Horizontal cell terminals lateral to the synaptic ribbons in goldfish cone pedicles give rise to 0.3 micron long, finger-like extensions in the light-adapted state. These structures, called spinules, disappear almost completely after dark adaptation. The ultrastructure of the horizontal cell terminals is characterized by the presence of occasional microtubules, microfilaments and sparse irregular vesicles; in the dark, large multivesicular bodies can also be found. Two types of membrane densities are described in the horizontal cell terminals, one of which is typically located at the tip of the spinules. Their positive reaction to E-PTA makes it probable that the spinules are synaptic structures. Reconstruction of serial sections shows about 12 spinules per terminal in the light but only two in the dark. Formation and disappearance of the spinules takes about 60 min and involves a transitional stage in the form of a spherical structure. Spinules can be found in five other teleost species, with a darkness-induced reduction in number, but not in the horizontal cell terminals of the mudpuppy, turtle and mammals.

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In the cone pedicules, the digitations of horizontal cell process lateral to the synaptic ribbon disappear after dark adaptation. This disappearance is correlated with the loss of color opponency and cone function shown in ganglion cell recordings in isolated retinas. Cone function and color-opponent responses are restored by reapplying background light.

Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis. Am J Physiol Heart Circ Physiol 287: H1039 -H1045, 2004. First published April 22, 2004 10.1152/ajpheart.01016.2003.-The creatine kinase (CK) system is involved in the rapid transport of high-energy phosphates from the mitochondria to the sites of maximal energy requirements such as myofibrils and sarcolemmal ion pumps. Hearts of mice with a combined knockout of cytosolic M-CK and mitochondrial CK (M/Mito-CK Ϫ/Ϫ ) show unchanged basal left ventricular (LV) performance but reduced myocardial high-energy phosphate concentrations. Moreover, skeletal muscle from M/Mito-CK Ϫ/Ϫ mice demonstrates altered Ca 2ϩ homeostasis. Our hypothesis was that in CK-deficient hearts, a cardiac phenotype can be unmasked during acute stress conditions and that susceptibility to ischemia-reperfusion injury is increased because of altered Ca 2ϩ homeostasis. We simultaneously studied LV performance and myocardial Ca 2ϩ metabolism in isolated, perfused hearts of M/Mito-CK Ϫ/Ϫ (n ϭ 6) and wild-type (WT, n ϭ 8) mice during baseline, 20 min of no-flow ischemia, and recovery. Whereas LV performance was not different during baseline conditions, LV contracture during ischemia developed significantly earlier (408 Ϯ 72 vs. 678 Ϯ 54 s) and to a greater extent (50 Ϯ 2 vs. 36 Ϯ 3 mmHg) in M/Mito-CK Ϫ/Ϫ mice. During reperfusion, recovery of diastolic function was impaired (LV end-diastolic pressure: 22 Ϯ 3 vs. 10 Ϯ 2 mmHg), whereas recovery of systolic performance was delayed, in M/Mito-CK Ϫ/Ϫ mice. In parallel, Ca 2ϩ transients were similar during baseline conditions; however, M/Mito-CK Ϫ/Ϫ mice showed a greater increase in diastolic Ca 2ϩ concentration ([Ca 2ϩ ]) during ischemia (237 Ϯ 54% vs. 167 Ϯ 25% of basal [Ca 2ϩ ]) compared with WT mice. In conclusion, CK-deficient hearts show an increased susceptibility of LV performance and Ca 2ϩ homeostasis to ischemic injury, associated with a blunted postischemic recovery. This demonstrates a key function of an intact CK system for maintenance of Ca 2ϩ homeostasis and LV mechanics under metabolic stress conditions. aequorin bioluminescence; transgenic mouse THE CREATINE KINASE (CK) system comprises a family of mitochondrial (Mito-CK) and cytosolic (MM-, MB-, and BB-CK) isoenzymes that are critically involved in intracellular energy homeostasis. The primary role of CK is to catalyze the reversible transfer of a high-energy phosphoryl group between ATP and phosphocreatine (PCr; PCr ϩ ADP ϩ H ϩ 7 ATP ϩ creatine). The functional and physical coupling of certain members of the CK isoenzyme family to the sites of energy production and utilization has underscored the integrated properties of this important enzyme system in excitable tissue, particularly in muscle cells (26). MM-CK, for example, is present in membrane vesicles of the sarcoplasmic reticulum (SR) isolated from skeletal muscle (15), suggesting that an efficient and fast energy replenishing system is necessary for op...

One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

Continuous- or free-flow electrophoresis is based upon a thin film of fluid flowing between two parallel plates. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. Using the Octopus apparatus in a horizontal position, continuous preparative separation of methadone enantiomers in the presence of (2-hydroxypropyl)-β-cyclodextrin as a chiral selector was investigated under conditions of continuous-flow zone electrophoresis and continuous-flow isotachophoresis. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary electrophoresis and circular-dichroism spectroscopy. In both electrophoretic modes, partial separation of the two enantiomers with an enrichment of about 80% and a throughput of 10-20 mg of racemic methadone per hour was obtained. Operating the Octopus apparatus with interrupted buffer flow during electrophoresis, a process termed interval-flow electrophoresis, resulted in complete separation of milligram quantities of the two methadone enantiomers. Furthermore, commencing with racemic methadone, continuous multistage isotachophoretic processing is shown to be suitable to purify (R)-(-)-methadone, the enantiomer with higher pharmacological activity, on a mg/h scale and at a mM concentration in the collected product stream.

BackgroundIn the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks.ResultsWe generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired.ConclusionsTogether, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.