Hormogonia mass differentiation from Nostoc sphaeroides Kütz. (cyanobacterium) and the comparison of structural characteristics between hormogonia and vegetative filaments

It was found that, after colonies of Nostoc sphaeroides Kütz. in exponential phase of growth were transferred to fresh complete BG‐11₀ medium, the hormogonia differentiated independent of the type of preculture used. This provided evidence that the hormogonia differentiation was not directly related to phosphorus and potassium status, nor to the osmotic effect of the media. In contrast, all the cultures in the stationary growth phase had no hormogonium differentiation after being transferred to fresh medium. However, the incomplete media for preculture seemed to favor the liberation of hormogonia from colonies to the medium. The result showed that the morphology and ultrastruc‐tures of the vegetative filament, the main stage of Nostocacean life history, determine its adaptability in changing environments, while the hormogonium remains as a propagule of the species.     

Syntheses and characterization of mono- and di-N-hydroxyethylated tetraaza macrocycles containing eight C-methyl groups and their nickel(II) and copper(II) complexes

New partially N-hydroxyethylated 14-membered tetraaza macrocycles 1,8-bis(2-hydroxyethyl)-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L²) and 1-(2-hydroxyethyl))-3,5,7,7,10,12,14,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L³) have been synthesized selectively by the one-step reaction of 2,5,5,7,9,12,12,14-octamethyl-1,4,8,11-tetraazacyclotetradecane (L¹) with 2-hydroxyethyl bromide. The complexes [NiL³]²⁺, [CuL²]²⁺, and [CuL³]²⁺ have been prepared and characterized. The complex [CuL²](ClO₄)₂ has a square-pyramidal coordination geometry with one apical oxygen atom; only one of the two hydroxyethyl groups is coordinated to the metal ion. Electronic absorption spectra of [CuL³](ClO₄)₂ containing one hydroxyethyl pendant arm indicate that the geometry is similar to that of [CuL²](ClO₄)₂. Unexpectedly, the nickel(II) complex [NiL³](ClO₄)₂ has a severely distorted trigonal bipyramidal coordination geometry with the oxygen atom of the pendant arm at the equatorial position. The Ni---O bond distance of the nickel(II) complex is shorter, or not longer, than the Ni---N bond distances. The ligand in [CuL²]²⁺ is in the RRSS (trans-III) configuration, as usual, whereas that in [NiL³]²⁺ has the RRRR (trans-V) conformation. The coordination geometry and properties of [NiL³]²⁺ are quite different from those reported for other related nickel(II) complexes containing one functional pendant arm.     

Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.     

betaPak-interacting exchange factor-mediated Rac1 activation requires smgGDS guanine nucleotide exchange factor in basic fibroblast growth factor-induced neurite outgrowth

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.     

Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: functional analysis of conserved aromatic amino acids in Region 1

Soybean sterol methyltransferase (SMT) in the presence of AdoMet catalyzes the transmethylation of the delta24-bond of the sterol side chain to produce phytosterols with a methyl(lene) or ethyl(idene) group at C-24. The function of six aromatic amino acids associated with the putative active center of the SMT, i.e., Region 1 that extends from Phe82 to Phe93 in soybean SMT, was studied by site-directed mutagenesis and heterologous expression in BL21(DE3) bacterial cells. The enzyme-generated products were characterized kinetically and by GC-MS analysis. Substitution of the aromatic amino acids at positions 82, 83, 85, 87, 91, and 93 with a leucine residue produced mutant SMTs with varying activities. The mutants converted cycloartenol to 24(28)-methylene cycloartanol [C1-activity] from a few percent to as much as 95% of the control activity. In contrast, none of the leucine mutants were found to catalyze 24(28)-methylene lophenol [C2-activity], suggesting a loss of function associated with the second C1-transfer activity. In contrast to the loss of the second C1-transfer activity of the Phe82Leu, replacement of the Phe82 residue to isoleucine had minimal effect on the first or second C1-transfer activities, suggesting that the increased bulk (branching) in the leucine side chain contributes to significant perturbations in the active site that generate inaccurate positioning of the substrate side chain disfavoring the second C1-transfer activity. Replacement of Tyr83 to phenylalanine resulted in an increase of the specificity constant (kcat/Km) for the substrate of the second C1-transfer activity by a factor of 5 compared to control and an increase of delta24(28)Z-ethylidene sterol formation in the 24-ethyl sterol product set, suggesting that loss of steric bulk from the phenolic hydroxyl group on tyrosine generates a less precise fit of the delta24(28) sterol side chain into the active site favoring the second C1-transfer activity and prompting reaction channeling during catalysis. Circular dichroism spectra, equilibrium dialysis studies of AdoMet, and chromatographic information of the wild-type and Tyr83 mutants confirmed retention of the overall conformation of the enzyme during the experiments. Together, these findings suggest that the amino acids of Region 1 provide a tight substrate orientation imposed by hydrophobic interactions between the sterol side chain and the SMT active site contacts and control the production and processing of the transmethylation pathways governed by the first and second C1-transfer activities.     

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.     

A concise and practical synthesis of antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc

A concise and practical synthesis of the antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc (13), was achieved by coupling of a monosaccharide donor, 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate (4) with a disaccharide acceptor, p-methoxyphenyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (8), followed by deprotection. In spite of the existence of a C-2-ester substituent capable of neighboring-group participation in the donor, the coupling gave exclusively the alpha-linkage in satisfactory yield. The acceptor 8 was readily obtained from selective 3-O-benzoylation of the galactosyl ring of p-methoxyphenyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (7), which was prepared from p-methoxyphenyl beta-D-lactoside (5) via isopropylidenation, benzoylation, and deisopropylidenation. Donor 4 was obtained from p-methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside (1) via selective 4,6-di-O-debenzoylation, oxidative removal of 1-O-MP, benzylidenation, and trichloroacetimidate formation.     

Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases

We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.