Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors

1. Download high-res image (143KB)
2. Download full-size image

     

Influence of ryanodine on the mechanical restitution and on the post-extrasystolic potentiation of the guinea-pig ventricular myocardium

This paper records the results of an investigation into potentiation and staircase phenomena in rightventricular guinea-pig papillary muscles with particular reference to the sarcoplasmic Ca²⁺-channel. As a tool to isolate the second (late, 1tonic) component of isoproterenol-induced biphasic contractions ryanodine was used. On the evidence at present available the monophasic ryanodine-resistant component of the twitch represents that portion of the activator calcium which reaches the troponin C directly, that is, not taking the roundabout way through the intracellular storage structures. In order to avoid functional instabilities of the isolated muscle preparation a short-time double rest stimulation programme was used which combines a number of different tests and gives information on (1) the post-rest potentiation, (2) the post-extrasystolic potentiation, (3) the mechanical post-rest recovery, (4) the interval-strength relationship, and (5) the mechanical restitution. The results of the present work show that under the influence of ryanodine (1) the BOWDITCH staircase, a typical feature of normodynamic mammalian ventricular preparations as well as of hypodynamic frog heart preparations, does not exist, (2) the post-extrasystolic potentiation disappears, (3) the curve reflecting the mechanical restitution, under normal in vitro conditions a monotonically increasing function, becomes biphasic within the relative refractory period, (4) the conspicuous depression of the isometric post-rest contraction for long iasting pauses interrupting the regular pacing rhythm, a typical feature of isolated guinea-pig ventricular tissue, is clearly diminished, and (5) the characteristic curve, reflecting the potentiation of the post-extrasystolic post-rest contraction as a function of the delay time preceding the extrastimulus, becomes displaced to the premature interstimulus interval. The concept of an extended 2-calcium-store model is supported by this work.     

Spin-state-selective coherence transfer via intermediate states of two-spin coherence in IS spin systems: Application to E.COSY-type measurement of J coupling constants

It is demonstrated that a new pulse sequence element, Spin-State-SelectiveCoherence Transfer (S³CT), via an intermediate state ofheteronuclear IS zero- or double-quantum coherence can transfer the twosingle-quantum coherences on one of the spins exclusively to any one of thetwo single-quantum coherences on the other spin. This fact is used for editinginto two subspectra that are most suitable for extraction of homo- orheteronuclear J coupling constants when S³CT is combined withhomonuclear coherence transfer during a mixing period. Experimentalconfirmation is obtained using a ¹⁵N-labeled 58-residue protein,the C-terminal Kunitz domain from human type VI collagen. The J coupling con-stants determined include ³J_HN-H and³J_N-H related to the and¹ angles, respectively.     

Integration of spin-state-selective excitation into 2D NMR correlation experiments with heteronuclear ZQ/2Q π rotations for1 JXH

Spin-State-Selective Excitation (S³E), which forexample selectively excites amide proton resonances corresponding toexclusively either the or the spin state of the covalentlybound ¹⁵N atom is employed for E.COSY-type extraction ofheteronuclear J coupling constants. Instead of having one spectrum with twopeaks (corresponding to the or spin state of¹⁵N), S³E generates two spectra, each with onlyone peak for each ¹⁵N nucleus. These two spectra are generatedfrom the same data set, so that there is no reduction in sensitivitycompared to conventional ¹J_NH-resolved methods.Another interesting feature in comparison with conventional methods is that¹J_NH can be suppressed during the evolutionperiod, meaning that no heteronuclear multiplet structure is visible in the₁ frequency dimension. The S³E pulsesequence element is combined with NOESY for measurement of³J_N-H and J_N-Hcoupling constants in either a hetero- or a homonuclear correlated version.Experimental confirmation is obtained using the protein RAP 17-;97(N-terminal domain of ₂-macroglobulin ReceptorAssociated Protein).     

Human bronchial smooth muscle responsiveness after in vitro exposure to oxidizing pollutants

The aims of this work were (1) to determine the dose-response relationship between ex vivo exposure to oxidizing pollutants such as nitrogen dioxide (NO₂), the aldehyde acrolein, and ozone (O₃), and the reactivity to agonists in isolated human bronchial smooth muscle; and (2) to investigate the alterations in the cellular mechanisms of human airway smooth muscle contraction induced by such exposures. Experiments were performed in isolated human bronchi obtained at thoracotomy. Isometric contraction in response to a variety of agonists was compared between pollutant-exposed preparations and paired controls. Short exposures to NO₂, acrolein, or O₃ altered the subsequent airway smooth muscle responsiveness in a dose-dependent manner. The cellular mechanisms producing the airway hyperresponsiveness observed in vitro are shared by the three pollutants and include alterations in airway smooth muscle excitation-contraction coupling as well as indirect effects on neutral endopeptidase activity.Abbreviations ACh acetylcholine - CCRC cumulative concentration-response curve - KH Krebs-Henseleit solution - NEP neutral endopeptidase - NKA neurokinin A - SP substance P     

Molecular dynamics simulation using weak-coupling NOE distance restraining

Summary Application of the weak-coupling scheme to restrain the configurations of a molecular system to a set of NOE distance restraints is investigated using two test systems: (i) a 15-atom chain molecule with one distance restraint; and (ii) a protein molecule with hundreds of NOE distance restraints. Atom-atom distance restraining by the weak-coupling technique is possible, but this method does not produce as good results as the penalty function method normally used to maintain NOE distance restraints.Abbreviations NOE nuclear Overhauser effect - MD molecular dynamics - PDB protein data bank     

DNA切除修复与转录偶联

细胞DNA受到某些环境理化因子损伤后,其中活性转录基因和DNA转录链上的损伤被优先切除修复,这种DNA选择性修复直接与基因转录过程偶联．在大肠杆菌中已分离到实现此功能的转录修复偶联因子（TRCF）,是由mdf基因编码的一种具有ATPase活性的DNA结合蛋白．在真核细胞中,发现某些DNA修复蛋白也在DNA转录中起作用,如人DNA切除修复基因ERCG－3编码产物,是转录因子TFⅡH中最大亚基p89,酵母切除修复基因RAD3就是编码因子b的最大亚基p85．     

Implication of the nucleus in excitation contraction coupling of heart cells

In the present study, Fluo-3 Ca²⁺ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]_c and nuclear [ ]_n free Ca²⁺ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca²⁺ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca²⁺ from 100–1600 nM gradually increased [Ca²⁺]_n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca²⁺ and the uptake and release of Ca²⁺ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca²⁺ levels remained elevated for several milliseconds even after cytosolic Ca²⁺ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca²⁺ concentration during excitation-contraction coupling in heart cells.     

Excitation-contraction coupling of the developing rat heart

Postnatal maturation of rat heart is characterized by increases in force production, velocity of shortening and heart rate. Simultaneously with the enhanced cardiac power production the size of ventricular myocytes markedly increases. Parallel increase in cardiac rate functions and cells size would be expected to require reorganization of cardiac Ca regulation so that adequate rate of Ca release and uptake can be maintained. In accordance with this the source of activator Ca shifts from extracellular space to intracellular stores within the first four or five weeks of postnatal life. Calcium handling of sarcoplasmic reticulum and sarcolemma change in complementary manner so that diminishing sarcolemmal Ca transport is compensated with enhanced Ca release and sequestration by the sarcoplasmic reticulum during the early postnatal development of rat heart. These functional changes are partly due to reciprocal alterations in surface area of sarcolemma and sarcoplasmic reticulum, partly due to age-dependent changes in the expression of different transport systems and their kinetic properties.     

Dihydropyridine Receptor-Ryanodine Receptor Uncoupling in Aged Skeletal Muscle

The mechanisms underlying skeletal muscle functional impairment and structural changes with advanced age are only partially understood. In the present study, we support and expand our theory about alterations in sarcolemmal excitation-sarcoplasmic reticulum Ca²⁺ release-contraction uncoupling as a primary skeletal muscle alteration and major determinant of weakness and fatigue in mammalian species including humans. To test the hypothesis that the number of RYR1 (ryanodine receptor) uncoupled to DHPR (dihydropyridine receptor) increases with age, we performed high-affinity ligand binding studies in soleus, extensor digitorum longus (EDL) and in a pool of several skeletal muscles consisting of a mixture of fast- and slow-twitch muscle fibers in middle-aged (14-month) and old (28-months) Fisher 344 Brown Norway F1 hybrids rats. The number of DHPR, RYR1, the coupling between both receptors expressed as the DHPR/RYR1 maximum binding capacity, and their dissociation constant for high-affinity ligands were measured. The DHPR/RYR1 ratio was significantly reduced in the three groups of muscles (pool: 1.03 ± 0.15 and 0.80 ± 0.11, soleus: 0.44 ± 0.12 and 0.26 ± 0.10, and EDL: 0.95 ± 0.14 and 0.68 ± 0.10, for middle-aged and old muscles, respectively). These data support the concept that DHPR-RYR1 uncoupling results in alterations in the voltage-gated sarcoplasmic reticulum Ca²⁺ release mechanism, decreases in myoplasmic Ca²⁺ elevation in response to sarcolemmal depolarization, reduced Ca²⁺ supply to contractile proteins and reduced contraction force with aging. Received: 26 August 1996/Revised: 30 December 1996