Gelastatins and their hydroxamates as dual functional inhibitors for TNF-alpha converting enzyme and matrix metalloproteinases: synthesis, biological evaluation, and mechanism studies

The hydroxamic acid analogues (2) of the natural product gelastatins (1) were prepared by 1 step conversion reaction. The synthetic analogues (2) showed potent enzymatic inhibitory activities against MMP-2, MMP-9, and TACE IC50's of 6, 23, and 28 nM, respectively. In addition, 2 were able to inhibit TNF-alpha production effectively in mice as well as in a macrophage cell line, RAW 264.7. The protective effect of 2 also was examined on LPS-induced acute septic shock model. The mechanism of TNF-alpha inhibition was examined by RT-PCR and Western blot analyses. The relation of TACE and alpha-secretase was examined using cellular alpha-secretase assays on IMR-32 and SH-SY5Y cell lines. The docking mode of 2 with the catalytic domain of TACE was illustrated to analyze the binding mode for the further analogue design.     

Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.     

G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is the first selenoprotein identified in the Golgi apparatus.     

Cryopreservation of somatic embryos of the herbaceous peony (Paeonia lactiflora Pall.) by air drying

This study was carried out to establish a suitable method for the cryopreservation of somatic embryos of the herbaceous peony. The somatic embryos were obtained from cotyledon and anther cultures on a MS medium supplemented with abscisic acid (ABA) and phenylacetic acid (PAA), respectively. The frequency of somatic embryo formation was the greatest (61%) from the cotyledons cultured on a MS medium supplemented with 1.0 mg l(-1) of ABA. Embryos were also obtained directly from anthers cultured on a MS medium with or without 2.0 mg l(-1) of PAA. For the cryopreservation of peony somatic embryos, the embryos were dried under a stream of sterile air and frozen by immersion in liquid nitrogen. Thawed embryos were germinated into plantlets after placing on a medium containing 0.3 mg l(-1) of gibberellic acid (GA(3)). The frequency of the post-thaw regrowth of cryopreserved somatic embryos was related to their size and desiccation time, the latter ranging from 0 to 2 h. When the somatic embryos were desiccated for 1 h, the frequency of post-thaw regrowth was greater than 66%. The frequency of post-thaw regrowth of the cryopreserved somatic embryos from anthers and cotyledon tissues was generally high when they were 2-3 mm in size. Desiccation may be a suitable method for the cryopreservation of somatic embryos of the herbaceous peony.     

Lipid membrane interaction and antimicrobial activity of GsMTx-4, an inhibitor of mechanosensitive channel

GsMTx-4, a polypeptide from the spider Grammostola spatulata, is an inhibitor of mechanosensitive channels. It is known to interact with lipid membranes, suggesting it partitions into the membrane to alter the channel gating, but the effect of the membrane charge on GsMTx-4 activity remains unknown. In this study, we found that GsMTx-4 more effectively interacts with anionic lipids than zwitterionic ones. The effect of GsMTx-4 on negatively charged membranes was similar to that of the antimicrobial peptide melittin, which led us to assess GsMTx-4's antimicrobial activity. Interestingly, we found that, in contrast to other neurotoxins, GsMTx-4 exhibited antimicrobial properties and was more active against Gram-positive than Gram-negative bacteria. These results suggest that GsMTx-4 exerts its antimicrobial effect by altering the packing of the membrane and/or inhibiting mechanosensitive channels. These findings could point the way towards a new class of antimicrobial peptides.     

Genetic Polymorphism of Interferon-γ, Interferon-γ Receptor, and Interferon Regulatory Factor-1 Genes in Patients with Hepatitis B Virus Infection

The natural history of hepatitis B virus (HBV) infection is probably related to host immune factors. Interferon-γ (IFN-γ) plays significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of IFN-γ, IFN-γ receptor (IFNGR)-1 and 2, and interferon regulatory factor (IRF)-1 genes. Between March 2002 and December 2002, 614 Korean patients were enrolled in two different groups: an HBV clearance group (n = 201), who were hepatitis B surface antigen (HBsAg) negative with antibodies to HBsAg and hepatitis B core antigen, and an HBV persistence group (n = 413), who were repeatedly HBsAg positive. We assessed polymorphisms in the IFN-γ gene at position +874, in the IFNGR-1 gene at positions −56 and +95, in the IFNGR-2 gene at the second position of codon 64 (Gln64Arg), and in the IRF-1 gene promoter (−410, −388), and the genotype distributions of the HBV clearance and persistence groups were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with the IFN-γ, IFNGR-1 and 2, and IRF-1 gene polymorphisms under the codominant, dominant, and recessive models.     

Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family

Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.