Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth

Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on G(s) and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.     

Early development in utero of bovine nuclear transfer embryos using early G1 and G0 phase cells

Bovine somatic cell nuclear transfer (NT) embryos can develop to normal calves, but the success rates are still quite low. Recently, enhanced development of bovine NT embryos to full term has been achieved using fibroblasts at the early G1 phase instead of cells at the quiescent (G0) phase. In the present study, we examined the morphological development in utero of NT embryos using early G1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). We produced eG1- and G0-NT blastocysts, and then they were transferred to recipient heifers for transient development in utero up to day 14 of gestation. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The rate of formation of embryonic disks of the recovered embryos was the same among the groups of eG1-NT, IVF, and AI embryos (p>0.05). The formation rate in eG1-NT embryos was significantly higher than that in G0-NT embryos (p<0.05). The lengths of eG1-NT embryos were the same as those of IVF, parthenogenetic, and AI embryos (p>0.05), but significantly shorter than those of G0-NT embryos (p<0.01). We conclude that the morphological development of day 14 embryos derived from eG1-NT embryos was mostly similar to that of AI embryos, but that the morphological development of G0-NT embryos was abnormally large and different from that of AI and eG1-NT embryos.     

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.     

Effect of gap junction-mediated intercellular communication on TGF-β induced epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is the process in which epithelial cells lose cell polarity and cell adhesion with surrounding cells to obtain migratory and invasive abilities. On the other hand, the expression of connexin is decreased or lacked in the many types of tumor cells. This study examined the effect of gap junctional intercellular communication (GJIC) on EMT induced by the transforming growth factor-β1 (TGF-β1). To investigate the effect of GJIC on EMT in U2OS cells, smooth muscle 22-α (sm22α) promoter-driven luciferase reporter gene was introduced into Cx43-expressing cells (U2OS-Luc Cx43) and into the control parental cell line (U2OS-Luc). TGF-β1 induced the expression of EMT markers and the sm22α promoter activity of U2OS-Luc cells. Sm22α promoter activity of U2OS cells was neither dependent on the expression of Cx43 nor on the establishment of GJIC among U2OS cells. Furthermore, we found that the homocellular communication among tumor cells did not affected the tumor cell growth and migration. However, we revealed that tumor cell density was an important factor for tumor cells to acquire metastatic phenotype. Interestingly, the co-culture of U2OS cells with osteoblasts revealed that sm22α promoter activity was inhibited only by the GJIC established between these two cell types. These results suggest that normal osteoblast cells negatively regulate the EMT of tumor cells, at least in part. Thus, Cx43-mediated GJIC may have anti-metastatic activity in tumor cells. Our findings provide a new insight into the role of GJIC in cancer progression and metastasis and identify potential therapeutic targets for the treatment of cancer.     

Apoptosis induction by epigallocatechin gallate involves its binding to Fas

Epigallocatechin gallate (EGCG) is known to induce apoptosis in various types of tumor cells, but the precise mechanism by which EGCG induces apoptosis remains to be elucidated. The Fas-Fas ligand system is one of the major pathways operating in the apoptotic cascade. The aim of this study was to examine the possibility that EGCG-binding to Fas triggers the Fas-mediated apoptosis. The EGCG treatment of human monocytic leukemia U937 cells resulted in elevation of caspase 8 activity and fragmentation of caspase 8. The DNA ladder formation caused by the EGCG treatment was inhibited by the caspase 8 inhibitor. These findings suggested the involvement of the Fas-mediated cascade in the EGCG-induced apoptosis in U937 cells. Affinity chromatography revealed the binding between EGCG and Fas. Thus, the results suggest that EGCG-binding to Fas, presumably on the cell surface, triggers the Fas-mediated apoptosis in U937 cells.     

Effect of 13-hydroperoxyoctadecadienoic acid on the supply of arachidonic acid for prostaglandin synthesis from arachidonoyl-CoA mediated by the cytosolic or microsomal acyl-CoA hydrolase in rabbit kidney medulla

The effects of 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the cytosolic or microsomal acyl-CoA hydrolase (ACH) activity in rabbit kidney medulla and on the ACH-mediated prostaglandin (PG) formation from arachidonoyl-CoA (AA-CoA) were examined. 13-HPODE (10, 20, and 50 microM) had no effect on the cytosolic ACH activity but significantly inhibited the activity of the microsomal enzyme (43-57% inhibition). PG formation was measured as follows: AA-CoA (20 nmol) was preincubated with the cytosolic or microsomal fraction (as the source of ACH) in the presence or absence of 13-HPODE for 5 min at 37 degrees C, followed by incubation with the microsomal fraction (as the source of PG-synthesizing enzymes), hydroquinone and reduced glutathione for 5 min at 37 degrees C, and the PGs formed were measured by HPLC, with use of 9-anthryldiazomethane for derivatization. 13-HPODE reduced the PG formation when the microsomal fraction, but not the cytosolic fraction, was used as the source of ACH (10, 20, and 50 microM; 28-55% inhibition). These results suggest that 13-HPODE may modulate PG levels in rabbit kidney medulla by inhibiting the microsomal ACH activity.     

Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact alphabeta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both alpha-helices, a beta-bulge in the second beta-strand, and an additional short antiparallel beta-sheet coupled with a beta-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded beta-sheet, loops, and the C-terminal region. It is suggested that non-specific interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.