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G Thiel

Publications and source records attributed to G Thiel.

At least 37 records · Page 2Linked to original sources
PubMed2001

Electrically triggered all-or-none Ca(2)+-liberation during action potential in the giant alga Chara.

Electrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)). The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt). When the strength of a pulse exceeded a narrow threshold (suprathreshold pulse) it evoked the full extent of the Ca(2)+(cyt) elevation. This suggests an all-or-none mechanism for Ca(2)+ mobilization. A transient calcium rise could also be induced by one subthreshold pulse if it was after another subthreshold pulse of the same kind after a suitable interval, i.e., not closer than a few 100 ms and not longer than a few seconds. This dependency of Ca(2)+ mobilization on single and double pulses can be simulated by a model in which a second messenger is produced in a voltage-dependent manner. This second messenger liberates Ca(2)+ from internal stores in an all-or-none manner once a critical concentration (threshold) of the second messenger is exceeded in the cytoplasm. The positive effect of a single suprathreshold pulse and two optimally spaced subthreshold pulses on Ca(2)+ mobilization can be explained on the basis of relative velocity for second messenger production and decomposition as well as the availability of the precursor for the second messenger production. Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.

Action Potentials↗
PubMed2001

Biological activity of mammalian transcriptional repressors.

Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'krüppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position'. Thus, these domains are 'general' transcriptional repressor domains. The 'krüppel-associated box' was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.

Animals↗
PubMed2001

The extracellular signal-regulated protein kinases Erk1/Erk2 stimulate expression and biological activity of the transcriptional regulator Egr-1.

The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation. We show here that a constitutive active mutant of mitogen-activated kinase kinase-1 (MAPKK-1) strongly stimulates the activity of the Egr-1 promoter, thus explaining the effects of mitogens upon Egr-1 mRNA and protein levels. Moreover, we show that a constitutive active MAPKK-1 leads to an increase in the biological activity of Egr-1 to activate transcription. We conclude that the signaling pathway involving mitogen-activated protein kinase/extracellular signal-regulated protein kinase has a dual impact on the biology of Egr-1 by controlling the transcription of the Egr-1 gene and the transcriptional activity of the Egr-1 protein.

DNA-Binding Proteins↗
PubMed2000

The human transcriptional repressor protein NAB1: expression and biological activity.

The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation. Egr-1 has a large activating domain and three zinc finger motifs that function as a DNA binding region. We show here that a third functional domain of the Egr-1 protein, localized between the extended activation domain and the zinc finger DNA binding region, acts as a transcriptional repressor domain when fused to a heterologous DNA binding domain (DBD). Through protein-protein interaction this inhibitory domain of Egr-1 brings the transcriptional corepressor NAB1 in close proximity to the transcription unit. NAB1 is expressed ubiquitously in human cell lines as shown by RNase protection mapping. Overexpression studies revealed that NAB1 is able to completely block transcription mediated by Egr-1. In addition, the transcriptional repression activity of a fusion protein containing the inhibitory domain of Egr-1 and the DBD of the yeast transcription factor GAL4 was increased by overexpression of NAB1. A fusion protein consisting of the DBD of GAL4 and the coding region of human NAB1 repressed transcription from model promoters with engineered upstream GAL4 binding sites. The GAL4-NAB1 fusion protein functioned from proximal and distal positions indicating that NAB1 displays transcriptional repressor activity at any position within the transcription unit. Thus, the biological function of the inhibitory domain of Egr-1 is solely to provide a docking site for NAB1 via protein-protein interaction.

Binding Sites↗
PubMed2000

A common set of at least 11 functional genes is lost in the majority of NF1 patients with gross deletions.

Large deletions of the NF1 locus occur in 5 to 10% of patients with neurofibromatosis and are commonly associated with specific additional abnormalities characterized by mental retardation, dysmorphic features, and intellectual impairment. To characterize the extent of codeleted genes we constructed a long-range physical BAC/PAC map around the NF1 locus between D17S117 and D17S57 and determined the deletion boundaries in seven unrelated patients. Surprisingly, the proximal and distal breakpoints in five of seven patients fall at almost identical positions, resulting in the loss of at least 11 functional genes. Five of six patients investigated showed a de novo deletion on the maternally derived chromosome. Since D17S117 and D17S57 were previously reported as the outer limits for the great majority of NF1 deletions, we suggest that most NF1 patients with deletion of the entire NF1 gene are hemizygous for the same set of at least 10 additional genes, including SHGC-37343, SHGC-2390, SHGC-34232, OMG, EVI2B, EVI2A, WI-9521, WI-6742, SHGC-34334, and KIAA0160, and thus present with a relatively uniform clinical phenotype.

Cell Culture Techniques↗
PubMed2000

Testing for polyomavirus type BK DNA in plasma to identify renal-allograft recipients with viral nephropathy.

BACKGROUND: Reactivation of polyomavirus type BK (BK virus) is increasingly recognized as a cause of severe renal-allograft dysfunction. Currently, patients at risk for nephropathy due to infection with the BK virus are identified by the presence of cells containing viral inclusion bodies ("decoy cells") in the urine or by biopsy of allograft tissue. METHODS: In a retrospective analysis, we performed polymerase-chain-reaction assays for BK virus DNA in plasma samples from 9 renal-allograft recipients with BK virus nephropathy; 41 renal-allograft recipients who did not have signs of nephropathy, 16 of whom had decoy cells in the urine; and as immunocompromised controls, 17 patients who had human immunodeficiency virus type 1 (HIV-1) infection (stage C3 according to the classification of the Centers for Disease Control and Prevention) and who had not undergone transplantation. RESULTS: In all nine patients with BK virus nephropathy, BK virus DNA was detected in the plasma at the time of the initial histologic diagnosis (a mean [+/-SD] of 46+/-28 weeks after transplantation) and during the course of histologically diagnosed, persistent BK virus disease. In three of the six patients with nephropathy who were studied serially after transplantation, BK virus DNA was initially undetectable but was detected 16 to 33 weeks before nephropathy became clinically evident and was confirmed by biopsy. Tests for BK virus DNA in plasma became negative and the nephropathy resolved after the doses of immunosuppressive drugs were decreased in two patients and after removal of the renal allograft in three patients. BK virus DNA was found in the plasma of only 2 of the 41 renal-allograft recipients who had no signs of nephropathy and in none of the patients with HIV-1 infection. CONCLUSIONS: Testing for BK virus DNA in plasma from renal-allograft recipients with use of the polymerase chain reaction is a sensitive and specific method for identifying viral nephropathy.

BK Virus↗
PubMed2000

A potassium channel protein encoded by chlorella virus PBCV-1.

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

Amantadine↗
PubMed2000

Transient and permanent fusion of vesicles in Zea mays coleoptile protoplasts measured in the cell-attached configuration.

Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (Cm). Vesicle fusion was observed as (i) An irreversible step increase in Cm. (ii) Occasionally, irreversible Cm steps were preceded by transient changes in Cm, suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in Cm did not lead to an irreversible Cm step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction.

Cell Membrane↗
PubMed2000

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm.

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Cell Membrane↗
PubMed2000

Magnetic detection of injury-induced ionic currents in bean plants.

A superconducting quantum interference device (SQUID) multichannel magnetometer was used to measure the temporal and spatial evolution of the magnetic field accompanying stimulation by burning and/or cutting of Vicia faba plants. These magnetic fields are caused by ionic currents that appear after injury in different parts of the plant. All measured V. faba plants responded to the burning stimulation with detectable quasi-d.c. magnetic signals. In order to measure these signals, a suitable modulation had to be used. The covariance method was applied to analyse the measured data. The results demonstrate a dipolar-like magnetic signal, exponentially decreasing in time, above the cutting type of injury. After the burning stimulation, the magnetically detected activity was concentrated predominantly above the leaves/petioles and less above the stem. Possible mechanisms for this behaviour are suggested. A comparison with previously known electrical measurements of plant injury is given.

Electric Stimulation↗
PubMed2000

An unusual case of leukemic non-Hodgkin's lymphoma with blastic transformation.

We report on a patient who was diagnosed as having B-cell chronic lymphocytic leukemia (CLL) with atypical morphology. Flow cytometry disclosed CD5, CD19, and CD23 positivity, an immunophenotype seen mostly in B-CLL. Histology of the spleen and bone marrow suggested a diagnosis of small lymphocytic lymphoma. Upon blastic transformation, only 3 years after the diagnosis had been made, unusual clinical and laboratory features emerged. Lymphoid blasts appeared in the peripheral blood, and the patient developed nodular infiltrates consisting of these blasts at recent venous puncture sites. The patient did not respond to chemotherapy and died. The lymphoid blasts in the peripheral blood were CD5-, CD19+, and CD23+ and harbored t(11;14) (q13;q32) and t(11;21)(p11;q21) translocations. To account for the possibility of two independent lymphoid malignancies, molecular genetic analyses were performed on samples from the spleen, bone marrow and a lymph node with the large-cell lymphoma, which showed identical clones in these tissues. This unusual case supports the idea that in leukemic non-Hodgkin's lymphoma, in addition to morphology, an accurate diagnostic workup requires immunophenotypic, cytogenetic, and molecular studies.

CD5 Antigens↗
PubMed2000

Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel.

Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K(+) currents were reduced in a largely scalar fashion, the NH(4)(+) current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA(+)) to the bath. This stresses that both the mutation and MA(+) affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K(+) and NH(4)(+) passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is approximately 30 times smaller in T256Q compared to wt. It can be inhibited by external MA(+) and is stimulated strongly by K(+) and weakly by NH(4)(+). This sensitivity of gating to external cations may prevent K(+) leakage from cation-starved cells.

Amino Acid Substitution↗
PubMed2000

Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type 1 (NF1) gene.

Segmental neurofibromatosis (NF) is generally thought to result from a postzygotic NF1 (neurofibromatosis type 1) gene mutation. However, this has not yet been demonstrated at the molecular level. Using fluorescence in situ hybridisation (FISH) we identified an NF1 microdeletion in a patient with segmental NF in whom café-au-lait spots and freckles are limited to a single body region. The mutant allele was present in a mosaic pattern in cultured fibroblasts from a café-au-lait spot lesion, but was absent in fibroblasts from normal skin as well as in peripheral blood leukocytes. These findings prove the hypothesis that the molecular basis of segmental cutaneous NF is a mutation in the NF1 gene and that the regional distribution of manifestations reflects different cell clones, commensurate with the concept of somatic mosaicism.

Adolescent↗
PubMed2000

Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A.

UNLABELLED: Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A. BACKGROUND: Endothelins may play an important role in cyclosporine A (CsA)-induced renal vasoconstriction. Therefore, the effects of a mixed endothelin A and B receptor antagonist, bosentan (BO), on CsA were studied. METHODS: BO was given either alone or combined with CsA to healthy subjects in a double-blind, placebo-controlled, cross-over study. Standardized renal hemodynamics took place after a single dose of BO or placebo and after seven days of regular intake of CsA + BO or CsA + placebo. CsA was administered as a dose-adjusted regimen to achieve predetermined target trough levels. A pharmacokinetic study of CsA and BO was performed. RESULTS: A single dose of BO did not affect renal hemodynamics. After seven days of coadministration with CsA, BO significantly attenuated both the overall CsA-induced fall of renal plasma flow (RPF; placebo, 594 +/- 85; CsA + placebo, 490 +/- 93; CsA + BO, 570 +/- 106* mL/min, *P < 0.01) and the maximal RPF fall (P < 0.01) observed five hours after CsA intake. The CsA-induced rise of blood pressure and the decrease of glomerular filtration rate (GFR) were not influenced by comedication with BO. After seven days of CsA + BO, the area under the curve (AUC) of BO was nearly doubled compared with the AUC after a single dose of BO (P < 0.05). To reach the CsA target trough levels after seven days, the average CsA dose was increased by 35% when given with BO, as compared with placebo (P = 0.01). CsA exposure (trough levels, AUC) was not statistically different after CsA + placebo and after CsA + BO. CONCLUSIONS: Assuming CsA nephrotoxicity is mainly due to vasoconstriction, BO has the potential to attenuate the CsA renal toxicity by markedly blunting the renal hypoperfusion effect of CsA. A complex drug interaction between BO and CsA was observed.

Adult↗
PubMed2000

Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells.

PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell. We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se. Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription. First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK). This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF. The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction. Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters. We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription. The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type. EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.

Activating Transcription Factor 2↗
PubMed2000

BK-virus nephropathy in renal transplants-tubular necrosis, MHC-class II expression and rejection in a puzzling game.

We review BK-virus nephropathy (BKN) as a new complication that increasingly affects renal allografts and causes dysfunction. Since starting in 1996, we have seen 11 cases. Currently, the prevalence of BKN is 3% in our graft biopsies. The diagnosis can only be made histologically. The virus affects tubular epithelial cells that show characteristic intranuclear inclusion bodies. The major reason for impaired graft function and a possible way for viral particles to gain access to the blood via peritubular capillaries is necrosis of infected epithelial cells. BK-virus DNA in the plasma, which can be detected by PCR, is closely associated with nephropathy. BK-virus does not stimulate tubular MHC-class II expression as judged by immunofluorescence double labelling. The inflammatory response is inconsistent and the frequency of rejection episodes is not increased during disease. Clinical manifestation of viral nephropathy evolves in several stages. (i) Initial, asymptomatic and reversible activation of the virus, judged by the presence of inclusion bearing cells in the urine. (ii) High dose immunosuppressive drug regimens, often including tacrolimus. (iii) Tubular injury and viraemia as additional promoting conditions. BKN nephropathy was associated with graft loss in 45% of our patients. The remaining patients with persistent viral nephropathy showed renal dysfunction (serum creatinine levels on average 150% above baseline readings). Currently, no established antiviral therapy is available. We discuss attempts to lower immunosuppression as a means to control viral replication. We propose a diagnostic algorithm for screening and monitoring the disease.

Animals↗
PubMed2000

Ca(2)+-stimulated exocytosis in maize coleoptile cells.

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.

Calcium↗