SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene

Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribosomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. The results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.