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American Sign Language (ASL) has a group of verbs showing agreement with the subject or/and object argument. There has not been analysis on especially number agreement. This paper analyzes person and number agreement within the HPSG framework. I discuss person and number hierarchy in ASL. The argument of agreement verbs can be omitted as in languages like Italian. The constraints on the type agreement-verb have the information on argument optionality.
Russian shows the mixed agreement with the polite pronoun vy and pluralia tantum nouns, both of which have plural number in form but either singular or plural number in meaning. Two different forms of adjectives – short form and long form – agree in different number with those number mismatch nominals.
I adopted the idea of Siegel (1976) etc. that when a long-form adjective appears in the predicate position, there is always a null head that it modifies, with the HPSG's agreement theory of Wechsler & Zlatic (2003). I propose that all predicates – verbs, SF and LF adjectives – except predicate nominals show CONCORD agreement. LF adjectives show CONC agreement with the null anaphor 'one'. The different number values of LF adjectives results from index agreement between the null anaphor and the subject of the sentence.
We study the formal and pragmatic properties of the 'reinforced negation construction' in Italian, which, unlike the regular negative sentence, contains both non and an n-word in preverbal position. On the one hand, this construction relies on a more general construction (positive or negative), which is pragmatically associated with reprise assertion, on the other hand, it uses non without the usual constraints attached to it. We propose that this unfaithful recycling is a pattern for creating a form dedicated to metalinguistic negation. Our analysis integrates both negative types of negative forms with their formal and pragmatic properties.
Since Pollard and Sag (1994) it has been assumed that raising involves full structure sharing, whereas a control verb merely shares the content of one of the lower verb's arguments. This has been considered a property of the phenomena, despite the fact that Pollard and Sag (1994) present this syntactic difference as a hypothesis confirmed for Icelandic only. In this paper we discuss the difference between raising and control from the perspective of Dutch and German passives. It has already been shown by Van Noord and Kordoni (2005) that the secondary object passives in these languages are raising structures, in which the case of the raised argument changes. In this paper we provide additional evidence for the raising analysis, and we propose a new analysis, which allows for a uniform account of Dutch and German passives as raising structures. Przepiorkowski and Rosen (2004) show that control may exhibit case transmission; the data presented in this paper shows that raising may not. Therefore, we claim that the distinction between raising and control is found in theta-role assignment. Syntactically they tend to behave differently, but they may also behave in the exact same way.
In Modern Greek there is a rich aspectual system, which involves both morphologically expressed grammatical aspect and eventuality types, carried primarily by the meaning of the verbal predicate. Particular emphasis is paid to the interaction between grammatical aspect and eventuality types, since it is due to this interaction that the verbal predicate acquires distinct meanings. In order to explain potential changes in the meaning of the eventualities caused by the interaction with grammatical aspect, I propose a formal analysis within HPSG, using Minimal Recursion Semantics (MRS) for the semantic representations. Following the MRS architecture, I introduce a number of relations, which represent both grammatical aspect and eventuality types. The close interaction between grammatical aspect and eventuality types triggers special meanings which traditionally can be explained by inserting contextual information into the representations. In this paper, I argue against such an analysis, providing an alternative which is based on the introduction of subeventual templates formulated by Michaelis (2003) and Pustejovsky (1995). In this context, grammatical aspect combines with eventuality types and selects eventualities or subeventualities appropriate to its selection restrictions, using information that is already there in the denotation of the eventualities.
Floating affixes in Polish
(2006)
The morphosyntactic status of Polish past tense agreement markers has been a matter of considerable debate in recent years (Spencer 1991, Borsley & Rivero 1994, Borsley 1999, Bański 2000, Kupść 2000, Kupść & Tseng 2005). Past tense agreement is expressed by a set of bound forms that either attach to the past participle, or else float off to a host further to the left. Despite this relative freedom of attachment, it is often noted in the literature, e.g., Borsley 1999, Kupść & Tseng 2005, that the combination of verbal host and agreement marker forms a word-like unit.
In this paper I will argue that these agreement markers are best analysed as affixes uniformly introduced on the verb whose inflectional features they realise. Building on the linearisation-based theory of morphology-syntax interaction proposed in Crysmann 2003, syntactic mobility of morphologically introduced material will be captured by mapping phonological contributions to multiple lexically introduced domain objects. It will be shown that this is sufficient to capture the relevant data, and connect the placement of floating affixes to the general treatment of Polish word order Kupść 2000.
Several analysis of Coordination of Unlikes have been proposed within the HPSG framework. In some of these approaches the possible combinations of 'unlike categories' are encoded in the grammar, while other accounts resort to an independently motivated ellipsis analysis. In this paper we provide further arguments in favor of the latter. However, some problematic cases of Coordination of Unlikes in certain S-adjoining constructions are left unaccounted for. We propose a general analysis of these S-adjoining constructions, and in doing so, the problematic coordination cases are predicted without the need for further assumptions.
The "flexibility" of gender in Tigrinya is uncovered by (i) setting a value for gender for each noun at the lexical level (i.e. bare controllers) and (ii) analysing gender shifts as signals for evaluations (i.e. evaluated controllers). The analysis is formalized as lexical rules which change the value of gend and add an elementary predication in the rels list.
In this paper, we present an analysis of noun phrases with elided nouns that dispenses with the positing of empty categories and preserves the NP structure assumed for NPs with overt nouns, modulo the absence of the head noun. On a par with existing traceless analyses of long distance dependencies, this is proposed as a further step towards a more lean theory of grammar, without phonetically null items.
Languages often require negation to be realized in a prominent position. A well known example is Italian, which seems to require a pre-verbal realization of negation. Some other languages require negation to be in a prominent position but do not require it to be pre-verbal. An example is Swedish. Working within Lexical Functional Grammar (LFG), Sells (2000) proposes that Swedish requires a negative element which is not inside VP and that Italian has the same constraint. Similar facts are found in the VSO language Welsh. However, Sellss approach cannot be applied to Welsh. Borsley and Jones (2005) develop a selectional approach to Welsh, in which certain verbs require a negative complement. This works well for Welsh but cannot be applied to Swedish or Italian. A similar approach to all three languages is possible within the linearization-based version of Head-driven Phrase Structure Grammar (HPSG) developed by Kathol (2000). It seems, then, that a linear approach is preferable to both a structural and a selectional approach.
Metrical phonology in HPSG
(2006)
This paper proposes a new approach to the prosody-syntax interface in HPSG. Previous approaches to prosody in HPSG (Klein, 2000; Haji-Abdolhosseini, 2003) represent prosodic information by constructing metrical constituent structure in the tradition of (Selkirk, 1980; Liberman and Prince, 1977). One drawback of this approach is that it does not allow for a direct representation of purely metrical constraints, which are relegated to an unformalized performance component. By contrast, so called 'grid only' approaches (Prince, 1983; Selkirk, 1984; Delais-Roussarie, 2000) use a single data structure, a metrical grid, to encode prosodic constraints resulting from syntax and constraints of a rhythmic nature.
We first review relevant data from French showing that prosodic constituency is much less constrained by syntactic structure than is predicted by existing approaches. In all but very short utterances, many different prosodic groupings are possible for a given sentence with a determinate information structure, and rhythmic factors determine a preference ordering on the possible groupings. We then present an HPSG implementation of the metrical grid, and propose minimal syntactic constraints on relative prominence, leaving room for noncategorical rythmic constraints to choose between alternatives. We finish by discussing the interaction of the metrical grid with the rest of the prosodic grammar.
Conventional wisdom holds that productive morphology is regular morphology. Drawing evidence from French, we argue that the description of many lexeme formation processes is simplified if we hold that a productive rule may give rise to inflectionally irregular lexemes. We argue that the notion of a stem space allows for a straightforward description of this phenomenon: each lexeme comes equipped with a vector of possibly distinct stems, which serve as bases for inflectional form construction. The stem space is structured by default relations which encode the regular pattern of inflection; (partial) irregularities occur when a lexeme specifies a stem space violating the default relations. Derived irregularity is then the effect of a productive lexeme formation rule which specifies an irregular stem space for its output.
Abstract: A functional typology of copular be in Russian allows us to systematically relate variants of predication with and without copula. The analysis sketched in this article does not need empty categories; neither does it have to stipulate categories, category changes or constituents that are not morphologically signalled. With regard to HPSG formalization, the presented approach independently motivates the use of features and mechanisms that are already available in this framework.
Comparative correlative (CC) constructions have received much attention in recent years. Major issues have been whether they involve special constructions and whether they have symmetric or asymmetric structures. Evidence from Romance suggests that they require special constructions and that they may be either symmetric or asymmetric. French has a single construction which is asymmetric for some speakers and symmetric for others. Spanish has two distinct constructions, one asymmetric and the other symmetric with quite different properties. The facts can be accommodated in a straightforward way within construction-based HPSG.
Ziel der "Historisch-Kritischen Ausgabe" war es, allererst die Texte soweit wie möglich zu sichern und in methodisch vertretbarer Weise zugänglich zu machen. Anstatt in einen wie auch immer interpolierten Lebenszusammenhang eingebettet zu werden, sollten die Texte im Kontext der realisierten oder auch nur geplanten Publikationen ("Gedichte und Phantasien" 1804, "Poetische Fragmente" 1805, "Melete" 1806) zu Wort kommen. Das Zweifelhafte wurde als solches gekennzeichnet. Manche Texte (vor allem die bisher nur in verstümmelter Gestalt wiedergegebenen) gewannen durch dieses Verfahren, andere dagegen verloren vielleicht an Wert. Die dreibändige Ausgabe, 1991 erschienen, ist längst vergriffen. 15 Jahre danach, zu Karoline von Günderrodes 200. Todestag, erscheint eine unveränderte, einzig durch eine Korrigenda ergänzte Neuauflage. Sie trägt dem Umstand Rechnung, daß die Ausgabe inzwischen zur Grundlage der neueren Forschung geworden ist.
Dieser Band enthält Angaben zu Enstehung, Überlieferung und Publikation der in den ersten beiden Bänden herausgegebenen Texte. Der Kommentarband enthält - neben einem Herausgeberbericht, Übersichtstabellen und Verzeichnissen - allgemeine einleitende Kommentare zu den Abteilungen und Einzelkommentare zu den Texten, entsprechend der Anordnung des Textbandes bzw. - für die "Studien" - des Variantenbandes. Es werden keine Wort- und Sacherläuterungen gegeben.
Ziel der "Historisch-Kritischen Ausgabe" war es, allererst die Texte soweit wie möglich zu sichern und in methodisch vertretbarer Weise zugänglich zu machen. Anstatt in einen wie auch immer interpolierten Lebenszusammenhang eingebettet zu werden, sollten die Texte im Kontext der realisierten oder auch nur geplanten Publikationen ("Gedichte und Phantasien" 1804, "Poetische Fragmente" 1805, "Melete" 1806) zu Wort kommen. Das Zweifelhafte wurde als solches gekennzeichnet. Manche Texte (vor allem die bisher nur in verstümmelter Gestalt wiedergegebenen) gewannen durch dieses Verfahren, andere dagegen verloren vielleicht an Wert. Die dreibändige Ausgabe, 1991 erschienen, ist längst vergriffen. 15 Jahre danach, zu Karoline von Günderrodes 200. Todestag, erscheint eine unveränderte, einzig durch eine Korrigenda ergänzte Neuauflage. Sie trägt dem Umstand Rechnung, daß die Ausgabe inzwischen zur Grundlage der neueren Forschung geworden ist.
Dieser Band enthält die Varianten zu den im Textband gedruckten dichterischen Texten (in analoger Anordnung) und die hauptsächlichsten Studien von Karoline v. Günderrode.
Ziel der "Historisch-Kritischen Ausgabe" war es, allererst die Texte soweit wie möglich zu sichern und in methodisch vertretbarer Weise zugänglich zu machen. Anstatt in einen wie auch immer interpolierten Lebenszusammenhang eingebettet zu werden, sollten die Texte im Kontext der realisierten oder auch nur geplanten Publikationen ("Gedichte und Phantasien" 1804, "Poetische Fragmente" 1805, "Melete" 1806) zu Wort kommen. Das Zweifelhafte wurde als solches gekennzeichnet. Manche Texte (vor allem die bisher nur in verstümmelter Gestalt wiedergegebenen) gewannen durch dieses Verfahren, andere dagegen verloren vielleicht an Wert. Die dreibändige Ausgabe, 1991 erschienen, ist längst vergriffen. 15 Jahre danach, zu Karoline von Günderrodes 200. Todestag, erscheint eine unveränderte, einzig durch eine Korrigenda ergänzte Neuauflage. Sie trägt dem Umstand Rechnung, daß die Ausgabe inzwischen zur Grundlage der neueren Forschung geworden ist.
Dieser Band enthält alle von Karoline von Günderrode zu Lebzeiten für die Publikation freigegebenen Texte in der Fassung der Erstdrucke (I-IV), alle selbstständigen Texte des dichterischen Nachlasses aufgrund der handschriftlichen Fassungen (V) und einige Texte, die bisher der Günderrode zugeschrieben wurden, deren Autorschaft aber nicht mit völliger Sicherheit feststeht (VI).
Infection with the SARS (Severe Acute Respiratory Syndrome)-associated coronavirus results in respiratory failure probably by immunological mechanisms in 10% of patients. Laboratory markers that predict subsequent respiratory failure would therefore be useful in patient management.
We describe the clinical course, hematologic parameters, lymphocyte subpopulations and markers of inflammation in two patients with SARS, i.e., one man with diabetes mellitus and one pregnant woman, infected by the same viral isolate.
The patient with underlying diabetes mellitus developed respiratory failure after admission in the second week of the illness while the second patient developed only a mild disease without respiratory failure. Subsequent respiratory dysfunction was associated with lown umbers of Natural Killer (NK) cells at presentation and elevated CRP levels during the illness.
NK cells and CRP levels at the end of the first week of the disease might be related to subsequent respiratory dysfunction and may link underlying conditions to disease severity.
We present the first measurements of charge-dependent correlations on angular difference variables η1 − η2 (pseudorapidity) and φ1 − φ2 (azimuth) for primary charged hadrons with transverse momentum 0.15 <= pt <= 2 GeV/c and |η| <= 1.3 from Au–Au collisions at √sNN = 130 GeV. We observe correlation structures not predicted by theory but consistent with evolution of hadron emission geometry with increasing centrality from one-dimensional fragmentation of color strings along the beam direction to an at least two-dimensional hadronization geometry along the beam and azimuth directions of a hadron-opaque bulk medium.
Chromaktiviert die Wirkung von Insulin. Bei Typ-1- undTyp-2-Diabetikern reflektierten verminderte Chromgehalte in Leukozyten eine verminderte Chromversorgung. Je schlechter die Diabeteseinstellung, umso niedriger ward er Chromstatus bei Typ-2-Diabetikern. Daher sollte bei diesen Patienten – besonders bei Typ-2-Diabetikern mit schlechter Einstellbarkeit – eine Chromsupplementation erwogen werden, wenn eine sichere Bestimmung der Chromversorgung nicht gewährleistet ist. Kupferionenbesitzen anti- und auch prooxidative Eigenschaften. Hohe Kupferplasmawerte korrelieren mit der Entstehung einer Arteriosklerose. Die Kupferkonzentrationen im Plasma waren bei beiden Diabetikergruppen erhöht, weitererhöhte Werte zeigten Typ-2-Diabetiker mit Hyperlipidämie oder diabetischen Folgeerkrankungen. Um das Risiko für Mikro- und Makroangiopathie oder Nephropathie zu vermindern, sollten Diabetiker eine hohe Kupferzufuhr vermeiden. Selen wirkt antioxidativ, immunstimulierend und antiatherogen. Der Selengehalt im Plasma reflektiert die Selenzufuhr. Die Selenplasmawerte waren bei beiden Diabetesgruppen geringfügig vermindert und auffällig vermindert bei Patienten mit Folgeerkrankungen. Eine selenreiche Ernährung oder Selengaben zur Prävention von Spätfolgen könnten bei Diabetikern daher sinnvoll sein. Patienten mit Nephropathie und eingeschränkter Zufuhr an tierischem, selen- und zinkreichem Protein profitieren möglicherweise von einer Selensupplementation. Zink ist an der Wundheilung, der Immunfunktion und der Aktivierung und Speicherung von Insulin beteiligt. Der Elementgehalt war beiTyp-1-Diabetikern in Leukozyten, dem bestem Indikator der Zinkversorgung, vermindert. Bei Patienten mit Nephropathie, Mikroangiopathie oder Neuropathie war das Zinkdefizit noch deutlicher ausgeprägt. Eine hochwertige, proteinreiche Ernährung könnte die Versorgungslage ver-bessern, auch eine Zinksupplementation wäre möglicherweise von Nutzen.
Point-of-Care-Tests (POCT) stellen eine Gruppe innerhalb der In-vitro-Diagnostika (IVD) dar. Die Verkehrsfähigkeit von IVD im gemeinsamen europäischen Markt wird durch das CE-Kennzeichen ausgedrückt, das die Übereinstimmung des Tests mit den Vorgaben der europäischen IVD-Richtlinie dokumentiert. POCT unterliegen prinzipiell denselben Anforderungen wie alle anderen Labor-IVD. Die CE-Kennzeichnung wird vom Hersteller angebracht, der damit bestätigt, dass das betreffende Produkt den grundlegenden Anforderungen der Richtlinie entspricht und einem in der Richtlinie vorgesehenen Konformitätsbewertungsverfahren unterzogen wurde. Der Hersteller wird bei der CE-Kennzeichnung bestimmter IVD, deren möglicherweise inkorrektes Testergebnis mit einem höheren Risiko für Patient oder Dritte verbunden sein kann, von einer benannten Stelle unterstützt. Die Marktüberwachung CE-gekennzeichneter IVD wird durch nationale Behörden wahrgenommen, die bei Vorkommnissen Maßnahmen festlegen können.
Infektionen mit dem Respiratory Syncytial Virus (RSV)sind weltweit die bedeutendsten Atemwegserkrankungenim Säuuglings- und Kindesalter. Die RS-Viren werdend urch Schmierinfektionen und Aerosole übertragen, der Mensch ist das einzige Erregerreservoir. Im Säuglings-und Kleinkindalter finden gehäuft RSV-Infektionen statt. Mit zwei Jahren sind bereits 95% der Kinder seropositiv. Maternale Antikörper gewährleisten im Säuuglingsalterkeinen ausreichenden Nestschutz. Es ist von keiner sicheren Immunität auszugehen, daher sind Reinfektionen die Regel. Der Haüfigkeitsgipfel der RSV-Infektionenliegt in den Winter- und Frühlingsmonaten. Frühgeborene, immundefiziente und immunsupprimierte Patienten können das Virus mehrere Wochen ausscheiden. RSV-Infektionen verursachen zumeist Bronchitis, Bronchiolitis oder Pneumonie. Die Methode der Wahl ist der Erregernachweis über eine Virusisolierung in der Zellkultur im akuten Erkrankungsfall. Benötigt wird Nasenspülwasser oder ein tiefer Rachenabstrich. Auf einen schnellen Transport unter gekühlten Bedingungen ist zu achten (48C). Die Antikörpernachweise (Serologie) sind die Methode der Wahl für die epidemiologischen Auswertungen und weniger für die Akutdiagnostik geeignet. Nachdem Infektionsschutzgesetz (IfSG) § 6 Abs. 3 sind dem Gesundheitsamt gehäuft auftretende RSV-Infektionen zu melden. Die Therapie erfolgt symptomatisch; in schweren Fällen kann Ribavirin als Aerosol eingesetzt werden. Eine passive Immunisierung mit humanen Antikörpern gegen RSV kann bei Kindern mit erhöhtem Infektionsrisiko i.v. verabreicht werden (RespiGam). Auch sind monoklonale Antikörper gegen RSV (Palivizumab) prophylaktisch wirksam.
Bis einschließlich 10. Januar 2006 infizierten sich in Asien rund 150 Menschen mit dem Erreger der Vogelgrippe H5N1. In sechs Ländern (Kambodscha, China, Indonesien, Thailand, Vietnam und Türkei) verstarben an der “Hühnergrippe” rund 80 Patienten. Eine Übertragung von Mensch zu Mensch scheint in Einzelfällen möglich. Eine Pandemie hat der Erreger bisher nicht ausgelöst: Er wurde nicht (effektiv) von Mensch zu Mensch übertragen.
Aktuell erscheint aber eine Ausweitung der Hühnergrippe auch in Europa denkbar. Meldungen aus Rumänien im Oktober 2005 lassen eine Ausbreitung des H5N1-Erregers bei Wasservögeln vermuten. Jetzt (Stand Januar 2006) wurden auch aus der Türkei mehrere Infektionen des Menschen, davon drei Todesfälle, bekannt.
Sorge bereitet Experten die Möglichkeit eines genetischen “Reassortment” durch eine gleichzeitige Doppel-Infektion eines Wirtes (Mensch, Schwein) mit humanen und aviären Influenza-A-Viren-Erregern. Der neue Subtyp könnte bei passender Adaption an die menschlichen Zellen zu einer neuen Pandemie führen.
To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
Unlike other eukaryotes, plants possess a complex family of heat stress transcription factors (Hsfs) with usually more than 20 members. Among them, Hsfs A4 and A5 form a group distinguished from other Hsfs by structural features of their oligomerization domains and by a number of conserved signature sequences. We show that A4 Hsfs are potent activators of heat stress gene expression, whereas A5 Hsfs act as specific repressors of HsfA4 activity. The oligomerization domain of HsfA5 alone is necessary and sufficient to exert this effect. Due to the high specificity of the oligomerization domains, other class A Hsfs are not affected. Pull-down assay and yeast two-hybrid interaction tests demonstrate that the tendency to form HsfA4/A5 heterooligomers is stronger than the formation of homooligomers. The specificity of interaction between Hsfs A4 and A5 was confirmed by bimolecular fluorescence complementation experiments. The major role of the representatives of the HsfA4/A5 group, which are not involved in the conventional heat stress response, may reside in cell type-specific functions connected with the control of cell death triggered by pathogen infection and/or reactive oxygen species.
The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.
The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a beta-strand that could bind in parallel or antiparallel orientation to the beta2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions.
Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα.
The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the α-subunit, which mediates ATP hydrolysis and ion translocation, as well as the β-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the α- and β-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the α- and β-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the α-subunit and the β-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the β-subunit in relation to the α-subunit and suggest that the α- and β-subunits move toward each other during the E2 to E1 conformational transition.
The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.
In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.
A new procedure for the synthesis of 2-(4-propylphenyl)ethanol is provided. This new procedure significantly reduces side-products as 1-(4-propylphenyl)ethanol and 2-bromoethanol, which are obtained when using the previously known procedure. Only with the new procedure an efficient purification on the large scale needed for avoided-level-crossing muon-spin resonance experiments was possible.
Structural details of the title compound could be derived from an X-ray structure analysis of a crystalline derivative, the nitrobenzoyl ester.
Pyrazolyl-substituted 1,4-dihydroxybenzene and 1,4-dihydroxynaphthene derivatives have been synthesized by reaction of 1,4-benzoquinone and 1,4-naphthoquinone, respectively, with pyrazole. Cyclovoltammetric measurements have shown that 1,4-benzoquinone possesses the potential to oxidize 2-(pyrazol-1-yl)- and 2,5-bis(pyrazol-1-yl)-1,4-dihydroxybenzene. The 2,5-bis(pyrazol-1-yl)- 1,4-dihydroxybenzene reacts with air to give quantitatively black insoluble 2,5-bis(pyrazol-1-yl)-1,4- quinhydrone. Black crystals of 2,5-bis(pyrazol-1-yl)-1,4-quinhydrone suitable for X-ray diffraction were grown from methanol at ambient temperature (monoclinic C2/c). The poor yields of pyrazolylsubstituted 1,4-dihydroxybenzene and 1,4-dihydroxynaphthene derivatives can be explained by the formation of insoluble black quinhydrons in the reaction of benzoquinone and naphthoquinone with pyrazole. The dianions of 2-(pyrazol-1-yl)- and 2,5-bis(pyrazol-1-yl)-1,4-dihydroxybenzene react with oxygen to give the corresponding semiquinone anions. 2,5-Bis(pyrazol-1-yl)-1,4-benzoquinone shows two reversible one-electron reduction processes in cyclovoltammetric measurements, whereas pyrazolyl-substituted 1,4-dihdroxybenzene and -naphthene derivatives undergo irreversibile electrontransfer processes.
In this contribution we present algorithms for model checking of analog circuits enabling the specification of time constraints. Furthermore, a methodology for defining time-based specifications is introduced. An already known method for model checking of integrated analog circuits has been extended to take into account time constraints. The method will be presented using three industrial circuits. The results of model checking will be compared to verification by simulation.
The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.
CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins
(2006)
The mechanisms leading to prostate cancer metastasis are not understood completely. Although there is evidence that the CXC chemokine receptor (CXCR) 4 and its ligand CXCL12 may regulate tumor dissemination, their role in prostate cancer is controversial. We examined CXCR4 expression and functionality, and explored CXCL12-triggered adhesion of prostate tumor cells to human endothelium or to extracellular matrix proteins laminin, collagen, and fibronectin. Although little CXCR4 was expressed on LNCaP and DU-145 prostate tumor cells, CXCR4 was still active, enabling the cells to migrate toward a CXCL12 gradient. CXCL12 induced elevated adhesion to the endothelial cell monolayer and to immobilized fibronectin, laminin, and collagen. Anti-CXCR4 antibodies or CXCR4 knock out significantly impaired CXCL12-triggered tumor cell binding. The effects observed did not depend on CXCR4 surface expression level. Rather, CXCR4-mediated adhesion was established by alpha5 and beta3 integrin subunits and took place in the presence of reduced p38 and p38 phosphorylation. These data show that chemoattractive mechanisms are involved in adhesion processes of prostate cancer cells, and that binding of CXCL12 to its receptor leads to enhanced expression of alpha5 and beta3 integrins. The findings provide a link between chemokine receptor expression and integrin-triggered tumor dissemination.
The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized “living” cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading complex.
The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP′ part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of “natively unfolded” proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.
Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRβ subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5–40 amino acids, are subject to alternative splicing (C1–C7, C4′–C6′). Since one of the cassettes, C5′, has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRβ loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6′, already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5′ was coisolated mainly from liver. Notably C5′-containing gephyrin bound to the GlyRβ loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.
The genome, antigens of human cytomegalovirus (HCMV) are frequently found in prostatic carcinoma. However, whether this infection is causative or is an epiphenomenon is not clear. We therefore investigated the ability of HCMV to promote metastatic processes, defined by tumor cell adhesion to the endothelium, extracellular matrix proteins. Experiments were based on the human prostate tumor cell line PC3, either infected with the HCMV strain Hi (HCMVHi) or transfected with cDNA encoding the HCMV-specific immediate early protein IEA1 (UL123) or IEA2 (UL122). HCMVHi upregulated PC3 adhesion to the endothelium, to the extracellular matrix proteins collagen, laminin, fibronectin. The process was accompanied by enhancement of β1-integrin surface expression, elevated levels of integrin-linked kinase, phosphorylation of focal adhesion kinase. IEA1 or IEA2 did not modulate PC3 adhesion or β1-integrin expression. Based on this in vitro model, we postulate a direct association between HCMV infection, prostate tumor transmigration, which is not dependent on IEA proteins. Integrin overexpression, combined with the modulation of integrin-dependent signalling, seems to be, at least in part, responsible for a more invasive PC3Hi tumor cell phenotype. Elevated levels of c-myc found in IEA1-transfected or IEA2-transfected PC3 cell populations might promote further carcinogenic processes through accelerated cell proliferation.
The D-meson spectral density at finite temperature is obtained within a self-consistent coupled-channel approach. For the bare meson–baryon interaction, a separable potential is taken, whose parameters are fixed by the position and width of the Λc(2593) resonance. The quasiparticle peak stays close to the free D-meson mass, indicating a small change in the effective mass for finite density and temperature. Furthermore, the spectral density develops a considerable width due to the coupled-channel structure. Our results indicate that the medium modifications for the D-mesons in nucleus-nucleus collisions at FAIR (GSI) will be dominantly on the width and not, as previously expected, on the mass.
We study the gluonic phase in a two-flavor color superconductor as a function of the ratio of the gap over the chemical potential mismatch, Δ/δμ. We find that the gluonic phase resolves the chromomagnetic instability encountered in a two-flavor color superconductor for Δ/δμ<2. We also calculate approximately the free energies of the gluonic phase and the single plane-wave LOFF phase and show that the former is favored over the latter for a wide range of coupling strengths.
We propose to use the hadron number fluctuations in the limited momentum regions to study the evolution of initial flows in high energy nuclear collisions. In this method by a proper preparation of a collision sample the projectile and target initial flows are marked in fluctuations in the number of colliding nucleons. We discuss three limiting cases of the evolution of flows, transparency, mixing and reflection, and present for them quantitative predictions obtained within several models. Finally, we apply the method to the NA49 results on fluctuations of the negatively charged hadron multiplicity in Pb+Pb interactions at 158A GeV and conclude that the data favor a hydrodynamical model with a significant degree of mixing of the initial flows at the early stage of collisions.
Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e−. This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.
Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.
Prostaglandin (PG) E2 (PGE2) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE2 is accomplished by conversion of the cyclooxygenase (COX) product PGH2 by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Δ12,14-PGJ2 and PGA2 downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor γ or PGD2 receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE2. Our data suggest that there is a feedback mechanism between PGE2 and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.