Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

Human bronchial smooth muscle cell proliferation via thromboxane A2 receptor

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.     

Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome. ischemia; cultured cardiomyocytes     

Plant-growth inhibitory activity of heliannuol derivatives

In addition to (+)-, (-)- and (+/-)-heliannuol E, growth-inhibitory activities of five synthetic chromans and four tetrahydrobenzo[b]oxepins were examined against oat and cress. All heliannuol E isomers exhibited similar biological activities against cress, whereas when tested against oat roots, the unnatural optical isomer (+) showed no inhibitory activity. Four brominated chromans and two tetrahydrobenzo[b]oxepin derivatives also showed apparent inhibition against both cress and oat.     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

Construction of reporter yeasts for mouse aryl hydrocarbon receptor ligand activity

Aryl hydrocarbons such as dioxins, polychlorinated biphenyls and polyaromatic hydrocarbons bind to the cellular aryl hydrocarbon receptor (AhR) in the initial step of their metabolism. The activation of intracellular signaling subsequent to the AhR binding is highly correlated with the toxicity and carcinogenicity of these chemicals. We produced Saccharomyces cerevisiae coexpressing mouse AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) protein in accordance with Miller III's method for constructing yeasts with human Ahr and Arnt [Toxicol. Appl. Pharmacol. 160 (1998) 297]. Ligand treatment induced a dose-dependent increase in beta-galactosidase activity from a reporter plasmid in the yeast. Then, we compared activities of several ligands in yeast having the mouse Ahr/Arnt genes with those in yeast having the human genes, both of which have the same genetic background. There was no significant difference in the EC50 values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone between the mouse and human genes. However, indirubin, which was recently found in human urine as a potent AhR ligand [J. Biol. Chem. 276 (2001) 31475], had a 35-140 times higher EC50 value in the yeast with human genes than mouse genes. This difference might reflect species-specificity between mouse and human AhR/Arnt.     

Somatostatin suppresses ghrelin secretion from the rat stomach

Ghrelin is an acylated peptide that stimulates food intake and the secretion of growth hormone. While ghrelin is predominantly synthesized in a subset of endocrine cells in the oxyntic gland of the human and rat stomach, the mechanism regulating ghrelin secretion remains unknown. Somatostatin, a peptide produced in the gastric oxyntic mucosa, is known to suppress secretion of several gastrointestinal peptides in a paracrine fashion. By double immunohistochemistry, we demonstrated that somatostatin-immunoreactive cells contact ghrelin-immunoreactive cells. A single intravenous injection of somatostatin reduced the systemic plasma concentration of ghrelin in rats. Continuous infusion of somatostatin into the gastric artery of the vascularly perfused rat stomach suppressed ghrelin secretion in both dose- and time-dependent manner. These findings indicate that ghrelin secretion from the stomach is regulated by gastric somatostatin.     

Characterization of nucleotide pyrophosphatase-5 as an oligomannosidic glycoprotein in rat brain

Membrane glycoproteins of neural cells play crucial roles in axon guidance, synaptogenesis, and neuronal transmission. We have here characterized membrane glycoproteins containing terminal alpha-mannose residues in rat brain membranes. Affinity purification using Galanthus nivalis agglutinin, that is highly specific for terminal alpha-mannose residues, revealed a 50-kDa protein as well as 80-kDa SHPS-1 and 45-kDa beta2 subunit of Na,K-ATPase in rat brain membranes. Combination of N-terminal peptide sequencing and mass spectrometry indicated that the 50-kDa protein was rat nucleotide pyrophosphatase-5 (NPP-5). In contrast to other NPPs, NPP-5 was a type-I transmembrane protein. Northern blot analysis showed that NPP-5 was highly expressed in brain, but also expressed in other peripheral tissues. However, we could not detect either the NPP activity or the lysophospholipase D activity in the immunoprecipitates with antibodies to NPP-5 from rat brain membranes. These data, therefore, suggest that NPP-5 is a neural oligomannosidic glycoprotein that may participate in neural cell communications.