Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Subcellular localization of secretogranin II and synaptophysin by immunoelectron microscopy in differentiated hypothalamic neurons in culture

Secretogranin II (SgII), a tyrosine-sulfated secretory protein, is a widespread component of endocrine and neuronal cells. In the present study we used mouse hypothalamic neurons differentiated in culture and studied the subcellular localization of SgII by two methods, i.e., by the use of immunoperoxidase or immunogold electron microscopy. By immunoperoxidase labeling, SgII was mainly detected in the matrix of large dense-core vesicles (LDCVs). In addition, usually in nerve terminals containing LDCVs, peroxidase reaction product was also found in association with the membrane of small synaptic vesicles (SSVs). By immunogold labeling, SgII was detected only in the matrix of LDCVs. We also compared the localization of SgII and synaptophysin (SY), an integral membrane protein of SSVs, by double labeling, using a combination of pre-embedding immunogold and -peroxidase techniques for SgII and SY, respectively. In perikarya, SgII-positive LDCVs were observed in the vicinity of the Golgi complex and scattered in the cytoplasm. In contrast, SY labeling was restricted to electron-translucent vesicles and tubular membranes in the Golgi area. Moreover, membrane structures positive for both SgII and SY were not found either in the Golgi zone or in other regions of the cytoplasm. In synaptic boutons, immunolabeling of LDCVs and SSVs with anti-SgII and anti-SY, respectively, was mutually exclusive. In summary, within the limitation of the methods used, our data are consistent with the notion that SgII and SY are segregated from each other on exit from the trans-Golgi network, than follow two distinct membrane traffic pathways, and that the presence of SgII on the membrane of some SSVs is due to endocytosis.     

Physiology and morphology of protocerebral olfactory neurons in the male moth Manduca sexta

1. We have used intracellular recording and staining with Lucifer Yellow, followed by reconstruction from serial sections, to characterize the responses and structure of olfactory neurons in the protocerebrum (PC) of the brain of the male sphinx moth Manduca sexta. 2. Many olfactory protocerebral neurons (PCNs) innervate a particular neuropil region lateral to the central body, the lateral accessory lobe (LAL), which appears to be important for processing olfactory information. 3. Each LAL is linked by its constituent neurons to the ipsilateral lateral PC, where projection neurons from the antennal lobe terminate, as well as to other regions of the PC. The LALs are also linked to each other by bilateral neurons with arborizations in each LAL. 4. Some PC neurons showed long-lasting excitation (LLE) that outlasted the olfactory stimuli by greater than or equal to 1 s, and as long as 30 s in some preparations. LLE was more frequently elicited by the sex-pheromone blend than by individual pheromone components. All bilateral neurons that showed LLE had arborizations in the LALs. LLE responses were also recorded in a single local neuron innervating the mushroom body. 5. In some other PC neurons, pheromonal stimuli elicited brief excitations that recovered to background firing rates less than 1 s after stimulation.     

Light- and electron-microscopic evidence of costoring of immunoreactive enkephalins and substance P in dorsal horn neurons of rat

Summary The topographical localization of substance P (SP) and methionine-enkephalin-octapeptide (Enk-8) was examined immunohistochemically in the surface layer of the dorsal horn of rat cervical spinal cord. Although a few neurons were immunoreactive for Enk-8 in the intact animals, after an intracisternal administration of colchicine, immunoreactive Enk-8 neurons were numerous, and half of them indicated immunoreactivity also for SP. Some immunoreactive SP neurons appeared to show no immunoreactivity for Enk-8. Immuno-reactive nerve fibers, on the other hand, were numerous, and many of them contained both peptides. Electron-microscopic examination of the nerve fibers in tissue prepared by a freeze-drying procedure and stained by a postembedding procedure, revealed the costoring of both peptides in the same cored vesicles. The physiological significance of this costoring is discussed.     

Gustatory synergism in ants mediates a species-specific symbiosis with lycaenid butterflies

Here we show that larvae of the lycaenid butterfly Niphanda fusca secrete droplets containing trehalose and glycine. These droplets attract the larva's host ants Camponotus japonicus, which collect and protect the larvae. We comparatively investigated gustatory preference for trehalose, glycine or a mixture of the two between host (C. japonicus) and non-host (Camponotus obscuripes) species of ants in behavioral and electrophysiological experiments. Glycine itself induced no taste sensation in either host or non-host ants. The mixture of trehalose plus glycine was chosen as much as pure trehalose by non-host ants. However, the host ants clearly preferred the mixture of trehalose plus glycine to trehalose alone. When we used sucrose instead of trehalose, the mixture of sucrose plus glycine was chosen as much as sucrose alone, in both species. These behavioral data are supported by the electrophysiological responsiveness to sugars and/or glycine in the sugar-taste receptor cells of the ants. Considering that lycaenid butterflies' secretions have species-specific compositions of sugar and amino acid; our results clearly showed that such species-specific compositions of larval secretions are precisely tuned to the feeding preferences of their host ant species, in which the feeding preferences are synergistically enhanced by amino acid.     

Abundance estimation of riverine macrophyte Egeria densa using environmental DNA: effects of sampling season and location

Invasive macrophytes can have a variety of effects on aquatic ecosystems. The early detection and abundance estimation of an invasive species is important to effectively control it and minimize the ecosystem impacts. It is imperative to develop more efficient sampling methods for the abundance quantification of aquatic plants in large riverine systems. We examined (1) relationships between the environmental DNA (eDNA) concentrations of the invasive macrophyte, Egeria densa, and the upstream coverage area on the multiple life-history stages (dormant, growing, and senescence seasons) in a large riverine system in Japan and (2) if the relationships between the eDNA concentrations and coverage area could vary with the lateral sampling locations (left bank, middle, and right bank). The eDNA concentrations had significant positive relationships with the upstream coverage area of E. densa at multiple spatial scales for the dormant and senescence seasons. These results suggest that the eDNA analysis could be useful to quantify the relative abundance of this aquatic macrophyte in the riverine system; however, the selection of the eDNA sampling season could be important to accurately estimate abundance. Our results also showed that the eDNA concentrations of E. densa did not significantly differ from the lateral sampling location, suggesting that the eDNA samples could reflect the relative abundance of E. densa upstream of the study sites regardless of the lateral sampling location.

     

Syntheses and evaluation of a homologous series of aza-vesamicol as improved radioiodine-labeled probes for sigma-1 receptor imaging

Sigma-1 receptor imaging probes for determining the expression levels are desirable for diagnoses of various diseases and companion diagnoses of therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to develop probes with higher affinity for the sigma-1 receptor. For this purpose, we synthesized and evaluated compounds, namely, vesamicol derivatives, in which alkyl chains of varying chain length were introduced between a piperazine ring and a benzene ring. The binding affinity of the vesamicol derivatives for the sigma-1 receptor tended to increase depending on the length of the alkyl chain between the benzene ring and the piperazine ring. The sigma-1 receptor of 2-(4-(3-phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5) (K_i?=?5.8?nM) exhibited the highest binding affinity; therefore, we introduced radioiodine into the benzene ring in 5. The radioiodine labeled probe [¹²⁵I]2-(4-(3-(4-iodophenyl)propyl)piperazin-1-yl)cyclohexan-1-ol ([¹²⁵I]10) showed high accumulation in the sigma-1 receptor expressing DU-145 cells both in vitro and in vivo. Co-injection of [¹²⁵I]10 with an excess level of a sigma receptor ligand, haloperidol, resulted in a significant decrease in the tumor accumulation in vitro and in vivo, indicating sigma receptor-mediated tumor uptake. These results provide useful information for developing sigma-1 receptor imaging probes.