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F Lang

Publications and source records attributed to F Lang.

At least 145 records · Page 8Linked to original sources
PubMed1999

Tyrosine phosphatase SHP-1 is involved in CD66-mediated phagocytosis of Opa52-expressing Neisseria gonorrhoeae.

Opa proteins of Neisseria gonorrhoeae bind to CD66 receptors on human phagocytes, thereby inducing efficient uptake of the bacteria in the absence of opsonins. The interaction of Opa proteins and CD66 receptors leads to activation of Src family tyrosine kinases, a process that is of critical importance for the efficient, CD66-mediated internalization. Here we show that during Opa-mediated stimulation of CD66 the activity of the host cell tyrosine phosphatase SHP-1 is strongly downregulated, concomitant with increases in the tyrosine phosphorylation of several cellular proteins. Since the SHP-1 tyrosine phosphorylation level itself is influenced by Opa-induced events, this phosphatase comprises an important regulatory checkpoint of the pathogen-triggered signaling cascade in human phagocytes.

Antigens, Bacterial↗
PubMed1999

Two consecutive phase II studies of 5-fluorouracil/leucovorin/mitomycin C and of gemcitabine in patients with advanced biliary cancer.

INTRODUCTION: Carcinoma of the biliary system is a rare tumor entity, and patients with advanced disease face a dismal prognosis. Because of the absence of standard chemotherapy for advanced biliary carcinoma, we have performed two consecutive studies to evaluate the clinical potential of 5-fluorouracil, leucovorin and mitomycin C as well as the novel antimetabolite gemcitabine in this disease. PATIENTS AND METHODS: A total of 39 consecutive patients suffering from locally inoperable or metastatic biliary cancer were enrolled in the study between March 1994 and October 1997. Twenty patients were treated with leucovorin 200 mg/m2 and 5-FU 400 mg/m2, both given as intravenous bolus on days 1-4, and mitomycin C 8 mg/m2 on day 1 (group A). Treatment cycles were repeated every 28 days. The second cohort included 19 patients, who received gemcitabine 1200 mg/m2 on days 1, 8 and 15 with a 2-week interval before the next treatment cycle (group B). Treatment was continued for a maximum of 6 cycles in the absence of progressive disease in both groups, and endpoints of the study were responses rates, survival and toxicity. RESULTS: In group A, 5 patients (25%) had a partial response (PR), 6 additional patients (30%) had stable disease (SD) and 9 patients (45%) progressed during treatment. The median survival was 9.5 months (range, 3-14.5) with the median time to progression being 4 months (range, 3-9). In group B, 3 patients achieved a PR (16%), 4 showed SD (21%), while the remaining 12 patients had progressive disease. A median survival of 6.5 months (range, 2-11.5) was obtained, and the median time to progression was 2.5 months (range, 1-6+). Toxicity was generally mild in both treatment arms, 6 patients in group A required dose reductions, while no dose adaptation had to be performed for gemcitabine. CONCLUSION: Our data suggest that treatment of advanced biliary cancer is feasible and can be safely performed with both regimens applied in our study. While administration of gemcitabine has resulted in only mild toxicities, its exact impact on the management of advanced biliary cancer should be evaluated in a controlled trial.

Adult↗
PubMed1999

Effect of verapamil enantiomers and metabolites on cardiac K+ channels expressed in Xenopus oocytes.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

Animals↗
PubMed1999

Effects of tetrabutylhydroperoxide on hepatocyte ion channels.

As shown previously, tetrabutylhydroperoxide (t-BOOH) shrinks hepatocytes by release of cellular K(+). The hyperpolarization, the increase of K(+) selectivity, and the Ba(2+) sensitivity of K(+) release have been taken as evidence of activation of K(+) channels. To further define the ion channels involved, patch-clamp studies have been performed on rat hepatocytes. The data show that 0.1 mmol/l t-BOOH inhibits an inwardly rectifying K(+) channel and simultaneously activates a 35-pS K(+) channel. It is suggested that the activation of the latter channel population contributes to the observed K(+) release of hepatocytes following exposure to t-BOOH.

Animals↗
PubMed1999

Automated prozone effect detection in ferritin homogeneous immunoassays using neural network classifiers.

The application of turbidimetric homogeneous immunoassays made the determination of several plasma components widely available. The sensitivity and accuracy of these assays are appropriate enough for routine laboratory use; however, in the case of many pathologically high concentration samples, prozone effect (high dose hook effect) can be observed, that leads to false-negative determination. Up to the present there are no cost-effective algorithms available for the safe detection of the prozone effect. Pathological serum ferritin values can be elevated up to 5000 ng/ml, while the measuring range covers only the 0-440 ng/ml range by a commercial assay. The determination of samples with ferritin concentration higher than 1500 ng/ml results in false-negative values because of the overlapping measuring range and prozone effect range. The prozone effect can be recognised by analysis of reaction kinetics after measurement. We have developed a neural network classifier system to analyse reaction kinetics of the measurements and check the prozone effect. One thousand five hundred determinations and 77 patient samples were used for neural network training and test. Using the trained neural networks, false-negative results can be filtered immediately after the determination, without re-run; thus, the sensitivity of plasma ferritin determination may become reliable enough, even in the case of high concentration samples. Applying this new technology, false-negative serum ferritin determinations can be avoided, thus even a relatively high hook effect rate (5-12% in different patient groups) can be handled safely.

Blood Chemical Analysis↗
PubMed1999

The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux.

Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14C]glutamine (Km = 70 microM) that was Na(+)-dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.

Amino Acid Sequence↗
PubMed1999

ICln, an ion channel-forming protein associated with cell volume regulation.

It is not resolved whether the anionic channel involved in volume regulation after cell swelling comprises one or more subunits. Moreover, it remains to be determined which of the different proteins cloned so far, for which an involvement in cell volume regulation has been postulated, is the ideal candidate. In this review, we consider the role of the ICln protein, cloned from MDCK cells, in cell volume regulation.

Animals↗
PubMed1998

L-selectin stimulates the neutral sphingomyelinase and induces release of ceramide.

Selectins have been shown to be crucial in the rolling process of leukocytes during lymphocyte homing and in the early phase of inflammatory processes. Recently, we and others have shown that binding of L-selectin to its ligands correlates with a rapid induction of several intracellular signaling molecules, in particular, Src-like tyrosine kinases, MAP-kinases, Jun NH2-terminal kinase, the small G-proteins Ras and Rac, and a release of Ca2+ in leukocytes. Here, we demonstrate the activation of a novel signaling pathway by L-selectin. Stimulation of Jurkat T-lymphocytes via L-selectin results in an increase of neutral sphingomyelinase activity. This activity correlates with a consumption of cellular sphingomyelin and a release of ceramide. The activation of the neutral sphingomyelinase by L-selectin does not depend on tyrosine kinase activity and, therefore, represents an alternative and novel pathway to stimulate lymphocytes via L-selectin.

Ceramides↗
PubMed1998

Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl-, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl-, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl- channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3- reabsorption along the proximal tubule when tubules are exposed to AII.

Angiotensin II↗
PubMed1998

Genomic organization and chromosomal localization of the human SGK protein kinase gene.

The SGK protein kinase is a novel member of the serine/threonine protein kinase family. Its corresponding gene belongs to the group of immediate-early genes. SGK transcription is controlled by cell volume alterations in different cell lines. To analyze the genomic structure and chromosomal location of the SGK gene, a human P1 clone was isolated by screening a human genomic library with a SGK cDNA probe. This clone was confirmed to encode the authentic SGK gene by the detection of exon-intron structures and the correspondence between the nucleotide sequences of exons and human cDNA. Using this P1 clone as a probe for fluorescence in situ hybridization, a single chromosomal locus for SGK was assigned to band 6q23, a region frequently affected by deletion in various human neoplasms.

Cell Line↗
PubMed1998

Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes.

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations </=3 mM. In summary, the present data suggest that Ip does not reflect charge transfer related to Pi-uptake, but a Pi-mediated modulation of GCl.

Animals↗
PubMed1998

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes.

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30-65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Apoptosis↗
PubMed1998

The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes.

Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.

Benzoquinones↗
PubMed1998

Ion channels involved in insulin release are activated by osmotic swelling of pancreatic B-cells.

Measurements of the membrane potential showed that osmotic swelling (-80 mosmol/l) of pancreatic B-cells led to a transient hyperpolarization followed by a more sustained depolarization of the cell membrane. Cell swelling triggers a transient activation of the K+ATP current and of an inward current, carried by Cl-. This current was inhibited by DIDS, D600, and by omission of extracellular Ca2+. The depolarization opens voltage dependent L-type Ca2+ channels, thereby increasing the intracellular Ca2+ activity ([Ca2+]i). This effect was blunted by D600 or abolished by omission of Ca2+. Moreover, osmotic swelling transiently increased the amplitude of the Ca2+ currents. Replacement of NaCl by d-mannitol proved that the observed effects are due to an increase in cell volume and not to a reduction of extracellular Na+ or Cl-. Our results suggest that regulatory volume decrease is achieved by activation of K+ and Cl- currents. The Cl- current is responsible for the previously described depolarization and increase in insulin release induced by osmotic cell swelling.

Adenosine Triphosphate↗
PubMed1998

CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

Antigens, CD↗
PubMed1998

Fas or ceramide induce apoptosis by Ras-regulated phosphoinositide-3-kinase activation.

We demonstrate a rapid and transient activation of phosphoinositide-3-kinase (PI-3-K) by Fas receptor triggering or cellular treatment with synthetic C6-ceramide. The stimulation of PI-3-K is critical for Fas or C6-ceramide-induced programmed cell death because transfection with a transdominant inhibitory PI-3-K construct or pre-treatment with the PI-3-K inhibitor wortmannin almost completely prevented Fas or C6-ceramide-mediated apoptosis. Treatment with the caspase inhibitor Ac-YVAD-cmk or cellular transfection with transdominant inhibitory N17Ras prevented PI-3-K stimulation by Fas, suggesting that Fas activates PI-3-K via caspases and Ras. N17Ras expression also prevented C6-ceramide-initiated PI-3-K stimulation. The notion of a PI-3-K regulation by Ras upon Fas receptor ligation or ceramide treatment is supported by co-immunoprecipitation experiments revealing an activation-dependent association of PI-3-K and Ras.

Androstadienes↗
PubMed1998

Regulation of ion channels in rat hepatocytes.

Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K+ channel with a mean inward conductance of 55 +/- 6.5 pS (n = 20) and a mean outward conductance of 25 +/- 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl- channel with a mean outward conductance of 30 +/- 4.5 pS (n = 8) and a mean inward conductance of 10 +/- 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca2+ activity on the intracellular side. Accordingly, the Ca2+ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K+ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K+ channel is inhibited by progesterone, while the outwardly rectifying Cl- channel is insensitive to progesterone.

Animals↗