Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance

Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of approximately 2:1 (molar ratio). In addition, we show that INI1/hSNF5 is not incorporated into related primate lentiviral and murine retroviral particles despite the abundance of the protein in producer cells. We have found that the specificity in incorporation of INI1/hSNF5 into HIV-1 virions is directly correlated with its ability to exclusively interact with HIV-1 integrase but not with other retroviral integrases. This specificity is also reflected in our finding that the transdominant mutant S6, harboring the minimal integrase interaction domain of INI1/hSNF5, blocks HIV-1 particle production but not that of the other retroviruses in 293T cells. Taken together, these results suggest that INI1/hNSF5 is a host factor restricted for HIV-1 and that S6 acts as a highly specific and potent inhibitor of HIV-1 replication.     

Extracellular signal-regulated kinase 1c (ERK1c), a novel 42-kilodalton ERK, demonstrates unique modes of regulation, localization, and function

Extracellular signal-regulated kinases (ERKs) are signaling molecules that regulate many cellular processes. We have previously identified an alternatively spliced 46-kDa form of ERK1 that is expressed in rats and mice and named ERK1b. Here we report that the same splicing event in humans and monkeys causes, due to sequence differences in the inserted introns, the production of an ERK isoform that migrates together with the 42-kDa ERK2. Because of the differences of this isoform from ERK1b, we named it ERK1c. We found that its expression levels are about 10% of ERK1. ERK1c seems to be expressed in a wide variety of tissues and cells. Its activation by MEKs and inactivation by phosphatases are slower than those of ERK1, which is probably the reason for its differential regulation in response to extracellular stimuli. Unlike ERK1, ERK1c undergoes monoubiquitination, which is increased with elevated cell density concomitantly with accumulation of ERK1c in the Golgi apparatus. Elevated cell density also causes enhanced Golgi fragmentation, which is facilitated by overexpression of native ERK1c and is prevented by dominant-negative ERK1c, indicating that ERK1c mediates cell density-induced Golgi fragmentation. The differential regulation of ERK1c extends the signaling specificity of MEKs after stimulation by various extracellular stimuli.     

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.     

Replacement of the alpha5 helix of Galpha16 with Galphas-specific sequences enhances promiscuity of Galpha16 toward Gs-coupled receptors

G(16) can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Galpha(16), three chimeras incorporating increasing lengths of G(s)-specific residues (25, 44 or 81 residues) into the C-terminus of Galpha(16) were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca(2+) mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to G(s)-linked receptors. 16s25 coupled productively to 10 different G(s)-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Galpha(16). Similar results were observed for agonist-induced Ca(2+) mobilizations. These results show that incorporating the alpha5 helix of Galpha(s) into Galpha(16) can increase the promiscuity of 16s25 towards G(s)-coupled receptors.     

2,3-diarylpyran-4-ones: a new series of selective cyclooxygenase-2 inhibitors

A new series of cyclooxygenase-2 (COX-2) inhibitors with gamma-pyrone as central scaffold unit has been synthesized and their biological activities were evaluated against cyclooxygenase inhibitory activity. The changes of physical properties of the molecules were performed according to the medicinal chemistry principles and moderate oral anti-inflammatory activity was obtained with this series of inhibitors.     

Molecular phylogeny and biogeography of the Korean woodroaches Cryptocercus spp

Phylogenetic relationships and biogeography of Northeast Asian Cryptocercus were inferred based on the DNA sequences of mitochondrial COII and 16S rRNA genes and nuclear 18S rRNA gene. The results suggest that two clades exist in Korean populations. The southwestern population (Cryptocercus from Jiri-san) was more closely related to the populations from Northeast China and eastern Russia than to all the other Korean Cryptocercus. According to molecular-based estimated divergence times, the divergence event occurred between Cryptocercus in Jiri-san, Northeast China and eastern Russia and those in the remaining South Korea during the Miocene (7.5-17.4Myr ago), and then the divergence event between Cryptocercus in Jiri-san and those in Northeast China and eastern Russia occurred 0.8-1.9Myr ago. In the Korean Peninsula, Jiri-san is located in the most southwestern region among the high mountains surveyed. The location is the farthest from Northeast China and eastern Russia among sampling localities in South Korea. Thus, it was unexpected that the southwestern populations are more closely related to those from Northeast China and eastern Russia rather than to the other Korean Cryptocercus. Based on Korean topography and estimated divergence times, possible scenarios are proposed for the current geographical distribution of Korean Cryptocercus.     

Real-time analysis of the activities of GnRH and GnRH analogs in alphaT3-1 cells by the Cytosensor microphysiometer

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived alphaT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC(50) = 1.81 +/- 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on alphaT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH(2) at the C-terminus by N-ethylamide dramatically reduced the EC(50) value, suggesting that substitution of the Gly-NH(2) moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC(50) value, whereas D-Lys at the same position slightly increased the EC(50) value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in alphaT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future.     

Inhibition of HIV-1 virion production by a transdominant mutant of integrase interactor 1

Integase interactor 1 (INI1), also known as hSNF5, is a protein that interacts with HIV-1 integrase. We report here that a cytoplasmically localized fragment of INI1 (S6; aa183-294) containing the minimal integrase-interaction domain potently inhibits HIV-1 particle production and replication. Mutations in S6 or integrase that disrupt integrase-INI1 interaction abrogated the inhibitory effect. An integrase-deficient HIV-1 transcomplemented with integrase fused to Vpr was not affected by S6. INI1 was specifically incorporated into virions and was required for efficient HIV-1 particle production. These results indicate that INI1 is required for late events in the viral life cycle, and that ectopic expression of S6 inhibits HIV-1 replication in a transdominant manner via its specific interaction with integrase within the context of Gag-Pol, providing a novel strategy to control HIV-1 replication.     

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.     

ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK

We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK.