Fear Is A Prison From Which Action Wins Freedom

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Part A: Introduction I. Rationale In order to become competent in a foreign language, it is important for language learners not only to acquire new vocabularies and a new set of phonological and syntactic rules but also to learn what Wilson (1986) calls the rules of speaking: the patterns of sociolinguistic behavior of the target language. The rules of speaking involve us in knowing when and how it is suitable to open a conversation, what topics are appropriate to particular speech events, how speech acts are to be given and interpreted. In many cases, this interpretation goes beyond what the language learners might intend to convey and includes assessments such as “polite” and “impolite”. In Vietnam, as the economy grows and international business develops, English proficiency becomes a master tool for young people to get a job. They encounter foreigners in everyday settings where communication is necessary. In the modern society, the need for communication is increasing, especially in the process of globalization, when communication spreads beyond the boundary of a country. During the last decades, linguistic researchers have broadened their focus of their interests from the development of grammatical competence to other areas of target language development, such as discourse and pragmatic competence, common speech routines, for example, requests, apologies, complaints, compliments, refusals, and the like have been most frequently studied in cross-cultural and interlanguage pragmatics. According to Tsui (1994), there seems to be little empirical research that has been conducted in responses to questions. For a long time, question- response has been considered one of the most basic structures of conversation (Schegloff, 1974) but as Tsui (1994; p. 160) points out: “responses have been given little attention in the speech acts literature. Most of the acts characterized and listed in the various taxonomies are illocutionary acts which are often done by making the function of utterance in discourse, and as many responding acts do not have a corresponding responding performative verb, this kind of analysis inevitably neglects responses” A characterization of utterances (based on observation of real-life discourse) is not likely to neglect the importance of responses. Let’s consider an example illustrated by Tsui (1994) A: What’s the time? B: (a) Eleven (b) Time for coffee (c) I haven t got a watch, sorry’ 1 (d) How hold I know (e) Ask Jack (f) You know bloody well what time it is (g) Why do you ask? (h) What did you say? (i) What do you mean? Various possible responses from (a) to (i) shows us the complicated relationship between question and a proper answer. For the same question, the speaker A may be replied in different ways with different intentions by the addressee. Obviously, a response can be a proper answer, an indirect or implicit reply, an evasive answer, a refusal or denial, an outright lie or even a challenge to the speaker’s questioning Fear Is A Prison From Which Action Wins Freedom Fear Is A Prison From Which Action Wins Freedom By: Joe Tye Paul smirked at the notion that fear was a coward and a liar; what would fear be afraid of, and why would it lie? Rafe was speaking as though fear were a real living being, some sort of demon that could take physical possessions of your body, make you things you didn’t want to Could it be that his fear really wasn’t part of him but some external thing trying to work its way in from the outside? “Have you ever seen the great Wall of China?” Something in the way he said it suggested to Paul that not only had Rafe seen the Great Wall, he’d watched it being built “Like most such walls, it was much better at keeping people in than at keeping invaders out The Great Wall was a prison wall So is fear “Fear is a prison It will no more keep frightening things out of your life than the Great Wall kept Mongol invaders out of China But it can destroy your freedom of action so effectively that you can’t the things that could prevent what you fear from happening.” Paul’s image looked at his watch and pushed away from the table Joan wasn’t ready to quit, though “Why don’t you call Bill Roberts? He might lend you money.” Paul snorted “Roberts thinks I’m nuts for spending all my time with this school instead of starting a real business with a real bottom line Nothing would give him more pleasure than to have me come begging for a handout.” “Then what about your father? He might give you a loan.” Paul rolled his eyes, remembering the early years when his father called almost every day with leads on a “real” job, and once a week sent a bundle full of newspaper help wanted ads “Dad’s got his own problems He doesn’t need to have a failed son come asking for handouts.” “Then I’m going back to work.” 1/3 Fear Is A Prison From Which Action Wins Freedom “No way! The kids need you at home now, especially with Jeff having so much trouble at school.” “Well, Paul, just what are we going to do?” Paul’s image picked up his briefcase and refi lled the coffee mug When Joan put her hands on her hips, he knew it was time to exit “Don’t worry, honey, everything will be okay It’ll work out fi ne The bank will give us a loan extension, but I have to get into the offi ce and get prepared for that meeting.” Paul watched his image slink out of the kitchen toward the front door Joan wiped her hands on her apron as she followed him out for a perfunctory kiss at the doorway “Are you telling Joan the truth, Paul, or is that just wishful thinking?” Paul marveled at Rafe’s penchant for asking questions that permitted only one truthful answer POSITIVE THINKING IS WORKING FOR SOMETHING AND BELIEVING IT WILL HAPPEN WISHFUL THINKING IS WAITING FOR SOMETHING AND HOPING IT WILL HAPPEN Never Fear, Never Quit “I’m surprised at you, Rafe Haven’t you ever heard of the power of positive thinking?” “Yes indeed In fact I’d like to think that I played some small part in the writing of a book by that name But you know the distinction between positive thinking and wishful thinking?” “Tell me.” “Positive thinking is believing something will happen, wishful thinking is hoping it will happen Positive thinking is working for something to happen, wishful thinking is waiting for it to happen “Wishful thinking is the lock that fear puts on the prison gate You know the good-cop/ bad-cop routine? Where the bad cop beats you up and then the good cop gets you to confess by treating you well? Well, fear is the bad cop, threatening you with doom and dread But fear needs a good cop, or else you might just get frightened enough to something constructive to chase fear away “So fear lets you indulge yourself for a while in fl ights of wishful thinking Somehow, you think, something will happen to make the problems go away By the time you wake up, it’s too late What you feared has happened, and fear has defeated you 2/3 Fear Is A Prison From Which Action Wins Freedom “The only way to escape from the prison of fear is action You cannot wish your way out, you cannot wait your way out You can only work your way out Every time you escape the prison of fear, you grow stronger and more confi dent It will always be there, trying to wall you in, but you will eventually grow so strong that you can just step right over the walls.” 3/3 The b-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions Ronald P. de Vries 1† , Lucie Par ˇ enicova ´ 1‡ , Sandra W. A. Hinz 2 , Harry C. M. Kester 1 , Gerrit Beldman 2 , Jacques A. E. Benen 1 and Jaap Visser 1§ 1 Molecular Genetics of Industrial Microorganisms and 2 Food Chemistry, Wageningen University, Wageningen, The Netherlands The Aspergillus niger b-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, D -galacturonic acid and L -arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site resi- dues identified previously for the Pseudomonas fluorescens b-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of ara- binogalactans of potato, onion and soy with GALA resulted initially in the release of D -galactotriose and D -galactotetra- ose, whereas prolonged incubation resulted in D -galactose and D -galactobiose, predominantly. MALDI-TOF analysis revealed the release of L -arabinose substituted D -galacto- oligosaccharides from soy arabinogalactan. This is the first report of the ability of a b-1,4-endogalactanase to release substituted D -galacto-oligosaccharides. GALA was not active towards D -galacto-oligosaccharides that were substi- tuted with D -glucose at the reducing end. Keywords: Aspergillus niger; b-1,4-endogalactanase; galac- turonic acid; expression; galactan degradation. Endogalactanases are involved in the degradation of plant cell wall polysaccharides, in particular pectin. Two types of arabinogalactan side chains are present in pectin. Type I consists of a chain of b-1,4 linked D -galactopyranose linkages, while type II contains a backbone of b-1,3-linked D -galactopyranose residues that can be substituted with b-1,6-linked D -galactopyranose residues [1]. Both types can be substituted with a)1-,3-linked L -arabinofuranose chains. Type I arabinogalactan is degraded by b-1,4-endogalacta- nase and b-galactosidase. b-1,4-Endogalactanases cleave within the galactan moiety of type I arabinogalactan, releasing D -galacto-oligosaccharides. Bacterial b-1,4-endo- galactanases release mainly galactotriose and galactotetra- ose [2–6], while some also release galactobiose [3,5,7]. Eukaryotic b-1,4-endogalactanases release predominantly galactobiose and galactose from galactan [8–10]. Genes encoding b-1,4-endogalactanase have been cloned from both bacteria and fungi [11–15]. Based on their derived amino acid sequence, the corresponding enzymes have been assigned to family 53 of the glycosyl hydrolases [16]. These enzymes have a retaining mechanism and for the Pseudo- monas fluorescens b-1,4-endogalactanase the catalytic resi- dues have been determined [11]. Recently, the Aspergillus aculeatus b-1,4-endogalactanase has been expressed in vivo in potato resulting in a 30% reduction of the galactosyl content of the pectin fraction of the A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for b -alanyl dipeptides Hidenobu Komeda and Yasuhisa Asano Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan Many different kinds of microbial hydrolases acting d-stereoselectively on amino acid amides or peptides have been characterized, and some of them have been applied to the production of optically active d-amino acids from the corresponding racemic amino acid amides [1]. The d-stereoselective amidases and peptidases known to date can be classified into four groups based on their primary structures. d-Aminopeptidase from Ochrobactrum anthropi C1-38 [2,3], d-amino-acid ami- dase from O. anthropi SV3 [4,5], alkaline d-peptidases from Bacillus cereus DF4-B [6,7] and AH559 [8], DmpB from O. anthropi LMG7991 [9] and MlrB from Sphingomonas sp. [10] are active site serine hydrolases, which are classified into the penicillin-recognizing Keywords amidase; b-alanine; dipeptidase; DmpA; Pseudomonas sp. Correspondence Y. Asano, Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939–0398, Japan Fax: +81 766 562498 Tel: +81 766 567500 E-mail: asano@pu-toyama.ac.jp (Received 18 March 2005, revised 12 April 2005, accepted 18 April 2005) doi:10.1111/j.1742-4658.2005.04721.x We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonas sp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity). The DmpA (called l-aminopeptidase d-Ala-esterase ⁄ amidase) hydrolyzes alanine-p-nitroani- lide, alaninamide, and alanine methylester with a preference for the d-con- figuration of the alanine, whereas the enzyme acts as an l-stereoselective aminopeptidase on a tripeptide Ala-(Gly) 2 , indicating a reverse stereoselec- tivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Fre ` re J-M (1999) Biochem J 341, 147–155]. A recombinant BapA exhibiting hydrolytic activity toward d-alanine- p-nitroanilide was purified from the cell-free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized. The purified enzyme contained two polypeptides corresponding to residues 1–238 (a-peptide) and 239–366 (b-peptide) of the precursor as observed for DmpA. On gel-filtration chromatography, BapA in the native form appeared to be a tetramer. It had maximal activity at 60 °C and pH 9.0– 10.0, and was inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, Zn 2+ ,Ag + ,Cd 2+ or Hg 2+ . The enzyme hydrolyzed d-alanine-p-nitroanilide more efficiently than l-alanine-p-nitro- anilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of b-alanyl dipeptides including b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (carnosine), b-Ala-l-Leu, and (b-Ala) 2 with high efficiency compared to d-alanine-p-nitroanilide. b-Alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing pro- teinogenic amino acids or their d-counterparts for N-terminal residues. Based on its unique substrate specificity, the enzyme should not be called l-aminopeptidase d-Ala-esterase ⁄ amidase but b-Ala-Xaa dipeptidase. Abbreviations ORF, open reading frame; SD, Shine–Dalgarno. FEBS Journal 272 (2005) 3075–3084 ª 2005 FEBS 3075 protein family together with dd-carboxypeptidases involved in peptidoglycan biosynthesis and b-lactamas- es for resistance against b-lactam antibiotics (group 1). Zinc-containing NblA from Anabaena sp. PCC 7120 is a mostly a-helical protein undergoing reversible trimerization in solution Holger Strauss 1 , Rolf Misselwitz 2 , Dirk Labudde 1 , Sabine Nicklisch 3 and Kerstin Baier 3 1 Forschungsinstitut fu ¨ r Molekulare Pharmakologie (FMP), Berlin, Germany; 2 Max-Delbru ¨ ck-Centre fu ¨ r Molekulare Medizin (MDC), Berlin, Germany; 3 Humboldt Universita ¨ t zu Berlin, Institut fu ¨ r Biologie/Biochemie der Pflanzen, Germany The nblA family of genes encodes for small proteins neces- sary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis. Genes homologous to nblA seem to occur in all phycobili- some-containing organisms. However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis. In this study we report on the first characterization of an NblA-homologous gene product. The chromosomal gene from the cyanobacterium Anabaena sp. PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity. This allowed the protein to be characterized by analytical ultracentrifugation and CD spectroscopy. These experiments show that the NblA protein has a mostly a-helical structure, undergoing an association reaction of folded monomers to form trimers in solution. No dimers are detectable. Keywords: phycobilisome; chlorosis; NblA; cyanobacteria; analytical ultracentrifugation. Cyanobacteria are a widespread group of photosynthetic prokaryotes performing a plant-type oxygenic photosyn- thesis. They are very adaptable organisms that can survive in a wide variety of environmental conditions [1,2]. One limiting factor for growth is the nitrogen supply and cyanobacteria have developed various mechanisms to cope with this nutrient stress. One of the first responses exhibited by cyanobacteria when they are starved for nitrogen is the degradation of their major light-harvesting complex, the phycobilisome. Phycobilisomes (PBS), which also represent light-harvesting antennae of red algae, are large, water-soluble multiprotein complexes associated with the thylakoid membranes. PBS consist mainly of the pigmented phycobiliproteins that can constitute up to 50% of the total cellular protein, thus representing a large nitrogen store [3]. Degradation of PBS is thought to provide substrates for protein synthesis required for the acclimatization process. In addition, PBS degradation minimizes the absorption of excess excitation energy under the stress situation. Nondiazotrophic cyanobacteria such as Synechococcus sp. PCC 7942 completely degrade their PBS when starved for combined nitrogen and differentiate into nonpigmented resting cells, able to survive prolonged periods of nutrient stress [4,5]. Diazothrophic filamentous cyanobacteria such as Anabaena sp. PCC 7120 adapt to nitrogen limitation (lack of combined nitrogen) by developing differentiated cells, called heterocysts. These are specialized for fixation of N 2 in an aerobic environment [6]. In a filament, approxi- mately 5–10% of vegetative cells undergo this differenti- ation process. However, during the first hours of nitrogen starvation all cells start to degrade their PBS [7]. When heterocysts mature and nitrogenase is active, vegetative cells resynthesize their light-harvesting complexes, while in heterocysts the PBS content remains very low [8,9]. Phycobilisome degradation is an ordered proteolytic process, visible by a colour change of the cyanobacterial cell from blue-green to yellow-green, a EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein Daizo Hamada 1 , Tomoaki Kato 1,2 , Takahisa Ikegami 3 , Kayo N. Suzuki 1 , Makoto Hayashi 2 , Yoshikatsu Murooka 2 , Takeshi Honda 4 and Itaru Yanagihara 1 1 Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan 2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan 3 Laboratory of Structural Proteomics, Institute for Protein Research, Osaka University, Japan 4 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Japan Several bacteria, including enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively), express type III secretion systems [1] consisting of various proteins encoded at the genetic locus of enterocyte effacement [2–5]. To date, type III secretion systems have been identified in more than 20 pathogenic bacterial species [6]. Type III secretion systems are multiprotein complexes that span the bacterial and host membranes, permitting the direct delivery of effector proteins, such as the EPEC pro- teins [7], Tir [8–10], EspF [11,12], EspG [13] and Orf19 [14]. In the case of EHEC and EPEC, such complexes are formed by proteins including EspA, EspB and EspD [15,16]. Thus, the type III system regulates effector secretion and delivery into host cells. Keywords circular dichroism; natively partially folded proteins; nuclear magnetic resonance; fluorescence quenching; multiangle laser light scattering Correspondence I. Yanagihara, Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1011, Japan Fax: +81 725 57 3021 Tel: +81 725 56 1220 (ext. 5302) E-mail: itaruy@mch.pref.osaka.jp (Received 20 August 2004, revised 17 November 2004, accepted 2 December 2004) doi:10.1111/j.1742-4658.2004.04513.x The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes a-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acryl- amide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedi- mentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15 N- 1 H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advan- tageous when EspB interacts with various biomolecules during the bacterial infection of host cells. Abbreviations ANS, 8-anilinonaphthalene-1-sulfonate; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; ... too late What you feared has happened, and fear has defeated you 2/3 Fear Is A Prison From Which Action Wins Freedom “The only way to escape from the prison of fear is action You cannot wish your.. .Fear Is A Prison From Which Action Wins Freedom “No way! The kids need you at home now, especially with Jeff having so much trouble at school.” “Well, Paul, just what are we going to do?” Paul’s... her hands on her apron as she followed him out for a perfunctory kiss at the doorway “Are you telling Joan the truth, Paul, or is that just wishful thinking?” Paul marveled at Rafe’s penchant