Free and ATP-bound structures of Ap(4)A hydrolase from Aquifex aeolicus V5

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 angstrom resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alpha beta alpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

Standardizing clinical trials workflow representation in UML for international site comparison.

BACKGROUND: With the globalization of clinical trials, a growing emphasis has been placed on the standardization of the workflow in order to ensure the reproducibility and reliability of the overall trial. Despite the importance of workflow evaluation, to our knowledge no previous studies have attempted to adapt existing modeling languages to standardize the representation of clinical trials. Unified Modeling Language (UML) is a computational language that can be used to model operational workflow, and a UML profile can be developed to standardize UML models within a given domain. This paper's objective is to develop a UML profile to extend the UML Activity Diagram schema into the clinical trials domain, defining a standard representation for clinical trial workflow diagrams in UML. METHODS: Two Brazilian clinical trial sites in rheumatology and oncology were examined to model their workflow and collect time-motion data. UML modeling was conducted in Eclipse, and a UML profile was developed to incorporate information used in discrete event simulation software. RESULTS: Ethnographic observation revealed bottlenecks in workflow: these included tasks requiring full commitment of CRCs, transferring notes from paper to computers, deviations from standard operating procedures, and conflicts between different IT systems. Time-motion analysis revealed that nurses' activities took up the most time in the workflow and contained a high frequency of shorter duration activities. Administrative assistants performed more activities near the beginning and end of the workflow. Overall, clinical trial tasks had a greater frequency than clinic routines or other general activities. CONCLUSIONS: This paper describes a method for modeling clinical trial workflow in UML and standardizing these workflow diagrams through a UML profile. In the increasingly global environment of clinical trials, the standardization of workflow modeling is a necessary precursor to conducting a comparative analysis of international clinical trials workflows.

Identifying Barriers and Practical Solutions to Conducting Site-Based Research in North America: Exploring Acute Heart Failure Trials As a Case Study.

Although the prognosis of ambulatory heart failure (HF) has improved dramatically there have been few advances in the management of acute HF (AHF). Despite regional differences in patient characteristics, background therapy, and event rates, AHF clinical trial enrollment has transitioned from North America and Western Europe to Eastern Europe, South America, and Asia-Pacific where regulatory burden and cost of conducting research may be less prohibitive. It is unclear if the results of clinical trials conducted outside of North America are generalizable to US patient populations. This article uses AHF as a paradigm and identifies barriers and practical solutions to successfully conducting site-based research in North America.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Ocupação indígena na foz do rio Tapajós (3260-960 AP): estudo do sítio Porto de Santarém, baixo Amazonas

Na arqueologia Amazônica, o período Formativo (4.000-2.000 AP) é definido pela existência de assentamentos sedentários de povos cuja subsistência baseava-se na agricultura, complementada pela caça, pesca e coleta. Esse período é importante, entre outras razões, porque precede o desenvolvimento de sociedades regionais a partir do início da Era Cristã. Entretanto, é pouco conhecido, seja pela pouca quantidade de sítios identificados, seja pela falta de pesquisas. Esta dissertação apresenta os resultados da investigação de contextos Formativos no sítio Porto de Santarém, no baixo Amazonas, uma região onde a longa sequência de ocupação se inicia no período Paleoíndio. A investigação visou observar essa ocupação no sítio Porto e questionando seu papel na dinâmica de ocupação regional de longo termo. As escavações revelaram evidências de ocupação humana no período pré-histórico tardio (cal. AD 1020 a 1160) conhecido como fase Santarém da Tradição Inciso-Ponteada, possibilitaram o estudo de uma ocupação anterior, localizada na base da camada cultural, que corresponderia cronologicamente ao período Formativo (cal. 3160 a 3090 AP).

Transcriptional repression of serum amyloid A1 by AP -2 contributes to its liver -specific expression

Studies on the transcriptional regulation of serum amyloid A1 (SAA1) gene, a liver specific acute-phase gene, identified a regulatory element in its promoter that functioned to repress (SAA1) gene transcription in nonliver cells. This silencer element interacts with a nuclear protein that is detectable in HeLa cells, fibroblasts and placental tissues but not in liver or liver-derived cells. As the expression pattern of this repressor is consistent with its potential regulatory role in repressing SAA1 expression, and that many other liver gene promoters also contain this repressor binding site, we sought to investigate whether this repressor may have a broader functional role in repressing liver genes. ^ We have utilized protein purification, cell culture, transient and stable gene transfection, and molecular biology approaches to identify this protein and investigate its possible function in the regulation of (SAA1) and other liver genes. Analyses of amino acid sequence of the purified nuclear protein, and western blot and gel shift studies identified the repressor as transcription factor AP-2 or AP-2-like protein. Using transient transfection of DNA into cultured cells, we demonstrate that AP-2 can indeed function as a repressor to inhibit transcription of SAA1 gene promoter. This conclusion is supported by the following experimental results: (1) overexpression of AP-2 in hepatoma cells inhibits conditioned medium (CM)-induced expression of SAA1 promoter; (2) binding of AP-2 to the SAA1 promoter is required for AP-2 repression function; (3) one mechanism by which AP-2 inhibits SAA1 may be by antagonizing the activation function of the strong transactivator NFκB; (4) mutation of AP-2 binding sites results in derepression of SAM promoter in HeLa cells; and (5) inhibition of endogenous AP-2 activity by a dominant-negative mutant abolishes AP-2's inhibitory effect on SAM promoter in HeLa cells. In addition to the SAM promoter, AP-2 also can bind to the promoter regions of six other liver genes tested, suggesting that it may have a broad functional role in restricting the expression of many liver genes in nonliver cells. Consistent with this notion, ectopic expression of AP-2 also represses CM-mediated activation of human third component of complement 3 promoter. Finally, in AP-2-expressing stable hepatoma cell lines, AP-2 inhibits not only the expression of endogenous SAA, but also the expression of several other endogenous liver genes including albumin, α-fetoprotein. ^ Our findings that AP-2 has the ability to repress the expression of liver genes in nonliver cells opens a new avenue of investigation of negative regulation of gene transcription, and should improve our understanding of tissue-specific expression of liver genes. In summary, our data provide evidence suggesting a novel role of AP-2 as a repressor, inhibiting the expression of liver genes in nonliver cells. Thus, the tissue-specific expression of AP-2 may constitute an important mechanism contributing to the liver-specific expression of liver genes. ^

Expression regulation of AP -2 gamma gene

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placenta development. In an effort to identify factors regulating AP-2γ gene expression we isolated and characterized the promoter and 5′ flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ RACE. Transient gene transfer studies showed that basal promoter activity resides within a highly conserved ∼200 by DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp and AP-2 binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and non-trophoblast cells, demonstrating the functional importance of these sites in AP-2γ gene expression. ^ Mutational analysis of the 5′-flanking region revealed a 117-bp positive regulatory region of the mouse AP-2γ gene located between −5700 and −5583 upstream of the transcription start site. This 117-bp positive regulatory element provided approximately 7-fold enhancement of reporter gene expression in cultured trophoblast cells. A C/EBP-Sp1 transcription factor-binding module is located in this DNA sequence. Electrophoretic mobility shift assays demonstrated that transcription factors Sp1, Sp3 and C/EBP bind to the enhancer element. Mutation of each protein-binding site reduced the enhanced expression significantly. Mutagenesis assays showed that two other protein-binding sites also contribute to the enhancer activity. In summary, we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter region and contribute to basal promoter activity. We have identified a 117-bp positive regulatory element of AP-2γ gene, and we have shown that Sp and C/EBP proteins bind to the cis -regulatory elements and contribute to the enhanced gene expression. ^

(Table 1) Composition of paraffin hydrocarbons at DSDP Site 56-434

Geochemical investigation of 18 samples of sediments from Site 434 involved determining the content of organic carbon, of bitumoid A (The chloroform A-chl and alcohol-benzene A-alb extracts) and its various fractions, and of individual hydrocarbons as well as the structural group composition of resins. We identified certain samples that differed sharply from the rest by their increased bitumen content and relatively low molecular hydrocarbons and by the fact that their resinous components were more neutral and aliphatic in composition. The distribution of bitumoid and its components seems to reflect migration processes in operation during the early stages of the transformation of organic matter.

Geochemistry and provenance of basaltic clasts within volcaniclastic debris flows at DSDP Site 89-585

Pebble-sized basaltic and glassy clasts were extracted from seamount-derived volcaniclastic debris flows and analyzed for various trace elements, including the rare earths, to determine their genetic relationships and provenance. All the clasts were originally derived from relatively shallow submarine lava flows prior to sedimentary reworking, and have undergone minor low-grade alteration. They are classified into three petrographic groups (A, B, and C) characterized by different phenocryst assemblages and variable abundances and ratios of incompatible elements. Group A (clast from Hole 585) is a hyaloclastite fragment which is olivine-normative and distinct from the other clasts, with incompatibleelement ratios characteristic of transitional or alkali basalts. Groups B and C (clasts from Hole 585A) are quartz-normative, variably plagioclase-clinopyroxene-olivine phyric tholeiites, all with essentially similar ratios of highly incompatible elements and patterns of enrichment in light rare earth elements (chrondrite-normalized). Variation within Groups B and C was governed by low-pressure fractionation of the observed phenocryst phases, whereas the most primitive compositions of each group may be related by variable partial melting of a common source. The clasts have intraplate chemical characteristics, although relative to oceanic hot-spot-related volcanics (e.g., Hawaiian tholeiites) they are marginally depleted in most incompatible elements. The source region was enriched in all incompatible elements, compared with a depleted mid-ocean-ridge basalt source.

Pollen and macroremains from four profiles from site Samerberg 1

Pollen and macrofossil analysis of lake sediments revealed the complete development of vegetation from Riss late-glacial to early Würm glacial times at Samerberg (12°12' E, 47°45' N, 600 m a.s.l) on the northern border of the Alps. The pollen bearing sediments overlie three stratigraphic units, at the base a ground-moraine, then a 13 m thick layer of pollen free silt and clay, and then a younger moraine; all the sediments including the pollen bearing sediments, lie below the Würm moraine. The lake, which had developed in an older glacial basin, became extinct, when the ice of the river Inn glacier filled its basin during Würm full-glacial time at the latest. One interglacial, three interstadials, and the interdigitating treeless periods were identified at Samerberg. Whereas the cold periods cannot be distinguished from one another pollenanalytically, the interglacial and the two older interstadials have distinctive characteristics. A shrub phase with Juniperus initiated reforestation and was followed by a pine phase during the interglacial and each of the three interstadials. The further development of the interglacial vegetation proceeded with a phase when deciduous trees (mainly Quercus, oak) and hazel (Corylus) dominated, though spruce (Picea) was present at the same time in the area. A phase with abundant yew (Taxus) led to an apparently long lasting period with dominant spruce and fir (Abies) accompanied by some hornbeam (Carpinus). The vegetational development shows the main characteristics of the Riss/Würm interglacial, though certain differences in the vegetational development in the northern alpine foreland are obvious. These differences may result from the existence of an altitudinal zonation of the vegetation in the vicinity of the site and are the expression of its position at the border of the Alps. A greater age (e.g. the Holsteinian) can be excluded by reason of the vegetational development, and is also not indicated at first sight from the geological and stratigraphical data of the site. Characteristic of the Riss/Würm vegetational development in southern Germany - at least in the region between Lake Starnberg/Samerberg/Salzach - is the conspicuous yew phase. According to absolute pollen counts, yew not only displaced the deciduous species, but also displaced spruce preferentially, thus indicating climatic conditions less favourable for spruce, caused by mild winters (Ilex spreading!) and by short-term low precipitation, indicated by the reduced sedimentation rate. The oldest interstadials is bipartite, as due to the climatic deterioration the early vegetational development, culminating in a spruce phase, had been interrupted by another expansion of pine. A younger spruce-dominated period with fir and perhaps also with hornbeam and beech (Fagus) followed. An identical climatic development has been reported from other European sites with long pollen sequences (see chapter 6.7). However, different tree species are found in the same time intervals in Middle Europe during Early Würm times. Sediments of the last interglacial (Eem or Riss/Würm) have been found in all cases below the sediments of the bipartite interstadial, and in addition one more interstadial occurs in the overlying sediments. This proves that Eem and Riss/Würm of the north-european plain resp. of the alpine foreland are contemporaneous interglacials although this has been questioned by some authors. The climax vegetation of the second interstadial was a spruce forest without fir and without more demanding deciduous tree species. The vegetational development of the third interstadial is recorded fragmentary only. But it has been established that a spruce forest was present. The oldest interstadial must correspond to the danish Brørup interstadial as it is expressed in northern Germany, the second one to the Odderade interstadial. A third Early Würm interstadial, preserved fragmentarily at Samerberg, is known from other sites. The dutch Amersfoort interstadial most likely is the equivalent to the older part of the bipartite danish Brørup interstadial.

Mineralogy of ODP Site 105-645

Cores from the upper 70 meters below seafloor (mbsf) (upper Pleistocene) at Ocean Drilling Program (ODP) Site 645 in Baffin Bay show dramatic meter-scale changes in color and mineralogy. Below this interval, mineralogical changes are more gradual to the top of the Miocene at about 550 mbsf. The Pliocene-Pleistocene section can be divided into five facies: Facies 1 - massive, poorly sorted, gravel-bearing muds; Facies 2 - gray silty clays and silty muds; Facies 3 - laminated detricarbonate silty muds; Facies 4 - silty sand and sandy silt; and Facies 5 - poorly sorted muddy sands and silty muds. Facies 4 and 5 are restricted to the Pliocene section below depths of about 275 mbsf. The mineralogical/color cycles in the upper 70 mbsf are the result of alternations between Facies 2 and three lithotypes of Facies 1: lithotype A - tan-colored, carbonate-rich, gravel-bearing mud; lithotype B - weak, red-colored, gravel-bearing mud rich in sedimentary rock fragments; and lithotype C - gray, gravel-bearing mud. A fourth lithotype, D, is restricted to depths of 168-275 mbsf and is dark gray, carbonate-poor, gravel-bearing mud. We believe that all lithotypes of Facies 1 and the sand and gravel fractions of Facies 2 and 3 were deposited by ice rafting. Depositional processes for Facies 4 and 5 probably include ice rafting and bottom- and turbidity-current transport. Data from petrographic analyses of light and heavy sand-sized grains and X-ray analyses of silt- and clay-size fractions suggest that tan-colored sediments (lithotype A of Facies 1; Facies 3) were derived mainly from Paleozoic carbonates of Ellesmere, Devon, and northern Baffin islands. Weak red sediments (lithotype B) contain significant red sedimentary clasts, reworked quartzarenite grains and clasts, and rounded colorless garnets, all derived from Proterozoic sequences of the Borden and Thule basins, and from minor Mesozoic red beds. Other sediments in the upper 335 mbsf at Site 645 contain detritus from a heterogeneous mixture of sources, including Precambrian shield terranes around Baffin Bay. Sediments from 335 to 550 mbsf (Facies 5) are rich in friable sedimentary clasts and detrital micas and contain glauconite and, in a few samples, reworked diatoms. These components suggest derivation from poorly consolidated Mesozoic-Tertiary sediments in coastal outcrops and beneath the modern shelves of northeastern Baffin Island and western Greenland. For the upper Pleistocene section (about 0-100 mbsf), marked mineralogical cyclicity is attributed to fluctuating glacial margins, calving rates, and iceberg melting rates, particularly around the northern end of Baffin Bay. Tan-colored, carbonate-rich units were derived at times of maximum advance of glaciers on Ellesmere and Devon islands, during relatively warm intervals induced by incursion of warm Atlantic surface water into the bay. At the beginning of these warmer episodes, most icebergs were contributed by glaciers near sea level around the Arctic channels, which resulted in deposition of weak red, ice-rafted units rich in Proterozoic sedimentary clasts.

Middle Eocene-Early Pliocene Subantarctic planktic foraminiferal biostratigraphy of ODP Site 1090 on the Agulhas Ridge, South Atlantic

The analysis of planktic foraminiferal assemblages from Site 1090 (ODP Leg 177), located in the central part of the Subantarctic Zone south of South Africa, provided a geochronology of a 330-m-thick sequence spanning the Middle Eocene to Early Pliocene. A sequence of discrete bioevents enables the calibration of the Antarctic Paleogene (AP) Zonation with lower latitude biozonal schemes for the Middle-Late Eocene interval. In spite of the poor recovery of planktic foraminiferal assemblages, a correlation with the lower latitude standard planktic foraminiferal zonations has been attempted for the whole surveyed interval. Identified bioevents have been tentatively calibrated to the geomagnetic polarity time scale following the biochronology of Berggren et al. (1995). Besides planktic foraminiferal bioevents, the disappearance of the benthic foraminifera Nuttallides truempyi has been used to approximate the Middle/Late Eocene boundary. A hiatus of at least 11.7 Myr occurs between V78 and V71 m composite depth extending from the Early Miocene to the latest Miocene-Early Pliocene. Middle Eocene assemblages exhibit a temperate affinity, while the loss of several planktic foraminiferal species by late Middle to early Late Eocene time reflects cooling. During the Late Eocene-Oligocene intense dissolution caused impoverishment of planktic foraminiferal assemblages possibly following the emplacement of cold, corrosive bottom waters. Two warming peaks are, however, observed: the late Middle Eocene is marked by the invasion of the warmer water Acarinina spinuloinflata and Hantkenina alabamensis at 40.5 Ma, while the middle Late Eocene experienced the immigration of some globigerinathekids including Globigerinatheka luterbacheri and Globigerinatheka cf. semiinvoluta at 34.3 Ma. A more continuous record is observed for the Early Miocene and the Late Miocene-Early Pliocene where planktic foraminiferal assemblages show a distinct affinity with southern mid- to high-latitude faunas.

The orientation of the AP-1 heterodimer on DNA strongly affects transcriptional potency

Activation of gene transcription in eukaryotes requires the cooperative assembly of an initiation complex containing many protein subunits. The necessity that these components contact each other and the promoter/enhancer in defined ways suggests that their spatial arrangement might influence the activation response. Indeed, growing evidence indicates that DNA architecture can profoundly affect transcriptional potency. Much less is known about the influence of protein architecture on transcriptional activation. Here, we examine the architectural dependence of activator function through the analysis of matched pairs of AP-1•DNA complexes differing only in their orientation. Mutation of a critical Arg residue in the basic-leucine zipper domain of either Fos or Jun yielded single point-mutant heterodimers that bind DNA in a single defined orientation, as determined directly by native chemical ligation/affinity cleavage; by contrast, the corresponding wild-type protein binds DNA as a roughly equal mixture of two isomeric orientations, which are related by subunit interchange. The stereochemistry of the point-mutant heterodimers could be switched by inversion of a C•G base pair in the center of the AP-1 site, thus providing access to both fixed orientational isomers. Yeast reporter gene assays consistently revealed that one orientational isomer activates transcription at least 10-fold more strongly than the other. These results suggest that protein architecture, especially the spatial relationship of the activation domain to the promoter, can exert a powerful influence on activator potency.

NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.