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The result of questionnaire studies are presented which shows (i) that conjuncts are scope islands in Japanese and (ii) that left-node raising can nullify such scope islands. This finding confirms the theory advanced in Yatabe (2001), in which semantic composition is almost entirely carried out within order domains, and arguably contradicts the theory proposed in Beavers and Sag (2004), which introduces a mechanism called Optional Quantifier Merger to deal with the fact that right-node raising and left-node raising can have semantic effects.
Based on Krifka (1992) and de Kuthy (2000), this paper develops an architecture for complex topic-comment structures in HPSG and applies it to predicate fronting in English with the goal of capturing the insights of Ward (1988) on this construction. We argue that predicate fronting is a distributed constructional form consisting of an auxiliary occurring in a predicate preposing phrase. The use of predicate preposing is a function of a combination of simultaneous constraints on its theme structure, its background-focus distribution, and its presuppositional structure. It is shown that these constraints can be made explicit within the HPSG architecture developed here.
This paper aims at making a general description of Chinese NPs using Head-Driven Phrase Structure Grammar. The paper introduces the basic and complex structures of Chinese NPs and then shed light on the noun-classifier matching problem when implemented in HPSG. To solve this problem, the paper tries to establish a basic grammar of Chinese NPs in the framework of HPSG, which is implemented in the LKB system. The implementation shows, although the matching problem between noun and classifier can be described in HPSG, especially by the MRS, it is still difficult to efficiently represent the semantic constrains in the LKB system.
The Big Mess Construction
(2007)
There is a construction in English, exemplified by 'how long a bridge', which is so irregular that it has been named the Big Mess Construction (Berman 1974). This paper first sketches its main characteristics and a treatment of the internal structure of the noun phrase which serves as a background for the analysis. It then presents three ways in which the Big Mess Construction can be analysed; two of them are lexicalist and are shown to be implausible; the third is constructivist and is argued to be superior. In a next step, the discussion is extended to two other types of constructions. The first concerns the English adnominal reflexives, as in 'the children themselves', and is shown to require a constructivist analysis which is similar but not identical to the one for the Big Mess Construction. The second concerns the combination of 'such' and 'what' with the indefinite article, as in 'such a pleasure'. In spite of its obvious resemblance with the Big Mess Construction this combination does not require a constructivist analysis; instead, it fits the lexicalist mould of most of the rest of HPSG.
An empirical overview of the properties of English prepositional passives is presented, followed by a discussion of formal approaches to the analysis of the various types of prepositional passives in HPSG. While a lexical treatment is available, the significant number of technical and conceptual difficulties encountered point to an alternative approach relying on constructional constraints. The constructional approach is argued to be the best option for prepositional passives involving adjunct PPs, and this analysis can be extended to create a hierarchy of constructions accommodating all types of prepositional passives in English, and the ordinary NP passive.
This paper aims to provide type hierarchies for Korean passive constructions on the basis of their forms within the HPSG framework. The type hierarchies proposed in this paper are based on the classification of Korean passives; suffixal passives, auxiliary passives, inherent passives, and passive light verb constructions. Verbs are divided into five subtypes in accordance with the possibility of passivization. We also provide type hierarchies for verbal nouns and passive light verbs.
In Sorani Kurdish dialects, the complement of a preposition can generally be realized either as a syntactic item (NP, independent pronoun or PP) or a bound personal morpheme (clitic/affix). However, the affixal realization of the complement gives rise to a range of specific phenomena. First, some prepositions display two different phonological forms depending on the realization of their complement: the variant combining with a syntactic item is referred to as ˋsimple', while the variant combining with an affixal complement is called ˋabsolute'. Furthermore, unlike syntactic complements, which are always realized locally, the affixal complement of an absolute preposition can have a non-local realization, attaching to a host with which it has no morphosyntactic relations. In order to deal with these facts, this paper proposes a classification of Sorani prepositions along two lines: the affixal versus non-affixal realization of the complement on the one hand and its local versus non-local realization on the other hand. All cases of non-local realization receive a lexical account, either in terms of argument composition or in terms of linearization constraints on domain objects.
Negative Polarity Items (NPI) are expressions such as English 'ever' and 'lift a finger' that only occur in sentences that are somehow negative. NPIs have puzzled linguists working in syntax, semantics and pragmatics, but no final conclusion as to which module of the grammar should be responsible for the licensing has been reached. Within HPSG interest in NPI has developed only relatively recently and is mainly inspired by the entailment-based approach of Ladusaw 1980 and Zwarts 1997. Since HPSG's CONTENT value is a semantic representation, the integration of such a denotational theory cannot be done directly. Adopting Discourse Representation Theory (DRT, Kamp and Reyle 1993, von Genabith et al. 2004) I show that it is possible to formulate a theory of NPI licensing that uses purely representational notions. In contrast to most other frameworks in semantics, DRT attributes theoretical significance to the representation of meaning, i.e. to a logical form, and not only to the denotation itself. This makes DRT particularly well-suited to my purpose.
Remarks on locality
(2007)
This paper proposes a modification of HPSG theory—Sign-Based Construction Grammar—that incorporates a strong theory of both selectional and constructional locality. A number of empirical phenomena that give the appearance of requiring nonlocal constraints are given a principled, localist analysis consistent with this general approach, which incorporates certain insights from work in the tradition of Berkeley Construction Grammar, as exemplified by Fillmore et al. (1988), Kay and Fillmore (1999), and related work.
Abeillé and Godard (2007) describe a variety of Spanish whose complex predicates differ structurally from the more familiar flat VP type of complex predicate common to other varieties of Spanish and Romance. I present a verb cluster analysis of this variety which both captures these structural differences, and at the same time preserves those features that are common across both construction types. Coupled with a simple morphological treatment of affixation, this analysis predicts the range of 'clitic climbing' facts. The parsimony of the affixation analysis is afforded by an alternative approach to the constraints on reflexive affix distribution in Spanish complex predicates. I depart radically from previous morpho-lexical approaches to the phenomenon, instead showing how the constraints follow from independently motivated binding principles. This approach not only handles more of the Spanish data, but also has the potential to provide a unified account of the phenomenon across Romance.
This paper is a follow up on Müller, 2006. It contains some comments on suggestions about the interaction of phrasal Constructions with constituent order that Adele Goldberg made at various occasions. In addition the paper discusses various HPSG analyses of particle verbs that assume lexical representations including phonologically specified parts of particle verb lexical entries. A recent phrasal analysis of resultatives (Haugereid, 2007) is discussed as well and it is pointed out that control constructions pose problems for phrasal analyses that do not assume empty elements but require that the subject is realized in a phrasal configuration.
Modern Hebrew is considered to be a 'partial pro-drop language'. Traditionally, the distinction between cases where pro-drop is licensed and those in which it is prohibited, was based on the person and tense features of the verb: 1st and 2nd person pronominal subjects may be omitted in past and future tense. This generalization, however, was found to be false in a number of papers, each discussing a subset of the data. Thus, contrary to conventional wisdom, dropped 3rd person pronouns subjects do occur in the language in particular contexts.
Identifying these contexts by way of a corpus-based survey is the initial step taken in this study. Subsequently, a careful syntactic analysis of the data reveals broad generalizations which have not been made to date. Thus, what was initially assumed to be a uniform phenomenon of 3rd person pro-drop turns out to be manifested in three distinct types of constructions. Finally, the proposed HPSG-based analysis incorporates insights concerning locality, correlations between finite and non-finite control, non-canonical elements, and binding.
The so-called floating quantifier constructions in languages like Korean display intriguing properties whose successful processing can prove the robustness of a parsing system. This paper shows that a constraint-based analysis, in particular couched upon the framework of HPSG, can offer us an efficient way of analyzing these constructions together with proper semantic representations. It also shows how the analysis has been successfully implemented in the LKB (Linguistic Knowledge Building) system.
Multiple nominative constructions (MNCs) in Korean have two main sub- types: possessive and adjunct types. This paper shows that a grammar allow- ing the interaction of declarative constraints on types of signs - in particular, having constructions (phrases and clauses) - can provide a robust and efficient way of encoding generalizations for two different MNCs. The feasibility of the grammar developed here has been checked with its implementation into the LKB (Linguistic Knowledge Building) system
The paper examines two verb sequencing constructions in Ga: the Serial Verb Construction (SVC) and the Extended Verb Complex (EVC). The former is an instance of a commonly recognized construction, the latter is typically found in the Volta Basin area of West Africa. EVCs are sequences of verbs functioning as single verb units relative to the syntax, but with an internal structure much like syntactic complementation. Both constructions show agreement of aspect and mood marking throughout the sequence, but with differences in exponence: in an SVC all Vs expose such marking, in an EVC only a limited (down to one) number of verbs, depending on the inflectional category. The paper presents the basic facts, based on works by Dakubu (2002, 2004, to appear), and gives an HPSG account of their morphology, syntax and semantics. The analysis is sustained by a grammar of the phenomena implemented with the 'Linguistic Knowledge Builder' (LKB), an engineering platform for natural language processing.
Licenser rules have originally been introduced in Müller (1999) as a part of a grammar based on discontinuous constituents. We propose licenser rules as a means to avoid underspecified empty elements in grammars with continuous constituents. We applied them to a verb movement analysis of the German main clause with right sentence bracket and to complement extraposition. To reduce the number of unnecessary hypotheses, we extended the licenser rule concept with a licenser binding technique. We compared the licenser rule approach to an approach based on underspecified traces with respect to processing performance. In our experiment, the use of licenser rules reduced the parse time by a factor of 13.5.
This paper examines the syntactic behavior of the Mauritian copula in predicative and extracted sentences. As it is the case in many languages, the Mauritian copula ete is absent in certain constructions: It only appears in extraction contexts. Our aim is to show that the postulation of a null copula, which has been proposed in various analyses, is inadequate for the Mauritian data. The phenomenon, as it is argued, rather lends itself to a strictly construction-based analysis within the framework of HPSG and is based on the distribution of weak pronouns and TAM markers.
In this paper I suggest an interface level of semantic representations, that on the one hand corresponds to morpho-syntactic entities such as phrase structure rules, function words and inflections, and that on the other hand can be mapped to lexical semantic representations that one ultimately needs in order to give good predictions about argument frames of lexical items. This interface level consists of basic constructions that can be decomposed into five sub-constructions (arg1-role, arg2-role ... arg5-role). I argue in favour of phrasal constructions in order to account for altering argument frames and maybe also coercion without having to use lexical rules or multiple lexical entries.
Townsend and Bever (2001) and Ferreira (2003) argue that simple templates representing the most commonly used orderings of arguments within a clause (e.g., NP-V-NP = Agent-Action-Patient) are used early in sentence comprehension to derive a preliminary interpretation before a full parse is completed. Sentences which match these templates (e.g., active sentences, subject clefts) are understood quickly and accurately, while sentences which deviate from the templates (e.g. passive sentences, object clefts) require additional processing to arrive at the correct interpretation. The present study extends the idea of canonical templates to the domain of noun phrases. I report on two experiments showing that possessive free relative clauses in English, which involve a non-canonical ordering of the head noun, are more difficult to understand than canonically headed noun phrases. I propose two reasons for this finding: (1) possessive free relatives deviate from the canonical template for interpreting noun phrases; and (2) the formal cues for interpreting possessive free relatives are relatively subtle. More generally I suggest that canonical templates help constrain mismatch in language by making certain kinds of mismatches costly for language users. Finally, I argue that evidence for canonical templates fits best within a parallel-architecture, constructionist theory of grammar.
In this paper, we report on an experiment showing how the introduction of prosodic information from detailed syntactic structures into synthetic speech leads to better disambiguation of structurally ambiguous sentences. Using modifier attachment (MA) ambiguities and subject/object fronting (OF) in German as test cases, we show that prosody which is automatically generated from deep syntactic information provided by an HPSG generator can lead to considerable disambiguation effects, and can even override a strong semantics-driven bias. The architecture used in the experiment, consisting of the LKB generator running a large-scale grammar for German, a syntax-prosody interface module, and the speech synthesis system MARY is shown to be a valuable platform for testing hypotheses in intonation studies.
This paper provides a background on the role of world knowledge in disambiguating modals and proposes treating the disambiguation of counterfactuals as a slightly more tractable sub-case of the general problem. Using a model theoretic possible worlds approach, counterfactuals are disambiguated with respect to a world of evaluation resembling classic Formal Semantic treatments (e.g., Kratzer 1977, 1981, 1989; Lewis 1973; Veltman 2005). The world, which provides a context of evaluation, is located through the interaction of the antecedent and consequent propositions with world knowledge axioms. This approach to modal disambiguation provides a connection between a grammar and the type of inferences typically handled in Knowledge Representation Systems (e.g., Hobbs et al. 1990) in a limited domain. The model theoretic semantics are linked with typed feature structures in an HPSG syntax (Pollard and Sag 1994). This grammar is implemented in TRALE, Penn's (2004) Prolog-based framework for typed feature structure grammar development. The compositional semantics in TRALE is specified in Penn and Richters' (2004, 2005) Constraint Language for Lexical Resource Semantics (CLLRS). This semantic component provides a semantic parse in which heads and arguments are combined systematically and the scope of negation or quantification can be accurately reflected. In the case of counterfactuals, the CLLRS semantic parse is passed to a model-theoretic interpreter. The mapping between the CLLRS semantic parse and the well-formed formulas of the model is defined by checking the parseability of the formula in the compositional semantics. Sets of possible worlds interact with constraints on world knowledge and constraints defining counterfactual validity. The truth value for a counterfactual is returned to the grammar relative to a context of evaluation. The results of counterfactual evaluation are returned in a form consistent with the grammar's internal compositional semantics. By the method described above, the interpreter provides a grammar-external component in which inferences involving world knowledge have the potential to be more efficiently evaluated. Through the development of model-checking techniques, for instance, it could be shown whether or not well-formed formulas and constraints hold in larger models and move towards capturing more fine-grained modal inferences in a larger domain.
This paper discusses a non-constituent coordination construction that occurs in Russian in which constituents with different syntactic functions and different thematic roles are conjoined. These conjuncts are co-arguments of the same head and are subject to a number of idiosyncrasies.
We consider several alternative analysis of the phenomena, and conclude that these are unable to account for the full range of the facts. Thus, even though these conjuncts do not form a semantic unit, there is evidence that they do form a kind of coordination structure. The phenomena are challenging for any theory of grammar, but the syntax-semantics account that we provide involves minimal changes to standard HPSG architecture.
Three distinctions seem relevant for the scope properties of adverbs: their function (adjuncts or complements), their prosody (incidental or integrated) and their lexical semantics (parenthetical or non parenthetical). We propose an analysis in which the scope of French adverbs is aligned with their syntactic properties, relying on a view of adjuncts as loci for quantification, a linearization approach to the word order, and an explicit modelling of dialogue.
Pseudocoordination in Danish
(2007)
In this paper we propose an analysis of Danish pseudocoordination constructions. The analysis is based on a hybrid phrase hierarchy where phrase types are assumed to be subtypes of types that cut across the traditional division of phrasal types, allowing the phrase type of pseudocoordinations to be a subtype of both coordinate phrases and headed phrases, and consequently inherit properties from both types. The analysis is linearization-based. We further develop a set of constraints on the phrasal types in the hierarchy.
The hybrid phrase hierarchy and the set of constraints on the various types in the hierarchy explain why, on the one hand, pseudocoordinations contain conjunctions and the conjuncts must have the same form and tense, and on the other, have a fixed order, allow extraction out of the second conjunct, do not allow overt subjects in the second conjunct and allow transitive verbs to appear in there-constructions.
The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.
Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.
Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.
Identification of a lysosomal peptide transport system induced during dendritic cell development
(2007)
The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
Thioredoxin 1 and thioredoxin 2 have opposed regulatory functions on hypoxia-inducible factor-1α
(2007)
Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.
The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK6) or 8-methylmenaquinone (MMK6) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.
By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.
Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-β (TGFβ) signaling pathway. Consequently, SPC is able to mimic TGFβ-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1β-stimulated prostaglandin E2 formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A2 (sPLA2) protein expression and activity. This effect is due to a reduction of sPLA2 mRNA expression caused by inhibited sPLA2 promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFβ signaling by a TGFβ receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA2 expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFβ, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.
2,5-Diformylbenzene-1,4-diol (5) is a well-suited starting compound for the preparation of ditopic hydroquinone-based ligands. Here, we report an optimized synthesis of 5 which improves the overall yield from published 7% to 42 %. Three new ditopic Schiff base ligands, 2,5-[iPr2N(CH2)2N=CH]2 - 1,4-(OH)2-C6H2 (8), 2,5-(pyCH2N=CH)2-1,4-(OH)2-C6H2 (9), and 2,5-[py(CH2)2N=CH]2-1,4- (OH)2-C6H2 (10), have been synthesized from 5 and structurally characterized by X-ray crystal structure analysis (py = 2-pyridyl).
The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
We present predictions for the pseudorapidity dependence of the azimuthal anisotropy parameters v1 and v2 of baryons and inclusive charged hadrons in Pb + Pb collisions at a LHC energy of sNN=5.5 TeV applying a microscopic transport model, namely the quark–gluon string model (QGSM) which has been recently extended for parton rearrangement and fusion processes. Pb + Pb collisions with impact parameters b=2.3 fm and b=8 fm have been simulated in order to investigate additionally the difference between central and semiperipheral configurations. In contrast to v1ch(η) at RHIC, the directed flow of charged hadrons shows a small normal flow alignment. The elliptic flow v2ch(η) turns out to be rather similar in shape for RHIC and LHC conditions, the magnitude however increases about 10–20% at the LHC, leading to the conclusion that the hydrodynamical limit will be reached.
Within a dynamical quark recombination model, we explore various proposed event-by-event observables sensitive to the microscopic structure of the QCD-matter created at RHIC energies. Charge ratio fluctuations, charge transfer fluctuations and baryon-strangeness correlations are computed from a sample of central Au + Au events at the highest RHIC energy available (sNN=200 GeV). We find that for all explored observables, the calculations yield the values predicted for a quark–gluon plasma only at early times of the evolution, whereas the final state approaches the values expected for a hadronic gas. We argue that the recombination-like hadronization process itself is responsible for the disappearance of the predicted deconfinement signals. This might explain why no fluctuation signatures for the transition between quark and hadronic matter was ever observed in the experimental data up to now.
Low concentrations of oxidized low density lipoprotein (OxLDL) are cytoprotective for phagocytes, although the underlying mechanisms remain unclear. We investigated signaling pathways used by OxLDL to attenuate apoptosis in monocytic cells. OxLDL at 25–50 μg/ml inhibited staurosporine-induced apoptosis in THP-1 cells and mouse peritoneal macrophages, and it was cytoprotective in human primary monocytes upon serum withdrawal. Attenuated cell demise was reversed by blocking extracellular signal-regulated kinase (ERK) signaling. Translocation of cytochrome c to the cytosol was attenuated by OxLDL, which again demanded ERK signaling. Analysis of Bcl-2 family proteins revealed phosphorylation of Bad at serine 112 as well as ERK-dependent inhibition of Mcl-1 degradation. Although the formation of reactive oxygen species (ROS) is an established signal generated by OxLDL, ROS scavengers did not interfere with cell protection by OxLDL. Thus, activation of the ERK signaling pathway by OxLDL is important to protect phagocytes from apoptosis.
The nuclear stopping, the elliptic flow, and the HBT interferometry are calculated by the UrQMD transport model, in which potentials for “pre-formed” particles (string fragments) from color fluxtube fragmentation as well as for confined particles are considered. This description provides stronger pressure at the early stage and describes these observables better than the default cascade mode (where the “pre-formed” particles from string fragmentation are treated to be free-streaming). It should be stressed that the inclusion of potential interactions pushes down the calculated HBT radius RO and pulls up the RS so that the HBT time-related puzzle disappears throughout the energies from AGS, SPS, to RHIC.
Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.