[Characteristics of hearing aid management in infancy, early childhood and preschool age].
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Auditory Threshold↗
Biomedical subjects
S Fleischer
Publications and source records attributed to S Fleischer.
[Forced inspiration. A useful criterium in diagnosis of Reinke edema?].
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Female↗
Electron tomography of frozen-hydrated isolated triad junctions.
Cryoelectron microscopy and tomography have been applied for the first time to isolated, frozen-hydrated skeletal muscle triad junctions (triads) and terminal cisternae (TC) vesicles derived from sarcoplasmic reticulum. Isolated triads were selected on the basis of their appearance as two spherical TC vesicles attached to opposite sides of a flattened vesicle derived from a transverse tubule (TT). Foot structures (ryanodine receptors) were resolved within the gap between the TC vesicles and TT vesicles, and some residual ordering of the receptors into arrays was apparent. Organized dense layers, apparently containing the calcium-binding protein calsequestrin, were found in the lumen of TC vesicles underlying the foot structures. The lamellar regions did not directly contact the sarcoplasmic reticulum membrane, thereby creating an approximately 5-nm-thick zone that potentially constitutes a subcompartment for achieving locally elevated [Ca(2+) ] in the immediate vicinity of the Ca(2+)-conducting ryanodine receptors. The lumen of the TT vesicles contained globular mass densities of unknown origin, some of which form cross-bridges that may be responsible for the flattened appearance of the transverse tubules when viewed in cross-section. The spatial relationships among the TT membrane, ryanodine receptors, and calsequestrin-containing assemblage are revealed under conditions that do not use dehydration, heavy-metal staining, or chemical fixation, thus exemplifying the potential of cryoelectron microscopy and tomography to reveal structural detail of complex subcellular structures.
Animals↗
Wetland management to reduce Baltic Sea eutrophication.
Seven regions with coastal eutrophication problems in the Baltic Sea, including the Kattegat, constitute the BERNET project (Baltic Eutrophication Regional Network). To counteract eutrophication and associated severe biological conditions the countries around this large brackish water body must all cooperate. The regions are characterized by large differences in land use, e.g. agricultural intensity, and losses of retention capacity in the catchments due to wetland reclamation. Initially it has been necessary to identify nutrient sources--especially nitrogen--and technical, economical and even administrative obstacles to initiate eutrophication management measures. Nitrogen retention in different types of wetlands in the Baltic Sea Region has been analysed. The wetlands generally have a positive effect on reduced nitrogen transport to aquatic environments and it is generally accepted that measures leading to decreased losses of nutrients to the aquatic environment must be combined with measures leading to increased retention of nutrients in catchments. Data analysed in the BERNET project show that the potential for such a measure is large. Therefore, conservation and restoration initiatives for wetlands is an essential part of the work in the BERNET project. Wetlands have been drained or totally eliminated due to intensive agriculture in some regions while large scale rehabilitation of wetlands occurs in regions with less intensive agriculture. Based on land use data from the seven regions, the working group for wetland management within the BERNET project has identified the possible use of wetlands as building blocks as a contribution to the management of the Baltic Sea eutrophication. Several recommendations are presented on the wise use of existing and constructed wetlands for water quality management in relation to non-point nutrient pollution.
Agriculture↗
Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes.
1. ATP-mediated Ca2+ signalling was studied in freshly isolated rat portal vein myocytes by means of a laser confocal microscope and the patch-clamp technique. 2. In vascular myocytes held at -60 mV, ATP induced a large inward current that was supported mainly by activation of P2X1 receptors, although other P2X receptor subtypes (P2X3, P2X4 and P2X5) were revealed by reverse transcription-polymerase chain reaction. 3. Confocal Ca2+ measurements revealed that ATP-mediated Ca2+ responses started at initiation sites where spontaneous or triggered Ca2+ sparks were not detected, whereas membrane depolarizations triggered Ca2+ waves by repetitive activation of Ca2+ sparks from a single initiation site. 4. ATP-mediated Ca2+ responses depended on Ca2+ influx through non-selective cation channels that activated, in turn, Ca2+ release from the intracellular store via ryanodine receptors (RYRs). Using specific antibodies directed against the RYR subtypes, we show that ATP-mediated Ca2+ release requires, at least, RYR2, but not RYR3. 5. Our results suggest that, in vascular myocytes, Ca2+ influx through P2X1 receptors may trigger Ca2+-induced Ca2+ release at intracellular sites where RYRs are not clustered.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
FKBP binding characteristics of cardiac microsomes from diverse vertebrates.
FK506 binding protein (FKBP) is a cytosolic receptor for the immunosuppressive drug FK-506. The common isoform, FKBP12, was found to be associated with the calcium release channel (ryanodine receptor 1) of different species of vertebrate skeletal muscle, whereas 12.6, a novel FKBP isoform was found to be associated with canine cardiac ryanodine receptor (ryanodine receptor 2). Until recently, canine cardiac sarcoplasmic reticulum was considered to be the prototype for studying heart RyR2 and its interactions with FKBP. In this study, cardiac microsomes were isolated from diverse vertebrates: human, rabbit, rat, mice, dog, chicken, frog, and fish and were analyzed for their ability to bind or exchange with FKBP isoforms 12 and 12.6. Our studies indicate that RyR2 from seven out of the eight animals contain both FKBP12 and 12.6. Dog is the exception. It can now be concluded that the association of FKBP isoforms with RyR2 is widely conserved in the hearts of different species of vertebrates.
Animals↗
Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes.
Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).
Animals↗
[Noninvasive evaluation of laryngeal function].
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Deglutition↗
Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1).
The skeletal muscle Ca2+ release channel (ryanodine receptor 1, RYR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type 1 from rabbit white muscle and adsorbed it to mica sheets with the cytoplasmic side facing up. Single receptors of uniformly distributed size and shape of 10-12 nm height and 40-50 nm width, and occasionally some aggregates were seen in contact mode AFM images. These immobilized RYR1 were specifically recognized by rabbit anti-RYR1 (antibody#8) with at least 30% efficiency, as measured by an enzyme immunoassay with goat-anti-rabbit. Single specific antibody-antigen recognition events were detected with AFM tips to which an antibody#8 was tethered. In linear scans, the occurrence of antibody-antigen binding showed significant lateral dependence, which allowed for the localization of binding sites with nm resolution. Variation of the loading rate in force spectroscopy experiments revealed a logarithmic dependence of the unbinding forces, ranging from 42 to 73 pN. From this dependence, a bond width of the binding pocket of L = 0.2 nm and a kinetic off-rate of koff = 12.7s(-1) was determined.
Animals↗
Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors.
The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.
Adenosine Diphosphate Ribose↗
Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein.
(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters). Six cysteine mutants each have an increased K(m)(NADH) (2-6-fold) but an unchanged K(m)(NAD)+. The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site. Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.
Catalytic Domain↗
Expression and subcellular localization of cpn60 protein family members in Leishmania donovani.
We have identified two diverged members of the cpn60 gene family in Leishmania donovani, causative agent of Indian Kala Azar. One of the genes, cpn60.1, although actively transcribed, is not expressed to detectable levels of protein in cultured L. donovani. The other gene, cpn60.2, which, compared with cpn60.1, shows a higher sequence conservation with the hsp60 genes from Trypanosoma brucei and Trypanosoma cruzi is expressed constitutively in cultured promastigotes. The abundance of the gene product, Cpn60.2, increases by 2.5-fold under heat stress and in axenic amastigotes of L. donovani. Cpn60.2 is also found enriched in mitochondrial cell fractions and localizes to the mitochondrial matrix. We conclude that Cpn60.2 is the major mitochondrial chaperonin in Leishmania.
Amino Acid Sequence↗
Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy.
Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.
Animals↗
Cardiac sarcalumenin: phosphorylation, comparison with the skeletal muscle sarcalumenin and modulation of ryanodine receptor.
Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca2+ binding protein (HCP) with respect to: (i) Ca2+ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg2+ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La3+; (iv) phosphorylation is obtained with [gamma-32P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La3+. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca2+- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified.
Adenosine Triphosphate↗
Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.
FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.
Amino Acid Sequence↗
[Disabilities and handicaps due to hip fractures in the elderly].
OBJECTIVE: to examine mortality, mobility, pain, institutionalization rates six month after hip fractures. DESIGN: observational study, non-selected consecutive patients, time-set protocol. SETTING: urban area (population n = 116,5000), five hospitals. PARTICIPANTS: hip fracture patients (n = 214), age 65 + years (mean 82.4 years). MEASUREMENTS: age, gender, prefracture status, operative procedures, complications, comorbidity, cognition (MMSE), depression (GDS), fear of falling, ADL (Barthel) and mobility and pain status (SAHFE protocol). RESULTS: the incidence for this age group was 636.9/100,000, 31.8% were institutionalized elderly. 84% of the patients were female. Pre-existing comorbidity showed a high prevalence of neurodegenerative (cognitive deficits 53. 6%, Parkinson's disease/syndrome 11.2%) and cerebrovascular diseases (16.8%). Six month postfracture, the mortality was 17.6%. From those surviving 76.2% did walk indoors, 58.5% did also walk outdoors. Independent to dress were 54.6%. Severe pain was reported by 10.2%, whereas 36.9% described no pain. The institutionalization rate at six month was 19.0%. CONCLUSIONS: the study showed considerable mortality, a significant loss in function and social disintegration. Considerable differences were observed for subgroups of patients. Future treatment should focus on risk stratification and include postdischarge training programs. Moreover, preventive strategies should be implemented for high risk groups, such as ambulating patients with a history of stroke. Parkinson's disease and syndrome, dementia and nursing home residents.
Activities of Daily Living↗
Isolated dysarthria due to extracerebellar lacunar stroke: a central monoparesis of the tongue.
OBJECTIVES: The pathophysiology of dysarthria can preferentially be studied in patients with the rare lacunar stroke syndrome of "isolated dysarthria". METHODS: A single study was carried out on seven consecutive patients with sudden onset of isolated dysarthria due to single ischaemic lesion. The localisation of the lesion was identified using MRI. The corticolingual, cortico-orofacial, and corticospinal tract functions were investigated using transcranial magnetic stimulation. Corticopontocerebellar tract function was assessed using 99mTc hexamethylpropylene amine oxime-single photon emission computerised tomography (HMPAO-SPECT) in six patients. Sensory functions were evaluated clinically and by somatosensory evoked potentials. RESULTS: Brain MRI showed the lesions to be located in the corona radiata (n=4) and the internal capsule (n=2). No morphological lesion was identified in one patient. Corticolingual tract function was impaired in all patients. In four patients with additional cortico-orofacial tract dysfunction, dysarthria did not differ from that in patients with isolated corticolingual tract dysfunction. Corticospinal tract functions were normal in all patients. HMPAO-SPECT showed no cerebellar diaschisis, suggesting unimpaired corticopontocerebellar tract function. Sensory functions were not affected. CONCLUSION: Interruption of the corticolingual pathways to the tongue is crucial in the pathogenesis of isolated dysarthria after extracerebellar lacunar stroke.
Cerebellum↗
FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals.
The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.
Animals↗