We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.
Abstract. The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesiclerich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or a-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.T hE nerve growth cone is a special structure at the leading edge of the extending axon and is responsible for axonal guidance and elongation. The growth cone consists of two distinct domains: the peripheral (p_)l and the central (C-) domains. P-domain, a microfilamentrich region including filopodia and lamellipodia, is responsible for motility (Dailey and Bridgman, 1993); C-domain, a vesicle-rich region (Forscher et al., 1987;Dailey and Bridgman, 1993), is the site where membrane expansion for axonal growth is suggested to occur (Goldberg and Address correspondence to Michihiro Igarashi, Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine, Maebashi, Gunma 371, Portions of this work have appeared in abstract form (1995. Cell Struct. Funct. 20[Suppl.]:566a).1. Abbreviations used in this paper: C-domain, central domain; CNS, central nervous system; DPD, 2,2'-dipyridyl; DRG, dorsal root ganglion; P-domain, peripheral domain; SLO, streptolysin O; SNARE, SNAP receptor; VEC-DIC, video-enhanced contrast~lifferential interference contrast. Burmeister, 1986;Forscher et al., 1987;Pfenninger and Friedman, 1993). The growth cone alters its shape between active (advancing) and inactive (ceasing) forms in response to an appropriate (and inappropriate) stimulus for axonal growth (Kater and Mills, 1991;Schwab et al., 1993). An active growth cone is large with several filopodia, while an inactive one, known as a collapsed growth cone, is very small with few or no filo...
Norcoclaurine synthase (NCS) catalyzes the stereoselective Pictet–Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde as the first step of benzylisoquinoline alkaloid synthesis in plants. Recent studies suggested that NCS shows relatively relaxed substrate specificity toward aldehydes, and thus, the enzyme can serve as a tool to synthesize unnatural, optically active tetrahydroisoquinolines. In this study, using an N-terminally truncated NCS from Coptis japonica expressed in Escherichia coli, we examined the aldehyde substrate specificity of the enzyme. Herein, we demonstrate the versatility of the enzyme by synthesizing 6,7-dihydroxy-1-phenethyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-propyl-1,2,3,4-tetrahydroisoquinoline in molar yields of 86.0 and 99.6% and in enantiomer excess of 95.3 and 98.0%, respectively. The results revealed the enzyme is a promising catalyst that functions to stereoselectively produce various 1-substituted-1,2,3,4-tetrahydroisoquinolines.
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