ABSTRACT
OBJECTIVES: While permanent retention is today the method of choice to stabilize orthodontic treatment outcomes, recent studies have increasingly reported posttreatment changes in tooth position during permanent retention. We conducted this study to analyze changes in the anterior mandible, whether the changes follow an underlying movement pattern, and, aiming for a preventive strategy, whether any risk factors could be identified comparing findings with the pretreatment situations. METHODS: We included 30 patients who had worn fixed Twistflex retainers (UK 3-3) extending from canine to canine in the mandible. Casts reflecting the intraoral situations before orthodontic treatment (T0), directly after completion of active therapy (T1), and 6 months later (T2) were scanned and superimposed using Imageware Surfacer software. Posttreatment changes (T2-T1) of tooth position within the retainer block were analyzed on 3D virtual models and were compared to pretreatment (T0) and treatment-related (T1-T0) findings to identify potential risk factors. RESULTS: Almost all analyzed patients revealed three-dimensional changes in tooth position within the retainer block. Comparing these movements, we repeatedly found rotated retainer blocks in labio-oral direction, while the center of rotation was located at the first incisors. This pattern was associated with intercanine expansion and excessive overjet correction during orthodontic treatment. The canines underwent the most pronounced (rotational and translational) movements. CONCLUSIONS: In general permanent lingual retainers are safe but in special clinical cases retainers can induce undesired tooth movement. Risk factors seem to be intercanine expansion and excessive overjet correction during orthodontic treatment. In specific cases an additional retention device might be needed.
Subject(s)
Dental Restoration Failure , Malocclusion/etiology , Malocclusion/prevention & control , Orthodontic Retainers/adverse effects , Tooth Mobility/pathology , Tooth Mobility/prevention & control , Adult , Cephalometry , Female , Humans , Male , Malocclusion/pathology , Mandible/pathology , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Tooth Mobility/etiology , Treatment OutcomeABSTRACT
In this study pentatricopeptide repeat (PPR) proteins in filamentous ascomycetes are identified and functionally characterized. PPR proteins, which have in common a degenerated 35 amino acid motif often arranged in multiple tandems, are known to be implicated in various steps of RNA metabolism in mitochondria and chloroplasts. In filamentous ascomycetes we identified a common set of nine PPR proteins. For seven of these proteins, which were not yet characterized, knockout mutants of Neurospora crassa were analyzed. The knockout of three genes appeared to be lethal while four mutants showed different degrees of alterations in respiratory chain complexes. Two mutants are specifically affected in the assembly of a functional complex I while the other enzymes of the respiratory chain are present. Both mutants demonstrate the presence of a peripheral arm and the absence of a detectable membrane arm. Analysis of the mitochondrial RNA revealed distinct alterations of the transcript patterns for certain complex I subunits. Synthesis and/or stability of the transcript for ND2-ND3 is grossly impaired in one mutant while in the other mutant splicing of the transcript for ND1-ND4 is hampered. Our analysis provides the basis for a comprehensive characterization of PPR proteins in filamentous ascomycetes.
Subject(s)
Ascomycota/metabolism , Electron Transport Complex I/metabolism , Mitochondrial Proteins/metabolism , Protein Interaction Domains and Motifs , Ascomycota/genetics , Electron Transport Complex I/genetics , Gene Knockdown Techniques , Genes, Mitochondrial , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mutation , Protein Subunits/genetics , Transcription, GeneticABSTRACT
The hydrochemistry and the microbial diversity of a pristine aquifer system near Garzweiler, Germany, were characterized. Hydrogeochemical and isotopic data indicate a recent activity of sulfate-reducing bacteria in the Tertiary marine sands. The community structure in the aquifer was studied by fluorescence in situ hybridization (FISH). Up to 7.3 x 10(5) cells/mL were detected by DAPI-staining. Bacteria (identified by the probe EUB338) were dominant, representing 51.9% of the total cell number (DAPI). Another 25.7% of total cell were affiliated with the domain Archaea as identified by the probe ARCH915. Within the domain Bacteria, the beta-Proteobacteria were most abundant (21.0% of total cell counts). Using genus-specific probes for sulfate-reducing bacteria (SRB), 2.5% of the total cells were identified as members of the genus Desulfotomaculum. This reflects the predominant role these microorganisms have been found to play in sulfate-reducing zones of aquifers at other sites. Previously, all SRB cultured from this site were from the spore-forming genera Desulfotomaculum and Desulfosporosinus.
Subject(s)
Desulfotomaculum/physiology , Ecosystem , Fresh Water/microbiology , Phylogeny , Water Microbiology , Archaea/genetics , Archaea/physiology , Desulfotomaculum/genetics , Fresh Water/chemistry , Germany , In Situ Hybridization, Fluorescence , Likelihood Functions , Models, Genetic , Oligonucleotides , Sequence Analysis, DNAABSTRACT
Proteins specifically involved in the biogenesis of respiratory complex I in eukaryotes have been characterized. The complex I intermediate associated proteins CIA30 and CIA84 are tightly bound to an assembly intermediate of the membrane arm. Like chaperones, they are involved in multiple rounds of membrane arm assembly without being part of the mature structure. Two biosynthetic subunits of eukaryotic complex I have been characterized. The acyl carrier subunit is needed for proper assembly of the peripheral arm as well as the membrane arm of complex I. It may interact with enzymes of a mitochondrial fatty acid synthetase. The 39/40-kDa subunit appears to be an isomerase with a tightly bound NADPH. It is related to a protein family of reductases/isomerases. Both subunits have been discussed to be involved in the synthesis of a postulated, novel, high-potential redox group.
Subject(s)
NADH, NADPH Oxidoreductases/biosynthesis , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/genetics , Electron Transport Complex I , Genes, Fungal , Mitochondria/enzymology , Models, Molecular , Mutation , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , Neurospora crassa/enzymology , Neurospora crassa/genetics , Oxidation-Reduction , Phylogeny , Protein SubunitsABSTRACT
1. We have used giant patch-clamp recording to investigate the interaction between pH gating and K(+)-dependent gating in rat K(ir)1.1 (ROMK) channels heterologously expressed in Xenopus oocytes. 2. Gating by intracellular protons (pH gating) and extracellular K(+) ions (K(+)-dependent gating) is a hallmark of K(ir)1.1 channels that mediate K(+) secretion and control NaCl reabsorption in the kidney. pH gating is driven by protonation of an intracellular lysine residue (K80 in K(ir)1.1). K(+)-dependent gating occurs upon withdrawal of K(+) ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterically. 3. K(+)-dependent gating was shown to be strictly coupled to pH gating; it only occurred when channels were in the pH-inactivated closed state, but not in the open state. Moreover, K(+)-dependent gating was absent in the non-pH-gated mutant K(ir)1.1(K80 M). 4. Channels inactivated by K(+)-dependent gating were reactivated upon addition of permeant ions to the extracellular side of the membrane, while impermeant ions failed to induce channel reactivation. Moreover, mutagenesis identified two residues in the P-helix (L136 and V140 in K(ir)1.1) that are crucial for K(+)-dependent gating. Replacement of these residues with the ones present in the non-K(+)-gated K(ir)2.1 abolished K(+)-dependent gating of K(ir)1.1 channels without affecting pH gating. 5. The results indicate that pH gating and K(+)-dependent gating are coupled to each other via structural rearrangements in the inner pore involving the P-helix.
Subject(s)
Hydrogen/metabolism , Ion Channel Gating/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Potassium/physiology , Amino Acid Sequence/genetics , Animals , Electric Conductivity , Hydrogen-Ion Concentration , Ions , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Oocytes , Patch-Clamp Techniques , Potassium Channels/chemistry , Potassium Channels/genetics , XenopusABSTRACT
Development of cytokine resistance is an important feature of melanoma cells during tumor progression. To study the mechanisms of interleukin-6 resistance, we examined an interleukin-6 sensitive (WM35) and an interleukin-6 unresponsive cell line (WM9). Interleukin-6 treatment resulted in rapid inhibition of cyclin-dependent kinase 2/cyclin E activity and accumulation of the hypophosphorylated retinoblastoma protein in WM35 but not in WM9 cells. In contrast to previous reports, no differences in the expression of the cyclin-dependent kinase 2 inhibitor p21Cip1/WAF1 upon interleukin-6 treatment were found in both cell lines. Interleukin-6-induced inhibition of cyclin-dependent kinase 2 was also not due to changes in protein expression of cyclin-dependent kinase 2, cyclin E, p27Kip1 and cdc25A, a phosphatase positively regulating cyclin-dependent kinase 2 activity. As it is established that interleukin-6 resistance of WM9 cells is not caused by differential interleukin-6 receptor expression, we studied whether this is due to defective interleukin-6 signaling in which activation of signal transducer and activator of transcription 3 is a critical step. WM9 cells showed reduced tyrosine phosphorylation, DNA binding, and delayed nuclear translocation of signal transducer and activator of transcription 3 as compared with WM35 cells. The kinase upstream of signal transducer and activator of transcription 3, Janus kinase 1, was constitutively tyrosine-phosphorylated in WM9 cells and did not respond to interleukin-6 with increased phosphorylation. As compared with WM35 cells, interleukin-6 treatment of WM9 cells was not paralleled by reduced activity of the mitogen-activated protein kinase kinase-1, which suppresses activation of signal transducer and activator of transcription 3. Our data suggest that resistance of advanced melanoma cells to interleukin-6 is associated with reduced inhibition of cyclin-dependent kinase 2, which appears to be a consequence of a complex alteration in interleukin-6 signal transduction.
Subject(s)
CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/metabolism , Interleukin-6/pharmacology , Melanoma , Skin Neoplasms , Trans-Activators/metabolism , Tumor Suppressor Proteins , Cell Cycle Proteins/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclins/metabolism , DNA-Binding Proteins/analysis , G1 Phase/drug effects , G1 Phase/physiology , Humans , Janus Kinase 1 , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Retinoblastoma Protein/metabolism , STAT3 Transcription Factor , Signal Transduction/drug effects , Signal Transduction/physiology , Trans-Activators/analysis , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology , Tyrosine/metabolism , cdc25 Phosphatases/metabolismABSTRACT
Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.
Subject(s)
Bicarbonates/metabolism , Chlorides/metabolism , Hair Cells, Auditory, Outer/physiology , Proteins/metabolism , Amino Acid Substitution , Animals , Anion Transport Proteins , Anions/pharmacology , Bicarbonates/pharmacology , CHO Cells , Cations/pharmacology , Cell Membrane/metabolism , Chlorides/pharmacology , Cricetinae , Electric Conductivity , Electrophysiology , Models, Biological , Mutation , Patch-Clamp Techniques , Protein Conformation , Proteins/chemistry , Proteins/genetics , Rats , Sulfate TransportersABSTRACT
As part of the German Neurospora crassa genome project, physical clone maps of linkage groups II and V of N. crassa were generated by hybridization-based mapping. To this end, two different types of clone library were used: (1) a bacterial artificial clone library of 15-fold genome coverage and an average insert size of 69 kb, and (2) three cosmid libraries--each cloned in a different vector--with 17-fold coverage and 34 kb average insert size. For analysis, the libraries were arrayed on filters. At the first stage, chromosome-specific sublibraries were selected by hybridization of the respective chromosomal DNA fragments isolated from pulsed-field electrophoresis gels. Subsequently, the sublibraries were exhaustively ordered by single clone hybridizations. Eventually, the global libraries were used again for gap filling. By this means, physical maps were generated that consist of 13 and 21 contigs, respectively, and form the basis of the current sequencing effort on the two chromosomes.
Subject(s)
Genetic Linkage , Neurospora crassa/genetics , Nucleic Acid Hybridization , Chromosomes/genetics , Contig Mapping , Cosmids , DNA/genetics , Gene Library , Physical Chromosome MappingABSTRACT
In a combined isotope geochemical and microbiological investigation, a setting of multiple aquifers was characterized. Biologically mediated redox processes were observed in the aquifers situated in marine sands of Tertiary age and overlying Quaternary gravel deposits. Intercalated lignite seams define the aquitards, which separate the aquifers. Bacterial oxidation of organic matter is evident from dissolved inorganic carbon characterized by average carbon isotope values between ?18.4 per thousand and ?15.7 per thousand (PDB). Strongly positive sulfur isotope values of up to +50 per thousand (CTD) for residual sulfate indicate sulfate reduction under closed system conditions with respect to sulfate availability. Both, hydrochemical and isotope data are thus consistent with the recent activity of sulfate-reducing bacteria (SRB). Microbiological investigations revealed the presence of an anaerobic food chain in the aquifers. Most-probable-number (MPN) determinations for SRB and fermenting microorganisms reached highest values at the interface between aquifer and lignite seam (1.5 x 103 cells/g sediment dry mass). Five strains of SRB were isolated from highest MPN dilutions. Spore-forming bacteria appeared to dominate the SRB population. Sulfate reduction rates were determined by the 35S-radiotracer method. A detailed assessment indicates an increase in the reduction rate in proximity to the lignite seam, with a maximum turnover of 8.4 mM sulfate/a, suggesting that lignite-drived compounds represent the substrate for sulfate reduction.
ABSTRACT
OBJECTIVE: The purpose of this work was to establish a low-cost device for simple positioning of several small animals within a whole-body PET scanner. METHODS: The device was designed as a stackable shelf for 3 x 3 animals, similar to a stackable shelf for wine bottles. It was constructed from ordinary PVC drain pipe and acrylic panes. RESULTS: The shelf simplified accurate and reproducible positioning of the animals and, therefore, supported automatic data processing. Deterioration of image quality by attenuation of photons within the shelf itself was rather small. CONCLUSION: The small-animal shelf is a useful, low-cost device for simultaneously assessing up to 9 small animals with a whole-body PET scanner.
Subject(s)
Environment, Controlled , Housing, Animal , Tomography, Emission-Computed/instrumentation , Acrylic Resins , Animals , Equipment Design , Fluorodeoxyglucose F18 , Housing, Animal/economics , Image Enhancement , Image Processing, Computer-Assisted , Polyvinyl Chloride , Posture , Radiopharmaceuticals , Rats , Reproducibility of Results , Tomography, Emission-Computed/economicsABSTRACT
The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.
Subject(s)
NADH, NADPH Oxidoreductases/chemistry , Electrochemistry , Electron Spin Resonance Spectroscopy , Electron Transport Complex I , Escherichia coli , Iron-Sulfur Proteins/chemistry , Mitochondria/chemistry , Neurospora crassa , Oxidation-Reduction , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform InfraredABSTRACT
Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.
Subject(s)
Hydrogen-Ion Concentration , Ion Channel Gating/physiology , Potassium Channels/physiology , Potassium/metabolism , Animals , Cations/metabolism , Chlorides/metabolism , Cloning, Molecular , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Infant, Newborn , Intracellular Fluid/chemistry , Ion Channel Gating/drug effects , Ion Transport/drug effects , Kidney/metabolism , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/metabolism , Models, Molecular , Point Mutation , Potassium Channels/chemistry , Potassium Channels/drug effects , Potassium Channels/genetics , Protein Conformation , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Sulfhydryl Reagents/pharmacology , Syndrome , XenopusABSTRACT
The proopiomelanocortin (POMC) products alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and In situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvascular endothelial cells and WM35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of sweat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated keratinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.
Subject(s)
Receptors, Corticotropin/immunology , Receptors, Corticotropin/metabolism , Skin/immunology , Skin/metabolism , Adult , Antigens/metabolism , Cell Line , Cells, Cultured , Fetus/immunology , Fetus/metabolism , Humans , Immunohistochemistry , Infant, Newborn , Keratinocytes/immunology , Keratinocytes/metabolism , Melanocytes/immunology , Melanocytes/metabolism , Receptors, Melanocortin , Skin/cytologyABSTRACT
Inward-rectifier K(+) channels of the ROMK (K(ir)1.1) subtype are responsible for K(+) secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K(ir) channels. Such anomalous titration of this lysine residue (Lys-80 in K(ir)1.1) is accomplished by the tertiary structure of the K(ir) protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K(ir)1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK(a) into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK(a) of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K(ir) channels as well as for the channel defects found in patients with antenatal Bartter syndrome.
Subject(s)
Arginine/genetics , Bartter Syndrome/metabolism , Ion Channel Gating , Lysine/genetics , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Bartter Syndrome/congenital , Bartter Syndrome/genetics , Binding Sites , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Lysine/chemistry , Mutation , Potassium Channels/genetics , TitrimetryABSTRACT
Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.
Subject(s)
Mitochondria/enzymology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , NADH, NADPH Oxidoreductases/chemistry , NADP/chemistry , Neurospora crassa/enzymology , Electron Spin Resonance Spectroscopy , Electron Transport , Electron Transport Complex I , Electrophoresis, Polyacrylamide Gel , Gene Deletion , Kinetics , NAD/chemistry , NADH, NADPH Oxidoreductases/genetics , Neurospora crassa/genetics , Oxidation-Reduction , Phylogeny , Protein Binding , SpectrophotometryABSTRACT
AIM: Lesion detection and localization of 2-[18F]fluoro-2-deoxy-D-glucose (F-18-FDG) Onco-PET-Investigations are usually performed on-line at the computer display. The aim of the present study was to evaluate the clinical efficacy of a standardized film documentation as an alternative approach. METHODS: 100 Onco-PET-investigations without attenuation correction were analyzed with regard to number and localization of lesions suspicious of malignancy. A standardized documentation on film was developed including 1. transversal slices of the brain, 2. coronal slices and maximum-intensity-projections (MIPs) of the head/neck region and 3. of the trunk and 4. MIPs of the legs. These transparencies were analyzed at the light box. An additional analysis on the computer display was performed slice by slice in coronal, transversal and sagittal directions for the whole body. RESULTS: A total of 315 lesions were detected in 100 patients. In 96/100 patients the two modalities agreed both in number and localization of tumor-suspicious lesions. 7 lesions in the legs of 3 patients didn't show when interpreting the films (MIPs only). In 2/100 patients additional analysis on the computer display caused a change in the localization of 9/315 lesions. 8 of these were located in the legs. When adding coronal slices for the documentation of the lower extremities all the lesions were shown. Moreover, all lesions were localized correctly except one clinically non-relevant change of localization out of a total of 322 lesions. CONCLUSION: The newly developed standardized documentation supports the concept of film reading and reporting of onco-PET investigations, restricting an additional on-line analysis to rare cases only. Furthermore, the intention of the "Arbeitsgemeinschaft Standardisierung" (work group standardisation) are met, i.e. to ease analysis of follow-up studies acquired at different places.
Subject(s)
Fluorodeoxyglucose F18 , Image Processing, Computer-Assisted/methods , Neoplasms/diagnostic imaging , Radiopharmaceuticals , Tomography, Emission-Computed , Documentation/methods , Female , Humans , Lymphoma/diagnostic imaging , Online SystemsABSTRACT
Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH.
Subject(s)
ATP-Binding Cassette Transporters , Myocardium/metabolism , Polyamines/pharmacology , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Receptors, Drug/metabolism , Animals , Cells, Cultured , Hydrogen-Ion Concentration , Mice , Mutagenesis/genetics , Mutation , Patch-Clamp Techniques , Potassium Channels/genetics , Spermidine/pharmacology , Sulfonylurea ReceptorsABSTRACT
Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.
Subject(s)
Dogfish/physiology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , Salt Gland/physiology , Animals , Blotting, Northern , Cloning, Molecular , Electric Stimulation , Electrophysiology , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Oocytes/metabolism , Patch-Clamp Techniques , Potassium Channels/biosynthesis , Potassium Channels/chemistry , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Salt Gland/metabolism , Xenopus laevisABSTRACT
Alpha-melanocyte-stimulating hormone (alpha-MSH) has evolved as a mediator of diverse biological activities in an ever-growing number of non-melanocytic cell types. One mechanism by which alpha-MSH exerts its effects is modulation of AP-1 and NF-kappa B. These two transcription factors also play an important role in fibroblasts, in extracellular matrix composition, and in cytokine expression. By use of electric mobility shift assays, we demonstrate that alpha-MSH (10(-6) to 10(-14) M) activates AP-1 in human dermal fibroblasts, whereas coincubation with interleukin-1 beta (IL-1 beta) results in suppression of its activation. alpha-MSH also induces activation of NF-kappa B but does not modulate DNA binding on costimulation with IL-1 beta. Since AP-1 and NF-kappa B are key elements in controlling interleukin-8 (IL-8) transcription, human fibroblasts were treated with alpha-MSH and IL-1 beta for 24 hours, and cytokine levels in the supernatants were measured by ELISA. alpha-MSH alone had little effect, whereas coincubation with IL-1 beta led to marked downregulation of IL-8 secretion (at most 288 +/- 152 ng/mL) when compared to treatment with IL-1 beta alone (919 +/- 157 ng/mL). Our results indicate that alpha-MSH exerts modulatory effects on the activation of NF-kappa B and AP-1, and that it can regulate chemokine secretion in human dermal fibroblasts. These effects of alpha-MSH may have important regulatory functions in extracellular matrix composition, wound healing, or angiogenesis.
Subject(s)
Fibroblasts/physiology , Interleukin-8/genetics , NF-kappa B/metabolism , Skin/cytology , Transcription Factor AP-1/metabolism , Transcription, Genetic/physiology , alpha-MSH/pharmacology , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Infant, Newborn , Interleukin-1/pharmacology , Interleukin-8/metabolism , Kinetics , Male , Transcription, Genetic/drug effectsABSTRACT
We have generated a polyclonal antibody raised against a synthetic peptide corresponding to the amino acids 2-18 of the extracellular, N-terminal domain of the human melanocortin-1 receptor (MC-1R). Specificity of the affinity-purified anti-MC-1R antibody was confirmed by dot blot analysis with the antigenic peptide. The antibody detected MC-1R antigenicity on the surface of normal human melanocytes and WM35 melanoma cells, as shown by FACS and immunofluorescence analysis. The antibody was suitable for immunoperoxidase staining of deparaffinized skin sections, revealing prominent MC-1R staining of a cutaneous melanoma as opposed to undiseased skin in which normal melanocytes were only occasionally immunoreactive. Distinct adnexal structures in normal skin also displayed MC-1R immunostaining. Specificity of the MC-1R immunoreactivity in each technique was confirmed by preabsorption with the immunogenic peptide, omission, or substitution of the primary antibody with preimmune serum. Our results provide a baseline for future studies on MC-1R expression in diseased human skin.