Effect of Phospholipid on Pea Cytochrome c Oxidase

Inhibitors of angiotensin I-converting enzyme were isolated from an enzymatic hydrolysate of bovine casein. The amino acid sequences of these inhibitors were Phe-Phe-Val-Ala-Pro-Phe-Pro- Glu-Val-Phe-Gly-Lys (CEI₁₂), Phe-Phe-Val-Ala-Pro (CEI₅), and Ala-Val-Pro-Tyr-Pro-Gln-Arg (CEI_β7). CEI₅ is a penta-peptide derived from the hydrolysate of CEI₁₂ with proline-specific endopeptidase, and CEI_β7 is a hepta-peptide derived from β-casein. These inhibitors potentiated bradykinin in the contraction of the uterus and the ileum of rats. The ileum was more sensitive to these inhibitors than the uterus. The bradykinin-potentiating activity of these inhibitors on the ileum lasted for more than 90 min even after washing the organ.     

Induced Synthesis of Mitochondrial RNA in Sweet Potato Root after Wounding

Ice structure was photographically analyzed for frozen soy protein curd and egg albumin gel frozen under various conditions. Dendritic ice structure was observed growing from the cooling plate parallel to the direction of the heat flux. The change in the ice structure size was analyzed at different locations from the cooling plate in the plane perpendicular to the direction of heat flux. In accordance with the theoretical relationship proposed by us before, the mean ice structure size was inversely proportional to the moving speed of the freezing front and the proportionality constant was not very much different from the diffusion coefficient of water, showing the important role of the molcular diffusion mechanism in the process of ice crystal growth. For the freezing accompanied with supercooling, the ice structure became very small, reflecting the very rapid moving speed of the freezing front when supercooling ceased. The theoretical model by us had advantages over the models proposed in the literature for its simple theoretical basis and wider applicability.     

Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer

A novel naphthylmethylimidazole derivative 1 and its related compounds were identified as 17,20-lyase inhibitors. Based on the structure-activity relationship around the naphthalene scaffold and the results of a docking study of 1a in the homology model of 17,20-lyase, the 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and identified as a potent and highly selective 17,20-lyase inhibitor. Biological evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked reductions in both serum testosterone and dehydroepiandrosterone concentrations. Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for clinical evaluation and is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer.     

β1-Adrenergic Receptor Recycles Via a Membranous Organelle, Recycling Endosome, by Binding with Sorting Nexin27

In cardiomyocytes, β₁-adrenergic receptor (β₁-AR) plays an important role in regulating cardiac functions. Upon continuous ligand stimulation, β₁-AR is internalized and mostly recycled back to the plasma membrane (PM). The recycling endosome (RE) is one of the membranous organelles involved in the protein recycling pathway. To determine whether RE is involved in the internalization of β₁-AR upon ligand stimulation, we evaluated the localization of β₁-AR after stimulation with a β-agonist, isoproterenol (Iso), in β₁-AR-transfected COS-1 cells. After 30 min of Iso treatment and cell surface labeling with the appropriate antibodies, β₁-AR was internalized from PM and translocated into the perinuclear region, the same location as the transferrin receptor, an RE marker. We then evaluated whether sorting nexin 27 (SNX27) participated in the β₁-AR recycling pathway. When β₁-AR and SNX27 were coexpressed, β₁-AR coimmunoprecipitated with SNX27. In addition, shRNA-mediated silencing of SNX27 compromised β₁-AR recycling and enhanced its delivery into lysosome. Overall, β₁-AR on PM was internalized into RE upon Iso stimulation and recycled by RE through binding with SNX27 in COS-1 cells.     

Mammalian cell cultivation using nutrients extracted from microalgae

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of “cultured meat.” Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes.     

Acrolein,an Environmental Toxin,Induces Cardiomyocyte Apoptosis via Elevated Intracellular Calcium and Free Radicals

Acrolein, an unsaturated aldehyde, is an environmental toxin known to inhibit mitochondrial electron transport chain in brain and induce lipid peroxidation and apoptosis. However, the nature of the effects of acrolein on cardiac function and myocardium is not known. The objective of this study is to examine whether acrolein induces apoptosis in cardiomyocytes and alters cytosolic calcium concentration and the intracellular oxygen free-radical levels. Adult mouse cardiomyocytes exposed to 1 μmol/l of acrolein showed a marked increase in the intracellular oxygen free-radicals and calcium concentration, by 12- and 2-fold, respectively, compared to the resting value. Moreover, the cardiomyocyte viability decreased significantly in a dose-dependent manner by treatment with 25, 50, and 100 μmol/l of acrolein compared to controls. Morphological changes and DNA laddering typical of apoptosis were found in acrolein-exposed cardiomyocytes. Our finding suggested that acrolein caused apoptotic death of adult mice cardiomyocytes by increasing intracellular oxygen free-radicals and calcium concentration.     

N-glycans of SREC-I (scavenger receptor expressed by endothelial cells): essential role for ligand binding, trafficking and stability

Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn(289), Asn(382) and Asn(393), which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn(393) regulates the intracellular sorting of SREC-I and that the N-glycan of Asn(382) controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn(289). A bisecting GlcNAc was also detected at Asn(382) and Asn(393). Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a β1-6GlcNAc branch at Asn(289) protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn(289)-, Asn(382)- and Asn(393)-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis.     

Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo

Background. Helicobacter pylori CagA is injected into the host cell and tyrosine‐phosphorylated. We examined tyrosine‐phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. Methods. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine‐phosphorylation sites were determined using quadropolar time‐of‐flight (Q‐TOF) mass spectrometry analysis. Specific anti‐pY CagA polyclonal and anti‐CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Results. Mass spectrometry identified five crucial tyrosine‐phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)‐infected AGS cells. CagA protein also had an immuno‐receptor tyrosine‐based activation motif (ITAM)‐like amino acid sequences in the 3′ region of the cagA, E PIY ATI x₂₇EIY ATI , which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX‐100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine‐phosphorylated GIT1/Cat1 (G protein‐coupled receptor kinase‐interactor 1/Cool‐associated tyrosine‐phosphorylated 1), substrate molecules of receptor type protein‐tyrosine phosphatase (RPTPζ/β), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA‐induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine‐phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. Conclusion. These findings suggest an important role for CagA containing ITAM‐like sequences in the pathogenesis of H. pylori‐related disease.     

Analysis of egg antigens inducing hepatic lesions in schistosome infection

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4⁺ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best-studied egg component is the Sm-p40 antigen. Sm-p40 elicits a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are characterized by severe egg-induced immunopathology. Two additional described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Other components, including moieties with molecular weights of 25 kDa (Sm-p25), 150/166 kDa (Sm-p155/166), and 29 kDa (Sm-GST29), are also found to stimulate specific T cells. These findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to ascertain whether such responses can be manipulated for the purpose of reducing pathology.     

Production of β-(pyrazol-1-yl)-l-alanine from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A

-(Pyrazol-1-yl)-l-alanine (-PA) was produced from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A. The amount of -PA increased with increasing l-serine concentrations up to 600 mM at 50 mM pyrazol while 100 mM pyrazol gave the highest -PA production with 50 mM l-serine. Under the optimized conditions, -PA accumulated in the broth at approximately 140 mM with a conversion of 90% with respect to the added amount of pyrazol.