting ATPases. In concert with the structurally related gastric H(+)-K(+)-ATPase, it plays a major role in K(+) reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK(1) renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na(+)-K(+)-ATPase beta-subunit or the gastric H(+)-K(+)-ATPase beta-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the alpha-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na(+)-K(+)-ATPase beta-subunit. Cotransfection of ATP1AL1 with the gastric H(+)-K(+)-ATPase beta-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by (86)Rb(+) uptake measurements. In contrast, cotransfected LLC-PK(1) cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the alpha-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

Animals↗
PubMed2000

Aldosterone and nuclear volume cycling.

The response of target cells to the steroid hormone aldosterone has been divided into acute nongenomic (< 10 min) and sustained genomic (> 10 min) action. In the light of recent experiments this nomenclature does not hold anymore and should be abandoned. By applying atomic force microscopy (AFM) we observed in living endothelial cells that aldosterone induces cell volume increase in less than 10 minutes. The cell nucleus was identified as the swelling site. Hormone-induced nuclear swelling can reach 15 to 28% of total cell volume dissipating within 30 minutes. This phenomenon could have functional impact on flow resistance in small blood vessels. AFM-investigation of the intracellular signal pathway in nuclear envelope of aldosterone-injected Xenopus laevis oocytes visualizes putative intracellular receptors (40 kD granules) bound to nuclear pores 2 minutes after hormone injection, with subsequent macromolecule translocation into the nucleus. 15 minutes later macromolecules (800 kD plugs) appear in the central channels of the nuclear pores. The plugs resemble ribonucleoproteins that carry the aldosterone-induced mRNA to the ribosomes. We postulate that steroid-induced nuclear swelling is caused by a shift of receptors/transcription factors from cytoplasm into nucleoplasm followed by gene transcription. Nuclear volume returns to normal when mRNA export through the nuclear pores is finished. Thus, steroid-induced net-movements of macromolecules between intracellular compartments initiate shifts in cell volume compensated by volume regulatory transporters and ion channels in the plasma membrane.

Aldosterone↗
PubMed1999

Cloning and characterization of the transport modifier RS1 from rabbit which was previously assumed to be specific for Na+-D-glucose cotransport.

Previously we cloned membrane associated polypeptides from pig and man (pRS1, hRS1) which altered rate and glucose dependence of Na+-d-glucose cotransport expressed by SGLT1 from rabbit and man. This paper describes the cloning of a related cDNA sequence from rabbit intestine (rbRS1) which encodes a gene product with about 65% amino acid identity to pRS1 and hRS1. Hybridization of endonuclease-restricted genomic DNA with cDNA fragments of rbRS1 showed that there is only one gene with similarity to rbRS1 in rabbit, and genomic PCR amplifications revealed that the rbRS1 gene is intronless. Comparing the transcription of rbRS1 and rbSGLT1 in various tissues and cell types, different mRNA patterns were obtained for both genes. In Xenopus oocytes the Vmax of expressed Na+-d-glucose cotransport was increased or decreased when rbRS1 was coexpressed with rbSGLT1 or hSGLT1, respectively. After coexpression with hSGLT1 the glucose dependence of the expressed transport was changed. By coexpression of rbRS1 with the human organic cation transporter hOCT2 the expressed cation uptake was not altered; however, the expressed cation uptake was drastically decreased when hRS1 was coexpressed with hOCT2. The data show that RS1 can modulate the function of transporters with non-homologous primary structures.

Amino Acid Sequence↗
PubMed1999

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume.

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87-R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume - possibly via [Ca2+]i - is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.

Actins↗
PubMed1999

The efficacy and safety of quinupristin/dalfopristin for the treatment of infections caused by vancomycin-resistant Enterococcus faecium. Synercid Emergency-Use Study Group.

A progressive increase in the incidence of vancomycin resistance in strains of Enterococcus faecium (VREF) has severely constrained treatment options for patients with infection caused by this emerging pathogen. Quinupristin/dalfopristin (Synercid), the first injectable streptogramin antibiotic, is active in vitro against VREF, with an MIC90 of 1.0 mg/L. We studied the clinical efficacy and safety of quinupristin/dalfopristin in the treatment of VREF infection. Two prospective studies were conducted simultaneously. The first enrolled only patients with VREF infection; the second included patients with infection caused by other gram-positive bacterial pathogens in addition to VREF. Patients were enrolled if they had signs and symptoms of active infection and no appropriate alternative antibiotic therapy. The recommended treatment regimen of quinupristin/dalfopristin was 7.5 mg/kg i.v. every 8 h for a duration judged appropriate by the investigator. A total of 396 patients with VREF infection were enrolled. The most frequent indications for treatment included intra-abdominal infection, bacteraemia of unknown origin, urinary tract infection, catheter-related bacteraemia, and skin and skin structure infection. This patient population had a high prevalence of severe underlying illness, including a history of diabetes mellitus, transplantation, mechanical ventilation, dialysis, chronic liver disease with cirrhosis and oncological disorders. The mean (+/- S.D.) duration of treatment was 14.5 +/- 10.7 days (range: 1-108). The majority of patients (82.1%) were treated every 8 h, as assessed on day 2 of treatment, while 15.9% were treated every 12 h. The clinical success rate was 73.6% [142/193 clinically evaluable patients; 95% confidence interval (CI): 67.4%, 79.8%], the bacteriological success rate 70.5% (110/156 bacteriologically evaluable patients; 95% CI: 63.4%, 77.7%) and the overall success (both clinical and bacteriological success) rate 65.8% (102/156 bacteriologically evaluable patients; 95% CI: 57.9%, 72.9%). VREF bacteraemia at entry, mechanical ventilation and laparotomy were associated with a worse outcome. Quinupristin/dalfopristin was generally well tolerated. The most common systemic adverse events related to treatment were arthralgias (9.1%) and myalgias (6.6%). Related laboratory abnormalities were infrequent. In these severely ill patients with VREF infection and no other clinically appropriate therapeutic alternatives, quinupristin/dalfopristin demonstrated substantial efficacy and a good nervous system, cardiovascular, gastrointestinal, renal and hepatic tolerability.

Adolescent↗
PubMed1999

K(+) channel-dependent migration of fibroblasts and human melanoma cells.

Previously, we showed that migration of transformed renal epithelial cells (MDCK-F cells) is a K(+) channel-dependent process [J Clin Invest 1994;93:1631]. In order to determine whether K(+) channel activity is a general requirement for locomotion, we extended our observations to NIH3T3 fibroblasts and human melanoma cells. Migration of both cell types and its dependence on K(+) channel activity was measured at the single cell level by time lapse photography in the absence and presence of the specific K(+) channel blocker charybdotoxin (CTX). Locomotion of both cell types is inhibited by K(+) channel blockade. CTX slows down migration of fibroblasts and of melanoma cells dose-dependently by up to 61 +/- 11%. These findings suggest that K(+) channel activity is a general prerequisite for migration. To determine whether CTX-induced inhibition of migration of fibroblasts and melanoma cells involves quantitative changes of actin filaments, we indirectly measured filamentous actin by quantitating binding of fluorescently labeled phalloidin. Whereas CTX elicits a decrease of bound phalloidin in fibroblasts there is an increase in melanoma cells. Since migration of tumor cells is required for invading surrounding tissue, we developed an assay to test whether CTX-induced inhibition of migration also impairs invasion of melanoma cells. Melanoma cells were seeded on a layer of high resistance renal epithelial cells (MDCK cells clone C7; transepithelial resistance R(te) >3,000 Omegacm(2)) and R(te) was measured daily. R(te) starts to decrease 2 days after seeding of melanoma cells onto MDCK-C7 cells. By day 7, R(te) has dropped to 24 +/- 1.5% of control. K(+) channel blockade with CTX (10 nmol/l) cannot prevent or delay this drop of R(te). R(te) reaches the same level with or without CTX. These results indicate that the disruption of an epithelial layer, unlike migration of melanoma cells, cannot be modulated by K(+) channel blockade.

3T3 Cells↗