Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

     

Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations

The small GTPase Ha-Ras and Rap1A exhibit high mutual sequence homology and share various target proteins. However, they exert distinct biological functions and exhibit differential subcellular localizations; Rap1A is predominantly localized in the perinuclear region including the Golgi apparatus and endosomes, whereas Ha-Ras is predominantly localized in the plasma membrane. Here, we have identified a small region in Rap1A that is crucial for its perinuclear localization. Analysis of a series of Ha-Ras-Rap1A chimeras shows that Ha-Ras carrying a replacement of amino acids 46-101 with that of Rap1 exhibits the perinuclear localization. Subsequent mutational studies indicate that Rap1A-type substitutions within five amino acids at positions 85-89 of Ha-Ras, such as NNTKS85-89TAQST, NN85-86TA, and TKS87-89QST, are sufficient to induce the perinuclear localization of Ha-Ras. In contrast, substitutions of residues surrounding this region, such as FAI82-84YSI and FEDI90-93FNDL, have no effect on the plasma membrane localization of Ha-Ras. A chimeric construct consisting of amino acids 1-134 of Rap1A and 134-189 of Ha-Ras, which harbors both the palmitoylation and farnesylation sites of Ha-Ras, exhibits the perinuclear localization like Rap1A. Introduction of a Ha-Ras-type substitution into amino acids 85-89 (TAQST85-89NNTKS) of this chimeric construct causes alteration of its predominant subcellular localization site from the perinuclear region to the plasma membrane. These results indicate that a previously uncharacterized domain spanning amino acids 85-89 of Rap1A plays a pivotal role in its perinuclear localization. Moreover, this domain acts dominantly over COOH-terminal lipid modification of Ha-Ras, which has been considered to be essential and sufficient for the plasma membrane localization.     

Identification of a neuron-specific human gene, KIAA1110, that is a guanine nucleotide exchange factor for ARF1

To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.     

Corticotropin-releasing factor and adrenocorticotropin stimulate ciliary motility in rabbit tracheal epithelium

To assess the effects of corticotropin-releasing factor (CRF) and adrenocorticotropin (ACTH) on airway ciliary activity, we measured ciliary beat frequency (CBF) by a photoelectric method in response to these peptides in cultured rabbit tracheal explants. When cumulatively added, both CRF and ACTH increased CBF in a dose-dependent fashion. Treatment of tissues with Ca2+-free medium or nifedipine abolished the effect of CRF but not of ACTH. The CRF- and ACTH-induced ciliostimulations were not affected by indomethacin or autonomic antagonists, but were attenuated by nordihydroguaiaretic acid and by their receptor antagonists, alpha-helical CRF (9-41) and ACTH (7-38). Intracellular cyclic AMP levels were significantly increased by CRF and ACTH. These results suggest that CRF and ACTH stimulate airway ciliary motility through the activation of adenylate cyclase and lipoxygenase by binding to their specific receptors, where the effect of CRF may be triggered by Ca2+ influx.     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. I. Investigations on Antibody Levels to Japanese Encephalitis Virus in Swine Sera and Vector Mosquito in Okinawa,Miyako and Ishigaki Islands

From 1985 to 1989, serum specimens of swine raised in the northern, central and southern areas in Okinawa island were examined for Japanese encephalitis (JE) virus antibody by ELISA and hemagglutination-inhibition test. The antibody positive rate was found to be higher in the north and central than in the south. The 2-mercaptoethanol sensitive antibody to JE was detected mostly in June and July, and occasionally in other months except February and March. There was no month when all specimens from three areas turned antibody-negative simultaneously, indicating that JE virus transmission to swine lasted longer in Okinawa island than in other temperate areas in Japan. From 1986 to 1991, the vector mosquitoes (Culex tritaeniorhynchus) were collected in a pig farm in the south of Okinawa island. A total of 153 strains of JE virus was isolated from the vector mosquitoes mainly in June. In Miyako and Ishigaki islands, the antibody positive rate in swine sera was found to be extremely low, compared with that in Okinawa island. In Miyako island, where no paddy rice field is cultivated, a few adults as well as larvae of the vector mosquito were collected, while in Ishigaki island, where there are many watered rice fields, a lot of adults as well as larvae were collected. Although the environmental situation is quite different between the two islands, JE virus transmission appeared to be very low in both islands.     

Host Factor SAMHD1 Restricts DNA Viruses in Non-Dividing Myeloid Cells

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.     

Hypoxia-induced phenotypic transition from highly invasive to less invasive tumors in glioma stem-like cells: Significance of CD44 and osteopontin as therapeutic targets in glioblastoma

The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence, GSCs with highly invasive and highly migratory activities must assume a less-motile state and proliferate to regenerate tumor mass. Elucidating the molecular mechanism underlying this transition from a highly invasive phenotype to a less-invasive, proliferative tumor could facilitate the identification of effective molecular targets for treating GBM. Here, we demonstrate that severe hypoxia (1% O₂) upregulates CD44 expression via activation of hypoxia-inducible factor (HIF-1α), inducing GSCs to assume a highly invasive tumor. In contrast, moderate hypoxia (5% O₂) upregulates osteopontin expression via activation of HIF-2α. The upregulated osteopontin inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, inducing GSCs to assume a less-invasive, highly proliferative tumor. These data indicate that the GSC phenotype is determined by interaction between CD44 and osteopontin. The expression of both CD44 and osteopontin is regulated by differential hypoxia levels. We found that CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. Mouse brain tumors generated from CD44-knockdown GSCs exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene decreased GSC proliferation. These results suggest that interaction between CD44 and osteopontin plays a key role in tumor progression in GBM; inhibition of both CD44 and osteopontin may represent an effective therapeutic approach for suppressing tumor progression, thus resulting in a better prognosis for patients with GBM.