Odontoblast processes in human dentin revealed by fluorescence labeling and transmission electron microscopy

In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.     

Differential expression of semaphorin 3A and its receptors during mouse retinal development

Semaphorins not only function in axon guidance during development but also contribute to various other biological processes. We have now examined the expression of semaphorin 3A (Sema3A) and its receptor components neuropilin 1 (Npn1) and plexin A (PlxA) during development of the mouse retina. Immunohistofluorescence analysis revealed that the expression patterns of Sema3A and Npn1 were similar during embryonic and postnatal development. The expression pattern of PlxA was also similar to those of Sema3A and Npn1 during embryonic and early postnatal (before eye opening) developments. However, the pattern of PlxA expression changed markedly after eye opening, with the expression disappearing from the optic nerve and increasing in intensity in the retinal pigment epithelium. Immunoprecipitation analysis showed that Sema3A interacted with PlxA in the retinal pigment epithelial cell line ARPE19 but not in the retinal ganglion cell line RGC5, whereas the opposite pattern of association was apparent for Sema3A and Npn1. Given that atmospheric oxygen is thought to play a role in the differentiation and maintenance of various ocular cell types, our results suggest that Sema3A-PlxA signalling activated by an effect of ambient oxygen on PlxA expression may contribute to differentiation of the retinal pigment epithelium. Copyright ? 2012 John Wiley & Sons, Ltd.     

Serum semaphorin 7A is associated with the risk of acute atherothrombotic stroke

Semaphorin 7A (Sema7A), a neural guidance cue, was recently identified to regulate atherosclerosis in mice. However, the clinical relevance of Sema7A with atherosclerotic diseases remains unknown. The aim of this study was to investigate the association between serum Sema7A and the risk of acute atherothrombotic stroke (AAS). We measured serum concentrations of Sema7A in 105 newly onset AAS cases and 105 age‐ and sex‐matched controls, showing that median Sema7A level in AAS cases was over three times of that in controls (5.86 vs 1.66 ng/mL). Adjusted for hypertension, body mass index, fasting blood glucose, total cholesterol, triglyceride, high‐density lipoprotein (HDL)‐cholesterol, low‐density lipoprotein (LDL)‐cholesterol, current smoking and alcohol consumption, multivariate logistic regression showed that higher Sema7A was independently associated with the odds of AAS (OR = 6.40, 95% CI: 2.88‐14.25). Each 1‐standard deviation increase in Sema7A was associated with a threefold higher odds of AAS (OR = 3.42, 95% CI: 1.84‐6.35). Importantly, adding Sema7A to a multivariate logistic model containing conventional cardiovascular risk factors improved the area under receiver operating characteristic curves from 0.831 to 0.891 for the association with AAS. In conclusion, elevated serum Sema7A is independently associated with the risk of AAS, suggesting that it may play a potential role in AAS.     

Ectopic expression of dentin sialoprotein during amelogenesis hardens bulk enamel

Dentin sialophosphpoprotein (Dspp) is transiently expressed in the early stage of secretory ameloblasts. The secretion of ameloblast-derived Dspp is short-lived, correlates to the establishment of the dentinoenamel junction (DEJ), and is consistent with Dspp having a role in producing the specialized first-formed harder enamel adjacent to the DEJ. Crack diffusion by branching and dissipation within this specialized first-formed enamel close to the DEJ prevents catastrophic interfacial damage and tooth failure. Once Dspp is secreted, it is subjected to proteolytic cleavage that results in two distinct proteins referred to as dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). The purpose of this study was to investigate the biological and mechanical contribution of Dsp and Dpp to enamel formation. Transgenic mice were engineered to overexpress either Dsp or Dpp in their enamel organs. The mechanical properties (hardness and toughness) of the mature enamel of transgenic mice were compared with genetically matched and age-matched nontransgenic animals. Dsp and Dpp contributions to enamel formation greatly differed. The inclusion of Dsp in bulk enamel significantly and uniformly increased enamel hardness (20%), whereas the inclusion of Dpp weakened the bulk enamel. Thus, Dsp appears to make a unique contribution to the physical properties of the DEJ. Dsp transgenic animals have been engineered with superior enamel mechanical properties.     

Cholinergic innervation of the human stomach.

A histochemical method for the acetylcholinesterase activity was used to establish the parasympathetic components of the gastric coats in man. The four gastric layers contain a rich cholinergic innervation. In the mucosa the positive nerve fibers are located around the gastric glands and between the muscles of the muscularis mucosae. In the submucosa rich interconnected nerve fibers, rare large nerve trunks, and scarce ganglia cells show a strong cholinergic reaction. The muscular layer contains the highest density of cholinergic nerve fibers, isolated or in large bundles. Auerbach's plexus has a strong acetylcholinesterase activity in the nerve cell bodies. The subserous layer is very rich in cholinergic nerve fibers, rarely isolated, but interconnected. The vessels of each gastric layer exhibit a rich cholinergic innervation in the adventitia and the outside part of media.     

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.     

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.     

Sustained up-regulation of semaphorin 3A, Neuropilin1, and doublecortin expression in ischemic mouse brain during long-term recovery

Strategies to provide neuroprotection and to promote regenerative axonal outgrowth in the injured brain are thwarted by the plethora of axon growth inhibitors and the ligand promiscuity of some of their receptors. Especially, new neurons derived from ischemia-stimulated neurogenesis must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. Therefore, characterization of the expression of inhibitory molecules in long-term surviving ischemic brains following stroke is important for designing selective therapeutics. Here, we describe a long-term recovery mouse model for cerebral ischemia in which a brief transient occlusion of the middle cerebral artery (30 min) was followed by up to 30 days of long-term reperfusion. Significantly decreased grip strength motor function and increased expression of one of the major repulsive guidance cues, Semaphorin 3A (Sema3A) and its receptor Neuropilin1 (NRP1) occurred in brains of these mice. Interestingly, increased Doublecortin (DCX) expression occurred only in the lateral ventricular wall zone, but not in the dentate gyrus granule cell layer on the ischemic side of the brain. Importantly, no DCX positive cells were detected in the infarct core region after 30 d ischemic recovery. Collectively, these studies demonstrated the sustained elevation of Sema3A/NRP1 expression in the ischemic territory, which may contribute to the inhibitory microenvironment responsible for preventing new neurons from entering the infarct area. This model will be of use as a platform for testing anti-inhibitory therapies to stroke.     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

Adrenergic innervation of the human gall bladder.

Adrenergic innervation of the human gall bladder was studied using two specific fluorescence histochemical methods. Blue-green fluorescing varicose nerves were scarce and mostly followed the course of blood vessels as typical perivascular plexuses. However, some adrenergic nerves not associated with the vessels were occasionally seen, as well as structures suggestive of a pericellular arrangement of varicose adrenergic nerve terminals on non-fluorescing ganglion cells. A few enterochromaffin cells were seen in the epithelial lining, also in the deep invaginations obviously representing the Aschoff-Rokitansky sinuses. Occasionally, small rounded cells with a rounded, relatively large nucleus, and exhibiting a weak yellow-green to blue-green granular cytoplasmic fluorescence, were observed in the wall of the gall bladder. The possible functional and evolutionary significance of these neural and endocrine elements was discussed against the data on physiological and pharmacological studies obtained from the literature. It was concluded that their significance is, in all probability, secondary to the influence of the intestinal polypeptide hormones, vagal innervation and circulating catecholamines upon the normal function of the gall bladder. The glyoxylic acid-induced fluorescence histochemical method was found to be superior to the conventional formaldehyde technique in studies on human tissue.     

Adrenergic innervation of human mesenteric blood vessels.

A fluorescent histochemical technique has been applied to study the adrenergic innervation of human superior mesenteric arteries obtained at autopsy. Specific catecholamine fluorescence was demonstrated in the smaller branches of this artery taken from three infants and one child. No specific fluorescence was seen in arteries from three adult subjects.     

Up‐regulation of semaphorin 4A expression in human retinal pigment epithelial cells by PACAP released from cocultured neural cells

Development and homeostasis of multicellular organisms require interactions between neighbouring cells. We recently established an in vitro model of cell–cell interaction based on a collagen vitrigel membrane. We have now examined the role of neural cells in retinal homeostasis by coculture of human retinal pigment epithelial (RPE) cells and neural cells on opposite sides of such a membrane. The neural cells (differentiated PC12 cells) induced up‐regulation of semaphorin 4A (Sema4A), a member of the semaphorin family of neural guidance proteins, in RPE (ARPE19) cells. This effect of the neural cells was mimicked by the neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) and was abolished by the PACAP antagonist PACAP(6–38). Coculture with neural cells or stimulation with PACAP also induced the phosphorylation of extracellular‐signal‐regulated kinase in ARPE19 cells, and this effect of the neural cells was inhibited by PACAP(6–38). Finally, among various cytokines examined, only the amount of interleukin‐6 released by cocultures of ARPE19 and neural cells differed from that released by ARPE19 cells cultured alone. Interleukin‐6 was not detected in culture supernatants of neural cells, and the reduction in the amount of interleukin‐6 released by the cocultures compared with that released by ARPE19 cells alone was prevented by PACAP(6–38). Our findings suggest that PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Development and homeostasis of multicellular organisms require interactions between neighbouring cells. With the use of a coculture system based on a collagen vitrigel membrane, we have now shown that neural cells induce up‐regulation of the neural guidance protein Sema4A in RPE cells. This effect of neural cells appears to be mediated by the neuropeptide PACAP. PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may thus regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Copyright © 2014 John Wiley & Sons, Ltd.     

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.     

Rapamycin inhibits aldolase A expression during human lymphocyte activation

Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.