mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan

Chloroplasts are believed to be descendants of ancestral cyanobacteria that had peptidoglycan layer between the outer and the inner membranes. Historically, the glaucophyte Cyanophora paradoxa and the rhizopod Paulinella chromatophora were believed to harbor symbiotic cyanobacteria having peptidoglycan, which were conventionally named “cyanelles”. In addition, the complete set of genes involved in the synthesis of peptidoglycan has been found in the moss Physcomitrella patens and some plants and algae. The presence of peptidoglycan-like structures was demonstrated by a new metabolic labeling technique in P. patens. However, many green algae and all known red algae lack peptidoglycan-related genes. That is the reason why we questioned the origin of peptidoglycan-synthesizing enzymes in the chloroplasts of the green algae and plants. We performed phylogenetic analysis of ten enzymes involved in the synthesis of peptidoglycan exploiting the Gclust homolog clusters and additional genomic data. As expected, all the identified genes encoded in the chromatophore genome of P. chromatophora were closely related to cyanobacterial homologs. In the green algae and plants, only two genes, murA and mraY, were found to be closely related to cyanobacterial homologs. The origins of all other genes were diverse. Unfortunately, the origins of C. paradoxa genes were not clearly determined because of incompleteness of published genomic data. We discuss on the probable evolutionary scenarios to explain the mostly non-cyanobacterial origins of the biosynthetic enzymes of chloroplast peptidoglycan: A plausible one includes extensive multiple horizontal gene transfers during the early evolution of Viridiplantae.     

Effect of antiperoxidative drugs on gastric damage induced by ethanol in rats

Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl radicals, contributes to lipid peroxidation and lesion formation in the gastric mucosa after absolute ethanol administration.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Improving the accuracy of predicting secondary structure for aligned RNA sequences

Considerable attention has been focused on predicting the secondary structure for aligned RNA sequences since it is useful not only for improving the limiting accuracy of conventional secondary structure prediction but also for finding non-coding RNAs in genomic sequences. Although there exist many algorithms of predicting secondary structure for aligned RNA sequences, further improvement of the accuracy is still awaited. In this article, toward improving the accuracy, a theoretical classification of state-of-the-art algorithms of predicting secondary structure for aligned RNA sequences is presented. The classification is based on the viewpoint of maximum expected accuracy (MEA), which has been successfully applied in various problems in bioinformatics. The classification reveals several disadvantages of the current algorithms but we propose an improvement of a previously introduced algorithm (CentroidAlifold). Finally, computational experiments strongly support the theoretical classification and indicate that the improved CentroidAlifold substantially outperforms other algorithms.     

Two isoforms of acid phosphatase secreted by tobacco protoplasts: differential effect of brefeldin A on their secretion

Summary The effects of brefeldin A (BFA) on the secretion of acid phosphatase (APase) by tobacco protoplasts were investigated. Secretion of APase was inhibited by BFA in a dose-dependent manner, with a concomitant intracellular accumulation of the enzyme. The secreted APase was composed of two isoforms. BFA (10/ g/ml) inhibited the secretion of one of the isoforms without inhibiting that of the other, and this phenomenon explains the partial inhibition of APase secretion as a whole. The inhibition of APase secretion was accompanied by changes in the morphology of the Golgi apparatus and also by an increment in massdensity of cells.Abbreviations APase acid phosphatase - BFA brefeldin A - CHX cycloheximide - PAGE polyacrylamide gel electrophoresis     

Crystallization and preliminary X-ray analysis of plant class I chitinase from rice

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.     

Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca(2+) signaling using CD38 knockout mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) using fura-2 microfluorimetry, we distinguished and characterized the Ca(2+) release mechanisms responsive to cADPR. The Ca(2+) response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca(2+) release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca(2+) spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca(2+) release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca(2+) response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca(2+) signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca(2+) release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca(2+) spikes.     

Solvent‐dependent conformation of a regioselective amylose carbamate: Amylose‐2‐acetyl‐3,6‐bis(phenylcarbamate)

Six amylose‐2‐acetyl‐3,6‐bis(phenylcarbamate) (AAPC) samples ranging in weight‐average molar mass M_w from 1.8 × 10⁴ g mol^?1 to 1.1 × 10⁶ g mol^?1 have been prepared from enzymatically synthesized amylose samples. Static light scattering, small‐angle X‐ray scattering, sedimentation equilibrium, and viscosity measurements were made for the samples in 1,4‐dioxane (DIOX), 2‐ethoxyethanol (2EE), and 2‐butanone (MEK) all at 25°C to determine particle scattering functions, z‐average radii of gyration, intrinsic viscosities, as well as M_w. The data were analyzed in terms of the wormlike cylinder model mainly to yield the helix pitch per residue h and the Kuhn segment length λ^?1, which corresponds to twice of the persistence length. The latter parameters (λ^?1) in 2EE (11 nm) and MEK (12 nm) are quite smaller than those for amylose tris(phenylcarbamate) (ATPC) in the same solvent (16 nm in 2EE and 18 nm in MEK) whereas those for AAPC (21 nm) and ATPC (22 nm) in DIOX are essentially the same as each other. This indicates that the chain stiffness of AAPC is more strongly influenced by the solvents since the number of intramolecular H‐bonds of AAPC is more changeable than that for ATPC. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1010–1017, 2012.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.