Neuroblasts produced in the ventricular zone of the neocortex migrate radially and form the cortical plate, settling in an insideout order. It is also well known that the tangential cell migration is not negligible in the embryonic neocortex. To have a better understanding of the tangential cell migration in the cortex, we disturbed the migration by making a cut in the neocortex, and we labeled the migrating cells with 1,1Ј-dioctodecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate (DiI) in vivo and in vitro. We also determined the birth dates of the cells.Disturbance of tangential cell migration caused an accumulation and disappearance of microtubule-associated protein 2 immunoreactive (MAP2-IR) cells on the ventral and dorsal side of the cut, respectively, which indicated that most of the MAP2-IR cells in the intermediate zone (IZ) were migrating toward the dorsal cortex. The DiI injection study in vivo confirmed the tendency of the direction of cell migration and suggested the origin of the cells to be in the lateral ganglionic eminence (LGE). DiI injection into the LGE in vitro confirmed that the LGE cells cross the corticostriatal boundary and enter the IZ of the neocortex. The migrating cells acquired multipolar shape in the IZ of the dorsal cortex and seemed to reside there. A 5-bromo-deoxyuridine incorporation study revealed that the migrating MAP2-IR cells in the IZ were early-generated neurons. We concluded that the majority of tangentially migrating cells were generated in the LGE and identified as a distinct population that was assumed not to have joined the cortical plate.
The ganglionic eminence (GE) supplies neurons containing gamma-aminobutyric acid (GABA) to the pallium of the telencephalon. We investigated the molecular guidance mechanisms of GE cell migration in the neocortex and found neuropilin-1 (Npn-1) or neuropilin-2 (Npn-2) on the GE cells. Ectopic Sema3A or -3F expression by COS1 cell clusters placed on embryo neocortical slices reduced the cell migration but did not block it completely. However, the cell migration was almost completely blocked by COS1 cell clusters expressing both Sema3A and -3F. The direction of cell migration could be reversed by placing Sema3A- and -3F-coexpressing COS1 cell clusters at the distal cut end of the neocortical slices. Further slice experiments revealed that migration of half of the GE cells in the neocortex was regulated by Sema3A and that migration of the other half of the GE cells in the neocortex was regulated by Sema3F. When the cells responding to Sema3A were diverted by ectopic Sema3A expression in vivo, Dlx2-positive cells were found predominantly in the lower intermediate zone (IZ). When the cells responding to Sema3F were diverted by ectopic Sema3F expression in vivo, Dlx2-positive cells were found predominantly in the upper IZ. It was speculated that the semaphorin-neuropilin interactions distribute the GABAergic GE cells evenly in the neocortex as well as guide the GE cells from the GE to the neocortex. The Sema3A expression site under the subplate extended dorsally as the embryo developed. The Sema3A expression seemed to block the Npn-1-positive GE cells in the neocortex from entering the cortical plate (CP) and guide them to the dorsal cortex and the hippocampus. Sema3F expression in the CP continued through the embryonic stages. The expression seemed to block Npn-2-positive GE cells in the neocortex from entering the CP and make them migrate into the lower IZ. Finally, the semaphorin-neuropilin interactions sorted GABAergic inteneurons into the CP and white matter neurons into the IZ.
Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL-8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil-IL-8 production in vitro; the causative role of NF-kappaB in its production, in association with the possible production of reactive oxygen intermediates that activate NF-kappaB; and the nicotinic acetylcholine receptors (nAChRs) involved in IL-8 production. Nicotine stimulated neutrophils to produce IL-8 in both time- and concentration-dependent manners with a 50% effective concentration of 1.89 mM. A degradation of IkappaB-alpha/beta proteins and an activity of NF-kappaB p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, nomega-Nitro-l-arginine methyl ester, prevented nicotine-induced IL-8 production, with an entire abrogation of DHR oxidation, IkappaB degradation, and NF-kappaB activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF-kappaB activity, thereby producing IL-8. The nAChRs seemed to be involved in IL-8 production. In smokers, blood IL-8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil-IL-8 production via nAChR by generating peroxynitrite and subsequent NF-kappaB activation, and the IL-8 appears to contribute to leukocytosis in tobacco smokers.
CpG DNA induces plasmacytoid dendritic cells (pDC) to produce type I IFN and chemokines. However, it has not been fully elucidated how the TLR9 signaling pathway is linked to these gene expressions. We examined the mechanisms involving the TLR9 and type I IFN signaling pathways, in relation to CpG DNA-induced IFN-α, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-κB subunits p65 and p50 were constitutively activated. pDC also constitutively expressed IRF-7 and CCL3, and the gene expressions seemed to be regulated by NF-κB. CpG DNA enhanced the NF-κB p65/p50 activity, which collaborated with p38 MAPK to up-regulate the expressions of IRF-7, CXCL10, and CCL3 in a manner independent of type I IFN signaling. We then examined the pathway through which IFN-α is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-α, in a NF-κB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-α in the presence of NF-κB/p38 MAPK inhibitor, and chloroquine abrogated this effect. With CpG DNA, IRF-7, both constitutively and newly expressed, moved to the nuclei independently of NF-κB/p38 MAPK. These findings suggest that, in CpG DNA-stimulated human pDC, the induction of IRF-7, CXCL10, and CCL3 is mediated by the NF-κB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-κB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-α.
1 We have studied the antagonist action of prazosin and KMD-3213 in a constitutively active mutant of the human alpha-1a adrenoceptor in which Ala 271 was substituted to Thr and was expressed in CHO cells. Inverse agonism was characterized by up-regulation of receptor density, a decrease in basal GTPgS binding, and a reduction in basal inositol-1,4,5-trisphosphate (IP 3 ) level. 2 According to the above criteria, prazosin acted as an inverse agonist, whilst KMD-3213 behaved as a neutral antagonist. 3 Compared with the wild-type receptor, mutant receptor exhibited single a nity sites for [ 3 H]-prazosin, [ 3 H]-KMD and the non-radioactive ligands tested, and displayed signi®cantly higher a nities for several agonists but not for the two antagonists. 4 Administration of KMD-3213 to prazosin-treated CHO cells expressing the mutant receptor reversed the inverse agonism of prazosin resulting in rapid increases in cellular IP 3 , in intracellular [Ca 2+ ] and in the rate of extracellular acidi®cation. 5 These results indicated that a neutral antagonist can reverse the action of an inverse agonist at the receptor site. The distinct properties of inverse agonist and neutral antagonist in a ecting receptor function may be important for the clinical use of such antagonists.
1 Two splice isoforms of rabbit a 1a -adrenergic receptor (AR), (named a 1a -OCU.2-AR and a 1a -OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (a 1a -OCU.1-AR). Although they have the identical splice position with human a 1a -AR isoforms, the C-terminal sequences are distinct from those of human isoforms. 2 Among these rabbit a 1a -AR isoforms, there are no signi®cant di erences in pharmacological properties: high a nity for prazosin, WB4101, KMD-3213 and YM617 and low a nity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. 3 Competitive reverse transcription-polymerase chain reaction (RT ± PCR) analysis revealed that mRNA of a 1a -ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; a 1a -OCU.1-AR was expressed most abundantly in those tissues. 4 CHO clones, stably expressing each isoforms with receptor density 740 fmol mg 71 protein in a 1a -OCU.1-AR, 1200 fmol mg 71 in a 1a -OCU.2-AR and 570 fmol mg 71 in a 1a -OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. 5 Noradrenaline elicited a concentration-dependent increase in extracellular acidi®cation rate (EAR) in the CHO clones with pEC 50 values of 6.19 for a 1a -OCU.1-AR, 6.49 for a 1a -OCU.2-AR and 6.58 for a 1a -OCU.3-AR, respectively. 6 Noradrenaline caused a concentration-dependent increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the CHO clones with pEC 50 values of 6.14 for a 1a -OCU.1-AR, 7.25 for a 1a -OCU.2-AR and 7.70 for a 1a -OCU.3-AR, respectively. 7 In conclusion, the present study shows the occurrence of three splice isoforms of rabbit a 1a -AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological pro®les in binding studies but a 1a -OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These ®ndings suggest that the structure of C-terminus of a 1a -ARs may give the characteristic functional pro®le.
Microtubule-associated protein 2 (MAP2) occurs in developing mammalian neuronal tissue as both high- and low-molecular-weight forms with temporally regulated expression. We studied the MAP2 expression in the developing rat telencephalon with monoclonal antibodies that recognized both the high- and low-molecular-weight forms of MAP2 variants or that specifically recognized high-molecular-weight forms of MAP2 variants. Differences in the staining patterns of these antibodies reflected differences in the distribution of the high- and low-molecular-weight MAP2s. The immunoreactive sites of high- and low-molecular-weight MAP2 had a more widespread distribution in the embryonic telencephalon than those of high-molecular-weight MAP2. Many bipolar cells in the ganglionic eminence (GE) and in the intermediate zone (IZ) of the neocortex showed low-molecular-weight MAP2 immunoreactivity, but they showed weak or no high-molecular-weight MAP2 immunoreactivity. Expression of mRNA containing exons common to high- and low-molecular-weight MAP2 was detected in the tangentially ellipsoidal cells in the IZ, but expression of mRNA containing an exon specific to high-molecular-weight MAP2 was not detected in these cells by in situ hybridization. We interpreted these observations as indicating that the bipolar cells contained MAP2c preferentially, but contained MAP2a and MAP2b (MAP2a/b) at a very low or negligible level. The cells that expressed MAP2c preferentially among the MAP2 splicing variants composed 50% of the preplate cells, most of the MAP2-positive cells in the hippocampus and the corpus callosum. Double labeling by DiI staining and Dlx2 immunohistochemistry, or by Dlx2 and MAP2 immunohistochemistry, revealed that most of the Dlx2-positive cells in the IZ expressed MAP2c preferentially at embryonic day 16. Another double-labeling study revealed that most GAD-positive cells in the preplate were MAP2a/b positive, whereas most GAD-positive cells in the IZ expressed MAP2c preferentially, with only a negligible level of MAP2a/b immunoreactivity. We conclude that MAP2 immunoreactivity in the IZ was localized in the tangentially migrating neurons. The tangentially migrating neurons seemed to acquire MAP2a/b immunoreactivity as they entered the preplate or cortical plate and developed into mature neurons. Radially migrating neurons in the IZ were MAP2 negative. After entering to the preplate or the cortical plate, they became MAP2a/b positive as they developed into mature neurons.
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