A chemical method for intracellular loading of the calcium indicator aequorin in mammalian skeletal muscle.

The bioluminescent calcium indicator aequorin was loaded into bundles of skeletal muscle fibers from the rat extensor digitorum longus by macroinjection, a technique previously applied only to cardiac muscle. After loading, the amplitude and time course of the twitch returned to control values, indicating lack of damage to the fibers. Individual light signals (i.e., calcium transients) were recorded during each twitch or tetanus without the need for signal averaging. The calcium transients obtained were qualitatively and quantitatively similar to those reported previously with microinjection of aequorin. Our data suggest that macroinjection may be the method of choice for loading aequorin into mammalian skeletal muscle.     

Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.     

Targeting of aequorin for calcium monitoring in intracellular compartments

We have recently developed a new method for monitoring Ca²⁺ concentrations in defined cell compartments. The cDNA encoding the Ca²⁺-sensitive photoprotein aequorin has been modified in order to include specific targeting sequences and expressed in eukaryotic cells; the recombinant protein, specifically located inside the cells, has allowed the direct study of mitochondrial and nuclear Ca²⁺ concentrations in living cells. The principles, and the application, of this new methodology are discussed in this article.     

A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels

A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.     

A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) has been considered a serious health threat because of its putative role in multiple neurodegenerative diseases. First reports on BMAA concentrations in cyanobacteria were alarming: nearly all cyanobacteria were assumed to contain high BMAA concentrations, implying ubiquitous exposure. Recent studies however question this presence of high BMAA concentrations in cyanobacteria. To assess the real risk of BMAA to human health, this discrepancy must be resolved. We therefore tested whether the differences found could be caused by the analytical methods used in different studies. Eight cyanobacterial samples and two control samples were analyzed by three commonly used methods: HPLC-FLD analysis and LC-MS/MS analysis of both derivatized and underivatized samples. In line with published results, HPLC-FLD detected relatively high BMAA concentrations in some cyanobacterial samples, while both LC-MS/MS methods only detected BMAA in the positive control (cycad seed sarcotesta). Because we could eliminate the use of different samples and treatments as causal factors, we demonstrate that the observed differences were caused by the analytical methods. We conclude that HPLC-FLD overestimated BMAA concentrations in some cyanobacterial samples due to its low selectivity and propose that BMAA might be present in (some) cyanobacteria, but in the low μg/g or ng/g range instead of the high μg/g range as sometimes reported before. We therefore recommend to use only selective and sensitive analytical methods like LC-MS/MS for BMAA analysis. Although possibly present in low concentrations in cyanobacteria, BMAA can still form a health risk. Recent evidence on BMAA accumulation in aquatic food chains suggests human exposure through consumption of fish and shellfish which expectedly exceeds exposure through cyanobacteria.     

Use of a metallochromic indicator to study intracellular calcium movements in skeletal muscle

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fibre is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We review here procedures recently developed in this laboratory for empirically characterizing the calcium removal processes in voltage-clamped fibres and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse.     

Regulation of intracellular calcium in human esophageal smooth muscles

We have investigated sources ofCa²⁺ contributing to excitation ofhuman esophageal smooth muscle, using fura 2 to study cytosolic freeCa²⁺ concentration([Ca²⁺]_i)in dispersed cells and contraction of intact muscles. Acetylcholine (ACh) caused an initial peak rise of[Ca²⁺]_ifollowed by a plateau accompanied by reversible contraction. Removal ofextracellular Ca²⁺ or addition ofdihydropyridine Ca²⁺ channelblockers reduced the plateau phase but did not prevent contraction.Caffeine also caused elevation of[Ca²⁺]_iand blocked responses to ACh. Undershoots of[Ca²⁺]_iwere apparent after ACh or caffeine. Blockade of the sarcoplasmic reticular Ca²⁺-ATPase bycyclopiazonic acid (CPA) reduced the ACh-evoked increase of[Ca²⁺]_iand abolished the undershoot, indicating involvement ofCa²⁺ stores. When contraction wasstudied in intact muscles, removal ofCa²⁺ or addition of nifedipinereduced, but did not abolish, carbachol (CCh)-induced contraction.Elevation of extracellular K⁺caused contraction that was inhibited by nifedipine, although CCh stillelicited contraction. CPA caused contraction and suppressed theCCh-induced contraction, whereas ryanodine reduced CCh-induced contraction. Our studies provide evidence that muscarinic excitation ofhuman esophagus involves both release ofCa²⁺ from intracellular stores andinflux of Ca²⁺.

     

Bradykinin stimulates increased intracellular calcium in papillary collecting tubules of the rabbit

The effect of bradykinin on cytoplasmic Ca2+ concentration in rabbit papillary collecting tubule cells was determined using the fluorescent indicator Quin 2. Bradykinin stimulated a rapid increase in intracellular Ca2+. The rise in Ca2+ was dose dependent, persisted for less than 90 seconds and was independent of extracellular calcium. The ED50 for bradykinin induced changes in [Ca2+]i paralleled that observed previously for hormone-induced PGE2 formation as well as for inositol trisphosphate labelling. These studies provide additional support for the role of Ca2+ as a second messenger for bradykinin in renal papillary collecting tubule cells.     

刺吸电位技术实验中固定昆虫的三种方法比较研究

刺吸电位技术(Electrical penetration graph,EPG)是用来记录刺吸式口器昆虫的口针在其寄主植物组织中刺探所引起的电信号变化特征的技术,其核心在于建立昆虫取食行为与EPG波形的对应关系。近年来,EPG技术在昆虫取食行为及其传毒行为,以及植物抗虫机制等研究中得到越来越广泛的应用。其中,昆虫固定是影响EPG电极连接成功与否的关键技术,进而影响EPG实验结果。本文以灰飞虱Laodelphax striatellus(Fallén)为例,就EPG实验中电极连接时常用的3种昆虫固定技术(麻醉法、冷冻法和负压法)进行了详细描述与比较。研究结果表明,固定灰飞虱最优的方法为负压法,其次为麻醉法和冷冻法。     

A comparative study of three methods for the estimation of total plasmalogens in lingual taste epithelium and other tissues

The total plasmalogen content of lingual and other tissues was analyzed using the iodine-addition (Method 1), the p-nitrophenylhydrazone (Method 2), and the two-dimensional thin layer chromatography procedure (Method 3). Methods 1 and 2 were simple, rapid and reproducible, yielding values usually in close agreement with each other, and values higher than those of Method 3. Method 3 exhibited poor reproducibility. All three methods were of comparable sensitivity (less than 20 nmol of total plasmalogen per sample). According to Methods 1 and 2, there was more total plasmalogen in lingual epithelium containing taste buds compared with lingual epithelium devoid of taste buds. Plasmalogen content of bovine and rat brain, heart and liver agreed with literature values.     

A study of the intracellular transport of calcium in rat heart

The distribution of in vivo injected ⁴⁵Ca⁺⁺ in the subcellular fractions of rat heart has been studied. Most of the radioactivity of the cell was found to be associated with the subcellular organelles; only a small fraction was recovered in the soluble phase. Mitochondria contained the greatest part of the total radioactivity associated with the subcellular organelles. After injection of ⁴⁵Ca⁺⁺ the specific activity of the mitochondrial calcium pool was several times higher than that of the calcium of the sarcoplasmic reticulum. Pentachlorophenol has been administered to rats to uncouple oxidative phosphorylation in heart mitochondria in vivo and its effect on the distribution of ⁴⁵Ca⁺⁺ in the heart studied. Under these conditions, it has been found that mitochondria contained much less ⁴⁵Ca⁺⁺ than the controls; this decrease was paralleled by an increase of the radioactivity associated with the microsomes and with the final supernatant. Experiments in which ⁴⁵Ca⁺⁺ was added to heart homogenates at 0° indicated that ⁴⁵Ca⁺⁺ also became bound to mitochondria and the other subcellular structures at 0°. However, PCP had no effect on the distribution of radioactivity among the subcellular fractions under these conditions. The results suggest that (1) energy-linked movements of Ca⁺⁺ take place in mitochondria of the intact rat heart, (2) a part of the uptake of ⁴⁵Ca⁺⁺ by mitochondria does not depend on metabolism, and, (3) the movements of Ca⁺⁺ in heart mitochondria in vivo are probably more active than those in the sarcoplasmic reticulum.     

A comparative study in methods of cryoprotection for human semen

A comparative study using either glycerol or an egg yolk-citrate-glycerol mix for cryoprotection under the conditions described showed the latter to give a significantly better post-thaw motility. The greatest drop was noted within the first hour, suggesting that freezability of a sample could be judged accurately by rethawing at that time.Controlled studies using scanning electron microscopy clearly showed a greater head disruption after freezing with glycerol and looped tails after centrifugation, which could account for some of the findings of the first part of the study.These findings are reviewed and it is recommended that seminal samples are not centrifuged and that a complex medium be employed for seminal freezing and storing.     

A comparative study of clustering methods for molecular data

The research aim is to use three clustering technologies for establishing molecular data model of large size sets by comparison between low energy samples (LES) and local molecular samples (LMS). Hierarchical cluster of multi-level tree distance relation, competitive learning network of similar inputs falling into the same cluster and topological SOM are used to analyze 6,242 LES and 5,000 LMS. Our experiments show that in SOM, there are 24 to 25 Davies-Boulding clustering index and color map cluster units in the LES more than 10 to 12 in the LMS, which is consistent with the results of hierarchical cluster and competitive learning network in the rough. The hierarchical cluster reflects the biggest inter-cluster distance about 30 for the LES is far larger than that of LMS about 10. The intra-cluster distance of LES about 15 is also far bigger than that of LMS about 3. In SOM, there are more cluster borders of high values (black) reflecting large distance and more clusters in the D-matrix and U-matrix of LES than that of LMS, due to the biggest standard deviation range from -8 to 10 of samples feature of the LES is bigger than that of LMS from -2.5 to 2.5.     

Lumbar loading during lifting: a comparative study of three measurement techniques.

Low back loading during occupational lifting is thought to be an important causative factor in the development of low back pain. In order to regulate spinal loading in the workplace, it is necessary to measure it accurately. Various methods have been developed to do this, but each has its own limitations, and none can be considered a "gold standard". The purpose of the current study was to compare the results of three contrasting techniques in order to gain insight into possible sources of error to which each is susceptible. The three techniques were a linked segment model (LSM), an electromyographic (EMG)-based model, and a neural network (NN) that used both EMG and inertial sensing techniques. All three techniques were applied simultaneously to calculate spinal loading when eight volunteers performed a total of eight lifts in a laboratory setting. Averaged results showed that, in comparison with the LSM, the EMG technique calculated a 25.5+/-33.4% higher peak torque and the NN technique a 17.3+/-10.5% lower peak torque. Differences between the techniques varied with lifting speed and method of lifting, and could be attributed to differences in anthropometric assumptions, antagonistic muscle activity, damping of transient force peaks by body tissues, and, specific to the NN, underestimation of trunk flexion. The results of the current study urge to reconsider the validity of other models by independent comparisons.     

Influence of adaptation to stressor effects on bioelectrical activity, contractile function and resistance of the papillary muscles to the excess of intracellular calcium

In experiments on papillary muscles of rats it was established that adaptation to stressor effects increases resistance of the myocardium to contracture effects and restricts depression of electrophysiological parameters caused by the excess of calcium. Such adaptation decreased contracture by 6.5 times. On the next stage it was established that adaptation restricts depression of electrophysiological parameters of cardiomyocytes with the action of large concentrations of calcium. Possible mechanism of cardiac protector effect of adaptation to stressor effects is being discussed.     

Monitoring presynaptic calcium dynamics in projection fibers by in vivo loading of a novel calcium indicator

Fluorometric calcium measurements have revealed presynaptic residual calcium (Ca(res)) to be an important regulator of synaptic strength. However, in the mammalian brain, it has not been possible to monitor Ca(res) in fibers that project from one brain region to another. Here, we label neuronal projections by injecting dextran-conjugated calcium indicators into brain nuclei in vivo. Currently available dextran conjugates distort Ca(res) due to their high affinity for calcium. Therefore, we synthesized a low-affinity indicator, fluo-4 dextran, that can more accurately measure the amplitude and time course of Ca(res). We then demonstrate the utility of fluo-4 dextran by measuring Ca(res) at climbing fiber presynaptic terminals. This method promises to facilitate the study of many synapses in the mammalian CNS, both in brain slices and in vivo.