Imatinib normalizes bone marrow vascularity in patients with chronic myeloid leukemia in first chronic phase.
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Benzamides↗
Biomedical subjects
Thomas Friedrich
Publications and source records attributed to Thomas Friedrich.
Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.
The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ( approximately 5%) on addition of extracellular K(+). When the enzyme was subjected to voltage jumps under Na(+)Na(+)-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E(1)P-E(2)P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na(+) by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K(+) concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K(+) concentrations. As a result, the stationary distribution between E(1) and E(2) states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na(+)Na(+) exchange as well as Na(+)K(+) turnover conditions.
Adenosine Triphosphate↗
Determination of temperature elevation in tissue during the application of the harmonic scalpel.
The temperature elevation in tissue during the application of the harmonic scalpel (UltraCision, operating frequency 55 kHz; Ethicon Endo-Surgery, Norderstedt, Germany) was determined using thermocouples of a specific design. In experiments with and without perfusion, two different scalpel blades were applied to three different kinds of pig tissue (lung parenchyma, tongue, parotid gland) in various configurations. Temperature elevations by more than 40 degrees C were found at 1 mm distance from the blade, whereas at distances of more than 5 mm, perfusion removed the heat very efficiently. The differences in the heating potential of the two blades were small and, at a distance of 2 mm, the temperature elevation did not exceed 6 degrees C at all. In histological investigations, the damaged area between blade and parenchyma was determined. No morphologic indications of thermal damage were found at a distance of more than 2 mm. It is concluded that, during application of the harmonic scalpel, a safety margin of 3 mm from sensitive structures should be kept.
Animals↗
A carboxy-terminal domain determines the subunit specificity of KCNQ K+ channel assembly.
Mutations in KCNQ K(+) channel genes underlie several human pathologies. KCNQ alpha-subunits form either homotetramers or hetero-oligomers with a restricted subset of other KCNQ alpha-subunits or with KCNE beta-subunits. KCNQ1 assembles with KCNE beta-subunits but not with other KCNQ alpha-subunits. By contrast, KCNQ3 interacts with KCNQ2, KCNQ4 and KCNQ5. Using a chimaeric strategy, we show that a cytoplasmic carboxy-terminal subunit interaction domain (sid) suffices to transfer assembly properties between KCNQ3 and KCNQ1. A chimaera (KCNQ1-sid(Q3)) carrying the si domain of KCNQ3 within the KCNQ1 backbone interacted with KCNQ2, KCNQ3 and KCNQ4 but not with KCNQ1. This interaction was shown by enhancement of KCNQ2 currents, testing for dominant-negative effects of pore mutants, determining its effects on surface expression and co-immunoprecipitation experiments. Conversely, a KCNQ3-sid(Q1) chimaera no longer affects KCNQ2 but interacts with KCNQ1. We conclude that the si domain suffices to determine the subunit specificity of KCNQ channel assembly.
Amino Acid Sequence↗
Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.
We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K(+) that were completely abolished by ouabain. When voltage jumps were applied under Na(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K(+) concentrations, that is, under turnover conditions. As a result, the distribution between E(1) and E(2) states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H(+)/K(+)-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na(+)/K(+)-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.
Amino Acid Sequence↗
Two-electrode voltage-clamp analysis of Na,K-ATPase asparagine 776 mutants.
Steady-state and pre-steady-state currents of Asn(776) mutants of Na,K-ATPase are presented. The stationary current generated by N776Q strongly depends on the membrane potential, but has a negative slope, opposite to that of the wild-type enzyme. The apparent rate constant of the reaction sequence E(1)P(Na(+)) <--> E(2)P + Na(+) of this mutant is rather independent of the membrane potential and is at resting and depolarizing membrane potential higher than that of the wild-type enzyme. Thus, the voltage-dependent increase of the rate coefficient of the wild type that is associated with extracellular Na(+) rebinding is almost absent in the N776Q mutant. These findings indicate that dislocating the carboxamide group of Asn(776) decreases the affinity of sodium at its extracellular binding site.
Alanine↗
Emergence of clonal cytogenetic abnormalities in Ph- cells in some CML patients in cytogenetic remission to imatinib but restoration of polyclonal hematopoiesis in the majority.
Chronic myelogenous leukemia (CML) is characterized by the presence of a Bcr-Abl fusion protein with deregulated tyrosine kinase activity that is required for maintaining the malignant phenotype. Imatinib, a selective inhibitor of Bcr-Abl, induces major cytogenetic remission (MCR) or complete cytogenetic remission (CCR) in the majority of patients with CML in first chronic phase. However, thorough re-evaluation of cytogenetics in a cohort of patients in MCR or CCR demonstrated clonal karyotypic abnormalities in more than 10% of cases, some of which were clinically associated with a myelodysplastic syndrome (MDS). Further analysis identified previous exposure to cytarabine and idarubicin as significant risk factors for the subsequent occurrence of abnormalities in Philadelphia chromosome-negative (Ph-) cells. To investigate if cytogenetically normal but clonal hematopoiesis might be present in other patients in cytogenetic remission, we studied X-chromosome inactivation as a marker of clonality by polymerase chain reaction analysis of the human androgen receptor (HUMARA). We find that imatinib restores a polyclonal pattern in most patients in CCR and MCR. Nonetheless, our results are consistent with the notion that targeted therapy of CML with imatinib favors the manifestation of Ph- clonal disorders in some patients. They indicate that patients on imatinib should be followed with conventional cytogenetics, even after induction of CCR.
Adult↗
Proteorhodopsin is a light-driven proton pump with variable vectoriality.
Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.
Animals↗
Complex cancer gene therapy in mice melanoma.
OBJECTIVE: We investigated the effects of continuous cancer gene therapy, including APC engineering and local stimulation of the immune system in a mice melanoma model. MATERIALS AND METHODS: B16 melanoma cells were injected into C57/Bl6 mice intradermally. The overlying dermis or the tumor were shot with different plasmids using a gene gun every 4th day starting 8 days after tumor implantation. Control groups were mice without any therapy or gene therapy as described above with the empty plasmid. Therapy was: group I, IL-12 and IL-2; group II, IFN-gamma/B7.1; group III, IFN-gamma/B7.1, IL-12, and IL-2. RESULTS: The median survival time of all therapy groups was significantly enhanced. The longest median survival was in the IL-12/IL-2 group. Tumor growth was reduced in all therapy groups. Control groups suffered a higher rate of metastasis and had fewer inflammatory cells at the tumor site. CONCLUSIONS: Continuous therapy with the gene gun is easy to handle and shows good results. Therapy with genes for IL-12 and IL-2 was superior to that with additional IFN-gamma/B7.1 or IFN-gamma/B7.1 alone. APC engineering is clearly less sufficient in the case of the B16 melanoma.
Animals↗
Cutaneous extramedullary hematopoiesis in idiopathic myelofibrosis.
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Aged↗
Pheochromocytoma and multifocal functioning paraganglioma in a 9-year-old boy with von Hippel-Lindau disease.
We report the case of a 9-year-old boy presenting with multifocal paraganglioma partially involving the right suprarenal gland, abdominal paraganglia, and the left para-aortal retropleural region. Von Hippel-Lindau disease (VHL) was diagnosed exclusively by VHL-gene analysis. Other clinical signs of VHL were lacking. After a complaint-free period of 7 months the boy developed a metachronous paraganglioma adjacent to the opposite adrenal gland. Patients with VHL disease should undergo adrenal-sparing surgery.
Adrenal Gland Neoplasms↗
Characterization of renal chloride channel (CLCN5) mutations in Dent's disease.
Dent's disease is an X-linked renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. The disease is caused by mutations in a renal chloride channel gene, CLCN5, which encodes a 746 amino acid protein (CLC-5), with 12 to 13 transmembrane domains. In this study, an additional six unrelated patients with Dent's disease were identified and investigated for CLCN5 mutations by DNA sequence analysis of the 11 coding exons of CLCN5. This revealed six mutations: four frameshift deletions involving codons 392, 394, 658, and 728, one nonsense mutation (Tyr617Stop), and an A to T transversion at codon 601 that would result in either a missense mutation (Asp601Val) or creation of a novel donor splice site. These mutations were confirmed by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis and were not common polymorphisms. The frameshift deletions and nonsense mutation predict truncated and inactivated CLC-5. The effects of the putative missense Asp601Val mutant CLC-5 were assessed by heterologous expression in Xenopus oocytes, and this revealed a chloride conductance that was similar to that observed for wild-type CLC-5. However, an analysis of the mutant CLCN5 transcripts revealed utilization of the novel donor splice site, resulting in a truncated CLC-5. Thus, all of the six mutations are likely to result in truncated CLC-5 and a loss of function, and these findings expand the spectrum of CLCN5 mutations associated with Dent's disease.
Adult↗