Temporal changes in prosaposin expression in the rat dentate gyrus after birth.

Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A-D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin. In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.

Lectin histochemistry of respiratory mucosa in the Pacific white-sided dolphin.

Sugars in the glycocalyx play an important role in the attachment of infectious agents to the respiratory mucosa. We examined the histochemistry of 23 lectins to survey the sugar expression in the glycocalyx of the respiratory mucosa of the Pacific white-sided dolphin, Lagenorhynchus obliquidens. The ciliated and basal cells were positive for all of the lectins studied. SBA, WFA, GSL-II, STL, S-WGA, and PNA staining in the cytoplasm showed different intensities between basal cells and ciliated cells. These results suggest that multiple terminal glycosylation occurs on ciliated and basal cells, such as GalNAc, GlcNAc, NeuNAc, galactose, glucose/mannose, oligosaccharide, and fucose, and that sugar residue expression changes during cell differentiation. The Pacific white-sided dolphin respiratory mucosa might express multiple sugar residues in the glycocalyx, to prevent the attachment and colonisation of infectious agents.

Morphological maturation level of the esophagus is associated with the number of circumesophageal muscle fibers during archenteron formation in the starfish Patiria (Asterina) pectinifera.

In echinoderms, the circumesophageal muscle is mesodermal in origin. Several studies of sea urchins have reported that the molecular events of myogenesis occur during the differentiation of the circumesophageal muscle in early embryogenesis. In contrast, few detailed reports have examined the differentiation of the circumesophageal muscle in larval starfish. Here, we examined the temporal-numeric distribution and differentiation of esophagus circular muscle fibers in the starfish Patiria pectinifera by using rhodamine-phalloidin staining. Muscle fibers were not detected in mouth-forming larvae, but a mean of about 10 muscle fibers was observed in 48-h larvae, and about 26 bundles were observed after 60 h. During the next 12 h, the number of muscle fiber bundles increased slightly to about 31 bundles and was stable until 96 h.

[The effects of a novel local ventilation system to reduce the health hazard to students during gross anatomy courses].

Formaldehyde or formalin is indispensable not only as a preservative but also as a disinfectant of cadavers for gross anatomy. It has recently attracted a great deal of attention as a health hazard for students and lecturers. To reduce the concentration of formaldehyde gas (FAG), we improved a novel local ventilation system of the push-pull type. This is the first report dealing with the effects of this ventilation system on the health of students before (over 1 ppm) and after (0.1 ppm) the installation. The percentages of students with lacrymal symptoms or airway irritation were reduced to a third of what they were before the installation. In particular, the number of those with continuously strong symptoms was reduced to a sixth of the pre-installation levels. This local ventilation system draws in fresh air from outside, and directs it to the breathing zone of the students, effectively reducing their symptoms.

Spatiotemporal distribution patterns of oligosaccharides during early embryogenesis in the starfish Patiria pectinifera.

To examine embryogenic mechanisms in the starfish Patiria (Asterina) pectinifera, we histochemically analyzed several larval stages using Alcian Blue (AB, which stains acidic mucins), Periodic Acid Schiff (PAS, which stains neutral mucins), and 21 types of lectins. Carbohydrate distribution patterns were observed in the cytoplasm, basement membrane, and blastocoel as follows: (1) The first group of lectins showed granular signals in the mesendodermal cells, and these lectins may be useful as mesendoderm markers. (2) The second class of lectins showed diffuse signals across the entire cytoplasm from the hatched blastula until the mid gastrula. These signals became localized to the basal cytoplasm of archenteron cells at the early bipinnaria. (3) Lectin reactivity in the basement membrane peaked at the early-to-mid gastrula and was nearly gone by the early bipinnaria. These results suggest the existence of various substances in the basement membrane and imply the importance of these substances during archenteron elongation and the induction of mesenchyme differentiation. (4) Signal colors with AB-PAS double staining in the blastocoel changed from magenta (by PAS staining) into blue (by AB staining) during these stages, thus, indicating that mucin located in the blastocoel changed from neutral to acidic. The most significant part of this report is the first description regarding temporal changes in the characteristics of intra- and extracellular components with the combination of many different lectins and stains.

Expression patterns in alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus after facial nerve transection.

Prosaposin acts as a neurotrophic factor, in addition to its role as the precursor protein for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. In rats, the prosaposin gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without. The expression of these mRNAs changes after brain injury. We examined the expression patterns of the alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus for 52 days following facial nerve transection. Pro+0 mRNA increased within 3 days of transection, peaked after 5-10 days, and remained significantly elevated for 21 days. In contrast, the expression of Pro+9 mRNA was constant throughout the regenerative period. Prosaposin mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. Our findings indicate that the saposin B domain of prosaposin, which is the domain affected by alternative splicing, plays an important role in both neurons and microglia during neuroregeneration.

Distribution of prosaposin in the rat nervous system.

Prosaposin is the precursor of four sphingolipid activator proteins (saposins A, B, C, and D) for lysosomal hydrolases and is abundant in the nervous system and muscle. In addition to its role as a precursor of saposins in lysosomes, intact prosaposin has neurotrophic effects in vivo or in vitro when supplied exogenously. We examined the distribution of prosaposin in the central and peripheral nervous systems and its intracellular distribution. Using a monospecific antisaposin D antibody that crossreacts with prosaposin but not with saposins A, B, or C, immunoblot experiments showed that both the central and peripheral nervous systems express unprocessed prosaposin and little saposin D. Using the antisaposin D antibodies, we demonstrated that prosaposin is abundant in almost all neurons of both the central and peripheral nervous systems, including autonomic nerves, as well as motor and sensory nerves. Immunoelectron microscopy using double staining with antisaposin D and anticathepsin D antibodies showed strong prosaposin immunoreactivity mainly in the lysosomal granules in the neurons in both the central and peripheral nervous systems. The expression of prosaposin mRNA, examined using in situ hybridization, was observed in these same neurons. Our results suggest that prosaposin is synthesized ubiquitously in neurons of both the central and peripheral nervous systems.

Localization of prosaposin in rat cochlea.

Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system.

Transient increase of TUNEL-positive cells on postnatal day 20 in the developing rat olfactory bulb.

In the developing central nervous system, apoptosis plays an important role in the normal organization of the neuronal circuit. The timing of neurogenesis, proliferation, and migration of the neurons in the developing olfactory bulb (OB) is well studied; however, the involvement of apoptosis in this process is not fully understood. In this study, we examined the changes in the distribution and the number of apoptotic cells in the rat OB during embryonic and postnatal periods, by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining. Although the number of TUNEL-positive cells was relatively small during the embryonic period, it gradually increased after birth, and peaked on postnatal day 20 with statistical significance, especially in the granule cell layer of the main OB. This transient increase of TUNEL-positive cells on postnatal day 20 may be involved in a critical event during maturation of the OB.

Rho-ROCK signal pathway regulates microtubule-based process formation of cultured podocytes--inhibition of ROCK promoted process elongation.

BACKGROUND: Podocytes, renal glomerular visceral epithelial cells, have two kinds of processes, namely major processes containing microtubules (MTs) and foot processes with actin filaments (AFs). The present study investigated how MTs are organized by the Rho-ROCK signal transduction pathway during process formation of podocytes. METHOD: After induction of differentiation, podocytes of the conditionally immortalized mouse cell line were treated with Y-27632, a specific inhibitor of ROCK, and exoenzyme C3, an inhibitor of RhoA, as well as with forskolin whose effects include inhibition of RhoA, in order to inhibit the Rho-ROCK pathway. RESULTS: Inhibition of ROCK significantly enhanced the formation of thick processes containing MT bundles. Y-27632 promoted process formation even in the presence of latrunculin A which disrupts AFs, strongly suggesting that ROCK directly regulates MT assembly. Treatment with Y-27632 increased MT stability, and stabilized MTs preferentially localized in podocyte processes. Moreover, when treated with a combination of Y-27632 and forskolin, and with Y-27632 and C3 as well, podocytes developed not only MT-based thick processes but also AF-based thin projections. CONCLUSIONS: These data indicate a contribution of ROCK in MT organization to promote podocyte process formation, although it was originally thought to regulate AF assembly. AF-based thin projections seem to be induced mainly by inhibition of RhoA and ROCK. The present study reveals a significant role of the Rho-ROCK signal pathway in the reorganization of both MTs and AFs during process formation of podocytes.

Process formation of the renal glomerular podocyte: is there common molecular machinery for processes of podocytes and neurons?

The renal glomerular podocyte exhibits a highly arborized morphology. In comparison with the neuron, which is the best studied process-bearing cell, the podocyte major processes share many cell biological characteristics with neuronal dendrites. Both podocytes and neurons develop microtubule-based thick processes with branching morphology and both have thin actin-based projections (i.e. podocyte foot processes and dendritic spines). Formation of podocyte processes and neuronal dendrites depends on the assembly of microtubules. Because the assembly of microtubules is regulated by phosphorylation of microtubule-associated proteins, inhibition of protein phosphatases abolishes and inhibition of protein kinases promotes process formation. Podocytes and dendrites also share the machinery of intracellular traffic of membranous vesicles, as well as cytoskeletal elements, which is indispensable for the elongation of these processes. Furthermore, these two cell types share expression of various molecules working for signal transduction, transmembranous transport and intercellular contacts. Such common gene expression implies a similar transcriptional regulation in these cells. Concerning the formation of podocyte foot processes and dendritic branches, actin filaments are thought to play a central role in orchestrating the function of various molecules and the regulation of actin assembly is necessary to establish and maintain such sophisticated cellular architecture. The molecular mechanism of foot process formation seems to include Rho family small GTP-binding proteins, which are known to be responsible for the establishment of dendritic branching morphology.

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera.

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.