PubMed Health⌕ Search

Biomedical subjects

R Nitschke

Publications and source records attributed to R Nitschke.

At least 19 recordsLinked to original sources
PubMed2006

A fusion tag enabling optical marking and tracking of proteins and cells by FRET-acceptor photobleaching.

Combined time-lapse imaging with optical marking of fluorescent proteins (FPs) is a widely used method in studies of the dynamic behaviour of proteins, organelles and cell populations. Most of the approaches have specific limitations as they do not permit simultaneous observation of marked and non-marked molecules, require co-expression of two FP-tagged proteins or rely on oligomerizing FPs. Here we provide a strategy to overcome such limitations with a fluorescence resonance energy transfer-competent tandem fusion tag composed of two FPs. We combine optical marking by acceptor photobleaching with spectral imaging to discriminate between marked and non-marked molecules. Such 'bleach-labelling' may be employed in a broad range of studies for robust real-time tracking of proteins, organelles and cells.

Amino Acid Sequence↗
PubMed2005

Synthesis of phospholipid-conjugated bile salts and interaction of bile salt-coated liposomes with cultured hepatocytes.

To examine the possibility of targeting liposomes to hepatocytes via bile salts, the bile salt lithocholyltaurine was covalently linked to a phospholipid. The isomeric compounds disodium 3alpha-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy)-5beta-cholan-24-oyl-2'-aminoethansulfonate and disodium 3beta-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy-5beta-cholan-24-oyl-2'-aminoethansulfonate (DSPE-3beta-LCT) were synthesized and incorporated into liposomal membranes. Confocal laser scanning microscopy studies showed that bile salt-bearing liposomes (BSLs) attach to the surface of rat hepatocytes in culture. Studies with radioactively labeled liposomes revealed that the bile salt linked via the 3beta-conformation resulted in a higher attachment efficiency than that with the 3alpha-derivative. In the presence of BSLs corresponding to 2 mM liposomal phosphatidylcholine, uptake of 50 microM cholyltaurine (CT) into hepatocytes was reduced by approximately 40% by the 3beta-derivative and by approximately 17% by the 3alpha-derivative. When added simultaneously with the liposomes, CT up to 75 microM inhibited the binding of DSPE-3beta-LCT-bearing liposomes. By contrast, increasing concentrations reversed this inhibition and resulted in an increased bile salt-mediated binding. The same was true when CT was added 10 min before the liposomes were added. The attachment of BSLs to the surface of hepatocytes opens up promising possibilities for hepatocyte-specific drug delivery. More generally, not only substrates for cellular endocytosing receptors but also substrates for cellular carrier proteins should be suitable ligands for the cell-specific targeting of nanoscale particles such as liposomes.

Animals↗
PubMed2005

How to improve quality assurance in fluorometry: fluorescence-inherent sources of error and suited fluorescence standards.

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

Journal Article↗
PubMed2002

The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.

Animals↗
PubMed2001

The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse.

KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.

Animals↗
PubMed2001

[Gastroenteropancreatic neuroendocrine/endocrine tumors. Current pathologic-diagnostic view].

Gastroenteropancreatic endocrine or neuroendocrine tumors (NETs) encompass a broad spectrum of neoplasms characterized by a heterogeneous biological behavior. They differ with regard to functional state, localization, and derivation from various segments of the primitive embryonal gut, growth pattern, degree of differentiation, expression of different neuroendocrine marker molecules, and prognosis. They occur generally as sporadic tumors but sometimes also in the setting of a familial cancer syndrome such as in type 1 multiple endocrine neoplasia or von Hippel-Lindau syndrome. The complex manifestations of NETs are reflected by the varying terms and classification systems. However, the terms "carcinoid" and islet cell tumor" do not indicate whether the tumors are benign, malignant, or of uncertain behavior. New clinical, morphological, and molecular developments in the field of neuroendocrine biology are contributing to the identification and characterization of NETs and are considered in the current World Health Organization classification.

Diagnosis, Differential↗
PubMed2001

The cardiac K+ channel KCNQ1 is essential for gastric acid secretion.

BACKGROUND & AIMS: Gastric H+ secretion via the H+/K+-adenosine triphosphatase is coupled to the uptake of K+. However, the molecular identity of luminal K+ channels enabling K+ recycling in parietal cells is unknown. This study was aimed to investigate these luminal K+ channels. METHODS: Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. RESULTS: We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a K+ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with H+/K+-adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. CONCLUSIONS: We identified KCNQ1 as the missing luminal K+ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.

Amino Acid Sequence↗
PubMed2000

Vasopressin V1a receptor signaling in a rat choroid plexus cell line.

A new cell line was derived from primary culture of rat choroid plexus (RCP) by immortalization with the TSOri minus adenovirus. The selected clone expressed vasopressin V1a receptors at a density of 64,000 sites per cell, and a K(d) of 7.2 nM. Addition of vasopressin to the RCP cells induced a transient calcium peak comparable to V1a receptor signalling in different expression systems. This [Ca(2+)](i) increase was dose-dependent with an EC(50) of 22 nM vasopressin. Similar [Ca(2+)](i) increase was elicited by addition of serotonin, angiotensin II, endothelin-1, and bradykinin. Heterologous desensitization of V1a receptor was observed in RCP cells exposed to the phorbol ester PMA or following stimulation of other receptors coupled to the phosphoinositide pathway. Positive immunolabelling with Factor VIII, Flt1 and CD 34 antibodies suggests that this new RCP cell line originated from endothelial cells of rat choroid plexus.

Animals↗
PubMed2000

Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel expressed in luminal membranes of secretory and reabsorptive epithelia. CFTR plays a predominant role in both cAMP- and Ca2+-activated secretion of electrolytes. Although Ca2+-dependent Cl- channels exist independent of CFTR in the airway epithelium, their physiological significance remains to be determined. However, CFTR seems to be the only relevant Cl- conductance in the colonic epithelium. Apart from its secretory function, CFTR also has a task in regulating the reabsorption of electrolytes by controlling the activity of the epithelial Na+ channel, ENaC. Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead to disturbances of both the secretion and absorption of electrolytes. Therefore, it is unclear what is pathophysiologically more important for the development of CF lung disease, the impaired secretion of Cl- or the enhanced reabsorption of Na+ and consecutive hyperabsorption of electrolytes. The mechanisms of how CFTR and ENaC interact are unknown. Previous work has given rise to several interesting working hypothesis, such as direct protein interaction or interaction via cytoskeletal proteins. Recent studies demonstrate the importance of the first nucleotide binding fold of CFTR, not only for the inhibition of ENaC but also for the interaction with other ion channels. Further studies are required to demonstrate whether regulation of other ion channels and membrane transport by CFTR occur by a common mechanism.

Animals↗
PubMed2000

KCNE1 reverses the response of the human K+ channel KCNQ1 to cytosolic pH changes and alters its pharmacology and sensitivity to temperature.

Previous studies have shown that heteromultimeric KCNQ1/KCNE1 (KvLQT1/minK) channels and homomultimeric KCNQ1 (KvLQT1) channels exhibit different current properties, e.g. distinct kinetics and different sensitivities to drugs. In this study we report on the divergent responses to internal pH changes and further characterize some of the current properties of the human isoforms of KCNQ1 and KCNE1 expressed in Chinese hamster ovary (CHO) cells or Xenopus laevis oocytes. Decreasing the bath temperature from 37 degrees C to 20 degrees C increased the half-activation time by a factor of 5 for KCNQ1/KCNE1 currents (IKs) but by only twofold (not significant) for KCNQ1 currents (IK) in CHO cells. Acidification of cytosolic pH (pHi) increased IKs but decreased 1K whereas intracellular alkalinization decreased I(Ks) but increased IK. pHi-induced changes in intracellular Ca2+ activity ([Ca2+]i) did not correlate with the current responses. At 20 degrees C mefenamic acid (0.1 mM) significantly augmented IKs but slightly decreased IK. It changed the slow activation kinetics of I(Ks) to an instantaneous onset. The form of the current/voltage (I/V) curve changed from sigmoidal to almost linear. In contrast, at 37 degrees C, mefenamic acid also increased I(Ks) but slowed the activation kinetics and shifted the voltage activation to more hyperpolarized values without markedly affecting the sigmoidal shape of the I/V curve. The potassium channel blockers clotrimazole and tetrapentylammonium (TPeA) inhibited I(Ks) with a lower potency than I(K). These results show that coexpression of KCNE1 reversed pH regulation of KCNQ1 from inhibition to activation by acidic pHi. In addition, KCNE1 altered the pharmacological properties and sensitivity to temperature of KCNQ1. The pH-dependence of I(Ks) might be of clinical and pathophysiological relevance in the pathogenesis of ischaemic cardiac arrhythmias.

Animals↗
PubMed2000

Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts.

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.

Algorithms↗
PubMed2000

Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms.

Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50(: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10(-6) mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10(-6) mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52+/-16% (n=10) and 13+/-10% (n=13) of control respectively. Diltiazem and verapamil (10(-6) mol/l) were also effective, but flufenamate (10(-4) mol/l), Gd3+ (10(-5) mol/l) and La3+ (10(-5) mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate-(InsP3-) sensitive Ca2+ stores by extracellular ATP (10(4) mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP3 concentration. Furthermore, inhibition of phospholipase-Cbeta (PLCbeta) by U73122 (2x10(-5) mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP3-sensitive Ca2+ stores, the InsP3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCbeta activity and InsP3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.

Adenosine Triphosphate↗
PubMed2000

Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus.

UNLABELLED: Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus. BACKGROUND: Knowledge about biological functions of podocytes in the glomerulus is limited because of its unique anatomical location. Here we introduce a new method for measuring the intracellular calcium activity ([Ca2+]i) in the podocyte in the intact glomerulus. METHODS: With the help of fluorescence high-resolution digital imaging and a recently developed ultraviolet laser-scanning microscope, [Ca2+]i was measured in fura-2-loaded glomeruli and single podocytes of intact microdissected rat glomeruli. RESULTS: Angiotensin II (Ang II) increased [Ca2+]i reversibly in a biphasic and concentration-dependent manner. In contrast to Ang II, bradykinin, thrombin, arginine vasopressin, and serotonin did not change [Ca2+]i in the glomerulus. At reduced extracellular Ca2+ activity, Ang II released [Ca2+]i from intracellular stores, but the second phase, corresponding to a Ca2+ influx from the extracellular space, was absent. The L-type Ca2+ channel blocker nicardipine did not influence the Ang II-mediated [Ca2+]i increase, and an increase of the extracellular K+ concentration did not change [Ca2+]i in the glomerulus. The angrotensin II type I (AT1) receptor antagonist losartan inhibited the Ang II-mediated [Ca2+]i increase. Confocal [Ca2+]i measurements using fura-2 or fluo-3 or fluo-4 on the single cell level show that some of the Ang II-mediated [Ca2+]i response originated from podocytes. Costaining with calcein allowed the identification of podocytes because of the characteristic morphology and location in relationship to the capillary network. CONCLUSIONS: These data suggest that podocytes in the intact glomerulus respond to Ang II with an increase of [Ca2+]i via an AT1 receptor.

Angiotensin II↗
PubMed2000

Intensification with intermediate-dose intravenous methotrexate is effective therapy for children with lower-risk B-precursor acute lymphoblastic leukemia: A Pediatric Oncology Group study.

PURPOSE: To determine whether early intensification with 12 courses of intravenous (IV) methotrexate (MTX) and IV mercaptopurine (MP) is superior to 12 courses of IV MTX alone for prevention of relapse in children with lower-risk B-lineage acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Six hundred fifty-one eligible patients were entered onto the study. Vincristine, prednisone, and asparaginase were used for remission induction therapy. Patients were randomized to receive intensification with IV MTX 1,000 mg/m(2) plus IV MP 1,000 mg/m(2) (regimen A) or IV MTX 1,000 mg/m(2) alone (regimen C). Twelve courses were administered at 2-week intervals. Triple intrathecal therapy was used for CNS prophylaxis. Continuation therapy included standard oral MP, weekly MTX, and triple intrathecal therapy every 12 weeks for 2 years. RESULTS: Six hundred forty-five patients (99.1%) achieved remission. Three hundred twenty-five were assigned to regimen A and 320 to regimen C. The estimated 4-year overall continuous complete remission for patients treated with regimen A is 82.1% (SE = 2.4%) and for regimen C is 82.2% (SE = 2.6%; P =.5). No significant difference in overall outcome was shown by sex or race. Serious grade 3/4 neurotoxicity, principally characterized by seizures, was observed in 7.6% of patients treated with either regimen. CONCLUSION: Intensification with 12 courses of IV MTX is an effective therapy for prevention of relapse in children with B-precursor ALL who are at lower risk for relapse but may be associated with an increased risk for neurotoxicity. Prolonged infusions of MP combined with IV MTX did not provide apparent advantage.

Antimetabolites, Antineoplastic↗
PubMed1999

The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.

Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.

Animals↗