Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

α7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at −1′ in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the −1′ position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken α7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing α7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the α7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.     

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.     

Studies on functions of the 63-kDa A- and 74-kDa B'(delta)-regulatory subunits in human erythrocyte protein phosphatase 2A: dissociation and reassociation of the subunits

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.