Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection

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Protein-bound conformation of a specific inhibitor against Candida albicans myristoyl-CoA:protein N-myristoyltransferase in the ternary complex with CaNmt and myristoyl-CoA by transferred NOE measurements.

Transferred nuclear Overhauser enhancement (trNOE) experiments have been performed to study the bioactive conformation(s) of Ro09-3472/000 derivatives in the ternary complex with Candida albicans myristoyl-CoA: protein N-myristoyltransferase (CaNmt) and myristoylCoA (MyrCoA). A critical step in the trNOE study is to identify 'true' trNOEs in the spectra. Nonspecific binding of ligands to target proteins and/or spin diffusion effects can give rise to 'false' trNOEs, which may lead to an incorrect conclusion if used to derive bound conformations. In this study for all ligands the observed trNOEs arose from specific binding interactions with the active site of CaNmt. This was shown by displacing the ligand with the known tightly binding active-site inhibitor 1 [Devadas, B., Zupec, M.E., Freeman, S.K., Brown, D.L., Nagarajan, S., Sikorski, J.A., McWherter, C.A., Getman, D. P. & Gordon, J.I. (1995) J. Med. Chem. 38, 1837-1840] and measuring the resonance linewidths in the NMR spectrum before and after addition of the competitive inhibitor. The compounds were also tested for nonspecific protein binding with bovine serum albumin (BSA) using the same METHOD: Of the six compounds tested, Ro09-3700/001 (racemate) and its optically pure enantiomers, Ro09-4764/001(S) and Ro09-4765/001(R), showed both specific binding to CaNmt and no interaction with BSA. The NMR data of these molecules in the ternary complex with CaNmt/MyrCoA could thus be used for a detailed structural analysis. Thereby, the conformation of the bound ligand was obtained from a conformational search using the observed trNOEs as a selection filter. The NMR-determined conformations are in good agreement with the recently solved CaNmt-bound X-ray structures of two similar Ro09-3472/000 derivatives.     

Coordination behavior of amine bases to the axial site of a (dibenzo[b,i][1,4,8,11]tetraazacyclotetradecinato)cobalt(II)

The mutual interaction of various amine bases with the (dibenzo[b,i][1,4,8,11] tetraazacyclotetradecinato)cobalt(II) (Co(II)-1) was investigated by measuring electronic spectra in methyl benzoate. The Co(II)-1 became the pentacoordinated complex by taking up an amine base in the axile site: Co(II)-1 + B ? BCo(II)-1. For the mutual interaction of substituted pyridines with the Co(II)-1, the general behavior of the equilibrium constants was explained on the basis of the amine basicity and the Hammett equation by reference to the corresponding behavior of the porphyrin, corrin and corrole complexes. Moreover, there exists a systematic correlation between log K and the chemical shift of the corresponding 4-position in the ¹³C-NMR spectra of substituted pyridines. The isoequilibrium temperature obtained from a plot of ΔH against ΔS was 260 K. The equilibrium is primarily controlled by entropy at the usual temperature. The weaker coordination tendency of some hindered pyridine such as 2-methyl- and 2,6-dimethylpyridine toward Co(II)-1 was attributed to the steric effect between the in-plane ligand of Co(II)-1 and the 2- and/or 6-methyl groups of substituted pyridines in the coordination process.     

Simultaneous spawning by female stream goby Rhinogobius sp. and the association with brood cannibalism by nesting males

A laboratory experiment was conducted by varying the undersurface area of nesting substratum and the number of females in an experimental tank to elucidate the determinants of the mating pattern in the stream goby, Rhinogobius sp. cross‐band type. Males with larger nests tended to attract two or more females to their nest in a tank. Moreover, males spawned simultaneously with multiple females and entire brood cannibalism by males was rarely observed under a female‐biased sex ratio. When males spawned with a single female with low fecundity, however, entire brood cannibalism occurred at a high frequency, suggesting that a male guarding a nest with fewer eggs consumes the brood. Therefore, spawning behaviour of females that leads to a large egg mass would decrease the risk of entire brood cannibalism. In this species, simultaneous spawning by multiple females in a nest serves as a female counter‐measure against entire brood cannibalism. These results suggest that a conflict of interest between the sexes through brood cannibalism is a major determinant of simultaneous spawning.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF(Fbs1) ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man3GlcNAc2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol.     

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.