A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.     

Therapeutic effect of anti-macrophage inflammatory protein 2 antibody on influenza virus-induced pneumonia in mice

We investigated the effect of anti-macrophage inflammatory protein 2 immunoglobulin G (aMIP-2 IgG) on the progression of influenza virus-induced pneumonia in mice. When mice were infected with a mouse lung-adapted strain of influenza A/PR/8/34 virus by intranasal inoculation, neutrophil counts in the bronchoalveolar lavage fluid (BALF) increased in parallel with the kinetics of MIP-2 production, which peaked 2 days after infection. After intracutaneous injection of a dose of 10 or 100 microg of aMIP-2 IgG once a day on days 0 and 1, neutrophil counts in BALF on day 2 were reduced to 49 or 37%, respectively, of the value in the control infected mice administered anti-protein A IgG. The antibody administration also improved lung pathology without affecting virus replication. Furthermore, by prolonged administration with a higher or lower dose for up to 5 days, body weight loss became slower and finally 40% of mice in both treatment groups survived potentially lethal pneumonia. These findings suggest that MIP-2-mediated neutrophil infiltration during the early phase of infection might play an important role in lung pathology. Thus, MIP-2 was considered to be a novel target for intervention therapy in potentially lethal influenza virus pneumonia in mice.     

Selective excretion of anti-inflammatory cytokine Interleukin-10 in a superantigen-inducing neonatal infectious disease

Neonatal toxic shock syndrome (TSS)-like exanthematous disease (NTED) is an emerging neonatal infectious disease caused by TSS toxin-1 (TSST-1). Although NTED and TSS are caused by the same superantigenic exotoxin, NTED is less severe than TSS. The mechanism of this reduced severity in NTED has not been elucidated. Thirteen patients with NTED were enrolled in the study. We investigated serum cytokine profile using a cytometric bead array system with a cytokine panel. Expression of Vβ2 and CD45RO in CD4⁺ T cells was investigated in mononuclear cells by using flowcytometry. Ten patients with other bacterial infections and eight patients without any infections were also enrolled as control groups. The mean serum level of IL-10 was 1209.9 pg/mL in patients with NTED at the time of admission into the study. The other inhibitory cytokine, IL-4, exhibited a minimum level. The high level of IL-10 rapidly decreased within 3–9 days of the onset of NTED. The cytokine profile of NTED, with its high IL-10 level, was clearly different from that of the other bacterial infections. The increased level of IL-10 seems to be related to the reduced severity of NTED. Th2 shift is not thought to be the cause of this IL-10 excretion.     

PI3K inhibition causes the accumulation of ubiquitinated presenilin 1 without affecting the proteasome activity

γ-Secretase is an enzymatic complex, composed of presenilin 1 (PS1), nicastrin, pen-2, and aph-1, and is responsible for the intramembranous cleavage of various type-I membrane proteins. The level of each component is tightly regulated in a cell via proteasomal degradation. On the other hand, it has previously been reported that PS1/γ-secretase is involved in the activation of phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. PI3K is inhibited in Alzheimer’s disease (AD) brain, whereas the effects of PI3K inhibition on the metabolism of PS1/γ-secretase have not been elucidated. Here, we demonstrate that the treatment of neurons with PI3K inhibitors leads to increased levels of PS1/γ-secretase components through an inhibitory effect on their degradation. Moreover, PI3K inhibition accelerated ubiquitination of PS1. We further show the evidence that the PS1 ubiquitination after PI3K inhibition is represented by the multiple mono-ubiquitination, instead of poly-ubiquitination. Accordingly, treatment of cells with PI3K inhibitor led to a differential intracellular redistribution of PS1 from the one observed after the proteasomal inhibition. These results suggest that PI3K inhibition may trigger the multiple mono-ubiquitination of PS1, which precludes the degradation of PS1/γ-secretase through the proteasomal pathway. Since PS1/γ-secretase is deeply involved in the production of Aβ protein, a deeper knowledge into its metabolism could contribute to a better elucidation of AD pathogenesis.