Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.     

Photo-movement of coral larvae influences vertical positioning in the ocean

Coral Reefs - Behaviour can have profound consequences for the dispersal potential of an organism. In the marine environment, larvae rely heavily on oceanic currents to migrate from one area to...     

Identification of a stable chymase inhibitor using a pharmacophore-Based database search

In general, serine protease chymase inhibitors readily decompose in plasma. We previously found that thiazolidine-2,4-dione and thiadiazole derivatives are also unstable. Using a pharmacophore-based database search, we identified a benzo[b]thiophen-2-sulfonamide derivative as a stable chymase inhibitor. Finding a lead compound with adequate activity and stability by a pharmacophore-based approach is more efficient than modifying an unstable compound to reduce its instability without simultaneously decreasing its inhibitory activity. Our pharmacophore model of chymase inhibitors suggests that the two hydrophobic interactions in the S1 and S1' regions and the two H-bonding interactions between them play important roles in chymase inhibitors.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway

Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1-JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1-JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE-JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1-JNK pathway.     

PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (Delta psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of Delta psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the Delta psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the Delta psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.     

Epidermal growth factor stimulates proliferation of mouse uterine epithelial cells in primary culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/- 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.     

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Summary Intermediates of DNA replication in the second half of the latent period after phage infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative froms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the genome. Replicating circles with a linear DNA tail longer than one unit length of genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.     

Incorporation of methionine by a soluble enzyme system from Escherichia coli

A soluble fraction from Escherichia coli B was found to incorporate methionine into 95°C CCl₃COOH-insoluble fraction. The incorporation required methionyl-tRNA synthetase, methionine tRNA, ATP, Mg²⁺ and bovine milk casein. The casein could be replaced by arginylated bovine serum albumin and arginylated bovine α-lactalbumin. A mixture of 19 amino acids other than methionine and GTP had no effect on the incorporation. KCl was rather inhibitory. Puromycin, RNase A and trypsin inhibited the incorporation, while DNase I did not. The soluble fraction also incorporated the methionyl moiety of methionyl-tRNA. This incorporation was not affected by the addition of free methionine.