Macrolide antibiotics inhibit prostaglandin E2 synthesis and mRNA expression of prostaglandin synthetic enzymes in human leukocytes

We investigated the action of macrolide antibiotics, which are considered to have anti-inflammatory activity, on lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 synthesis and the expression of mRNAs for cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX)-1, and COX-2 in human leukocytes. The production of LPS-stimulated PGE2 was significantly increased in peripheral polymorphonuclear leukocytes (PMNLs) and in mononuclear leukocytes (MNLs). Amounts of mRNAs for COX-2 and cPLA2, but not for COX-1, were enhanced by LPS in PMNLs and MNLs. The LPS-enhanced PGE2 synthesis and the expression of cPLA2 and COX-2 mRNAs were inhibited by clarithromycin, azithromycin and dexamethasone in PMNLs and MNLs. The mRNA expression of COX-1 in PMNLs was decreased by clarithromycin and azithromycin. Macrolide antibiotics inhibited PGE2 synthesis in human leukocytes by suppressing cPLA2, COX-1, and COX-2 mRNA expression. These data indicate one mechanism of macrolide anti-inflammatory activity.     

A novel metal-chelating inhibitor of protein farnesyltransferase

A novel metal chelator comprising a 4-(naphthalen-1-yl)pyridine and 2-aminoethanethiol was synthesized. This showed inhibitory activity against human protein farnesyltransferase with IC(50) 1.9 microM, induced morphological change in K-ras-NRK cells at 0.5 microg/mL and showed growth inhibition of K-ras-NRK cells with IC(50) 0.32 microg/mL.     

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.     

Evidence for a novel affinity mechanism of motor-assisted transport along microtubules

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (-) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that beta tubulin is exposed at the (+) end of the MT.     

Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Helical image reconstruction of the outward-open human erythrocyte band 3 membrane domain in tubular crystals

The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18 Å resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 × 110 Å in the membrane plane and a thickness of 70 Å across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface.     

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.     

Structural factors contributing to the Abl/Lyn dual inhibitory activity of 3-substituted benzamide derivatives

To investigate why 3-substituted benzamide derivatives show dual inhibition of Abl and Lyn protein tyrosine kinases, we determined their inhibitory activities against Abl and Lyn, carried out molecular modeling, and conducted a structure-activity relationship study with the aid of a newly determined X-ray structure of the Abl/Lyn dual inhibitor INNO-406 (formerly known as NS-187) bound to human Abl. We found that this series of compounds interacted with both kinases in very similar ways, so that they can inhibit both kinases effectively.