A New Synthesis of (±)-9-Demethylmunduserone Thermal Rearrangement of Phenyl 2-Propynyl Ether and Its Application

A new synthesis of (±)-9-demethylmunduserone (2) is described. Thermal rearrangement of l-(4-benzyloxy-2-hydroxyphenyl)-4-(3′,4′-dimethoxyphenoxy)-2-butyn-1-one (7) afforded 4-(4-benzyloxy-2-hydroxybenzoyl)-6,7-dimethoxy-2H-chromene (8), 3-(4-benzyloxy-2-hydroxyberrzoyl)-5,6-dimethoxy-2-methylbenzofuran (9) and 9-benzyloxy-2,3-dimethoxy-6a,12a-dihydrorotoxen-12(6H)-one (3). 4-Aroyl-2H-chromene (8) was smoothly converted to 3 in quantitative yield by the treatment with sodium acetate. The structure of 3 was confirmed by an alternative synthesis from methyl tephrosate (10). Debenzylation of 3 with aluminum bromide afforded (±)-9-demethylmunduserone (2) in high yield.     

A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Val50 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.     

Macrophage inflammatory protein-1α induces osteoclast formation by activation of the MEK/ERK/c-Fos pathway and inhibition of the p38MAPK/IRF-3/IFN-β pathway

Multiple myeloma (MM) is a bone disease that affects many individuals. It was recently reported that macrophage inflammatory protein (MIP)-1α is constitutively secreted by MM cells. MIP-1α causes bone destruction through the formation of osteoclasts (OCs). However, the molecular mechanism underlying MIP-1α-induced OC formation is not well understood. In the present study, we attempted to clarify the mechanism whereby MIP-1α induces OC formation in a mouse macrophage-like cell line comprising C7 cells. We found that MIP-1α augmented OC formation in a concentration-dependent manner; moreover, it inhibited IFN-β and ISGF3γ mRNA expression, and IFN-β secretion. MIP-1α increased the expressions of phosphorylated ERK1/2 and c-Fos and decreased those of phosphorylated p38MAPK and IRF-3. We found that the MEK1/2 inhibitor U0126 inhibited OC formation by suppressing the MEK/ERK/c-Fos pathway. SB203580 induced OC formation by upregulating c-fos mRNA expression, and SB203580 was found to inhibit IFN-β and IRF-3 mRNA expressions. The results indicate that MIP-1α induces OC formation by activating and inhibiting the MEK/ERK/c-Fos and p38MAPK/IRF-3 pathways, respectively, and suppressing IFN-β expression. These findings may be useful in the development of an OC inhibitor that targets intracellular signaling factors.     

Unique lowermost Cambrian small shelly fossils (SSFs) from eastern Yunnan,South China: implications for the early diversification history of animals

With a particular focus on the earliest Cambrian diversification of small shelly fossils (SSFs), stratigraphic analysis was conducted on the lower Cambrian Zhongyicun Member at the Hongjiachong section in the Chengjiang area, Yunnan, China. From ca. 3-m-thick bedded phosphorites (Unit A) in the lower part of the member, we recovered unique SSFs. This interval with 4 SSF-bearing horizons is characterized by the dominance in blade-shaped SSFs, including Halkieria spp., Brushenodus prionodes, Sinosachites delicatus, and Pteronus sp., and the absence of typical tube/cap-shape SSFs common in the Fortunian. This interval tentatively named “Halkieria-dominant interval” is stratigraphically positioned ca. 3 m below the previously known level of the oldest mollusks in the continuous bedded phosphorite sequence between the Anabarites trisulcatus-Protohertzina anabarica Assemblage Zone and the overlying Paragloborilus subglobosa-Purella squamulosa Assemblage Zone of the Fortunian (the earliest Cambrian). The “Halkieria-dominant interval” yields some SSFs likely affiliated with ostracods in view of size and morphology, suggesting that arthropod body fossils appeared in the Fortunian, considerably earlier than previously believed.     

Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.     

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO₄). Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn²⁺, Ca²⁺, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of β-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO₄, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn²⁺ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of β-hexosaminidase, ultrafine ZnO particles and ZnSO₄, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn²⁺ easily and increase intracellular Zn²⁺ concentration efficiently, thus decreasing FcεRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.     

Synergistic effect of basic residues at positions 14-15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.     

GRWD1 negatively regulates p53 via the RPL11–MDM2 pathway and promotes tumorigenesis

The ribosomal protein L11 (RPL11) binds and inhibits the MDM2 ubiquitin ligase, thereby promoting p53 stability. Thus, RPL11 acts as a tumor suppressor. Here, we show that GRWD1 (glutamate‐rich WD40 repeat containing 1) physically and functionally interacts with RPL11. GRWD1 is localized to nucleoli and is released into the nucleoplasm upon nucleolar stress. Silencing of GRWD1 increases p53 induction by nucleolar stress, whereas overexpression of GRWD1 reduces p53 induction. Furthermore, GRWD1 overexpression competitively inhibits the RPL11–MDM2 interaction and alleviates RPL11‐mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N‐terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage‐independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene.     

A Galanin Receptor Subtype 1 Specific Agonist

The chimeric peptide M617, galanin(1–13)-Gln¹⁴-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K_i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K_i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca²⁺, high Mg²⁺, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.