Identification of Valine- or Leucine-Containing Glycopeptidolipids from Mycobacterium avium–intracellulare Complex

Mycobacterium avium-intracellulare complex is a species of acid-fast microorganisms that cause opportunistic infections in immuno-compromised hosts. The cell wall of this microbe is rich in glycopeptidolipids (GPLs), which are composed of a fatty acyl moiety, several sugar moieties and a tripeptide-amino alcohol, D-Phe-D-alloThr-D-Ala-L-Alaninol. GPLs have molecular diversity in the hydrocarbon chain variety of the acyl moiety, and methyl and acetyl modifications of the sugar moiety, but there has been no report of any variety in the tripeptide-amino alcohol component. In this study, we showed two atypical GPL ions of 34 or 48 Da less than the dominant ions of GPLs by mass spectrometry. These ions could not be explained as resulting from conventional molecular diversity. To investigate the reasons why these ions appeared, we made a preparation of the lipopeptide component from intact GPLs and structurally analyzed the molecules. The results suggested that these atypical ions differed from the typical ions in amino acid composition. We further determined its composition by amino acid analysis, and the results showed that the tripeptide portion of the two atypical ions is composed of the Val-alloThr-Ala or the Leu-alloThr-Ala amino acid sequence. In this study, we present novel variations in the tripeptide portion of GPL molecules.     

Dichotomous organization of the external globus pallidus

Different striatal projection neurons are the origin of?a?dual organization essential for basal ganglia function. We have defined an analogous division of labor in the external globus pallidus (GPe) of Parkinsonian rats, showing that the distinct temporal activities of two populations of GPe neuron in?vivo are?underpinned by distinct molecular profiles and axonal connectivities. A first population of prototypic GABAergic GPe neurons fire antiphase to subthalamic nucleus (STN) neurons, often express parvalbumin, and target downstream basal ganglia nuclei, including STN. In contrast, a second population (arkypallidal neurons) fire in-phase with STN neurons, express preproenkephalin, and only innervate the striatum. This novel cell type provides the largest extrinsic GABAergic innervation of striatum, targeting both projection neurons and interneurons. We conclude that GPe exhibits several core components of?a dichotomous organization as fundamental as?that in striatum. Thus, two populations of GPe neuron?together orchestrate activities across all basal ganglia nuclei in a cell-type-specific manner.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.     

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH₂-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.     

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.     

Regulation of insulin signaling and glucose transporter 4 (GLUT4) exocytosis by phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, skeletal muscle, and kidney enriched inositol polyphosphate phosphatase (SKIP)

The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate PIP(3) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signaling in the skeletal muscle cells. Here, we compared its effects on insulin signaling by selective inhibition of SKIP, SHIP2, and phosphatase and tensin homologue on chromosome 10 (PTEN) by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signaling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis

Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.