Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1 : Functional Analysis of a Polycistronic Transcription Unit

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.

Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Biostratigraphy and paleobiogeographic affinities of the Jurassic brachiopod assemblages from Sierra Espuña (Maláguide Complex, Internal Betic Zones, Spain)

The assemblages of Early Jurassic brachiopods (Pliensbachian - Toarcian) from Sierra Espuña (Murcia Province, SE Spain) are described. This is the only area in the Internal Zones of the Betic Cordillera, corresponding to the margins of the Alborán Terrane, where Jurassic brachiopods are known to occur. In the tectonic Unit of Morrón de Totana (more southward located) assemblage MT1 of Late Pliensbachian age has been characterized. This assemblage has been subdivided into three successive sub-assemblages: MT1a (Algovianum Zone), MT1b (Emaciatum Zone, Solare Subzone) and MT1c (Emaciatum Zone, Elisa Subzone). Northward, in the Perona tectonic Unit two distinct assemblages, P1 (Latest Sinemurian - Early Pliensbachian) and P2 (Early Toarcian, Serpentinum Zone) have been recognized. Differences between the assemblages from the two tectonic units are evident after the paleobiogeographical analysis. In the Morrón de Totana Unit, taxa with Mediterranean affinities occur. MT1 assemblage is very similar to assemblages previously known in the Eastern Subbetic as well as in other areas of the Mediterranean Province. In the Perona Unit the Mediterranean affinity of the assemblages is not so evident. P1 Assemblage consists of widely distributed taxa, lacking in the most characteristic elements of the Mediterranean Province which, however, are present in neighbouring Betic areas. P2 Assemblage belongs to the Spanish Province that develops in Western Tethys after the Early Toarcian Mass Extinction Event. The occurrence in this assemblage of Prionorhynchia aff. msougari Rousselle, until now only found in North Africa, indicates a closer connection of the Perona Unit with the African paleomargin of the Tethys than with the South Iberian paleomargin. The paleobiogeographical data suggest a more southern and marginal (close to epicontinental areas) position of the Perona Unit than the Morrón de Totana Unit.

Topological structures of complex belief systems

The concepts of substantive beliefs and derived beliefs are defined, a set of substantive beliefs S like open set and the neighborhood of an element substantive belief. A semantic operation of conjunction is defined with a structure of an Abelian group. Mathematical structures exist such as poset beliefs and join-semilattttice beliefs. A metric space of beliefs and the distance of belief depending on the believer are defined. The concepts of closed and opened ball are defined. S′ is defined as subgroup of the metric space of beliefs Σ and S′ is a totally limited set. The term s is defined (substantive belief) in terms of closing of S′. It is deduced that Σ is paracompact due to Stone's Theorem. The pseudometric space of beliefs is defined to show how the metric of the nonbelieving subject has a topological space like a nonmaterial abstract ideal space formed in the mind of the believing subject, fulfilling the conditions of Kuratowski axioms of closure. To establish patterns of materialization of beliefs we are going to consider that these have defined mathematical structures. This will allow us to understand better cultural processes of text, architecture, norms, and education that are forms or the materialization of an ideology. This materialization is the conversion by means of certain mathematical correspondences, of an abstract set whose elements are beliefs or ideas, in an impure set whose elements are material or energetic. Text is a materialization of ideology.

Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2δ1

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L.

Geochemistry and stable isotope record of the hydtrothermally altered lower sheeted dike complex in ODP Hole 140-504B

Drilling during Legs 137 and 140 of the Ocean Drilling Program deepened Hole 504B, the only hole to penetrate through the volcanic section and into the underlying hydrothermally altered sheeted dike complex, by 438.1 m to a total depth of 2000.4 meters below seafloor. This paper presents the secondary mineralogy, bulk-rock sulfur contents, and stable isotopic (O, S) compositions, plus oxygen isotopic compositions of secondary minerals from the lower sheeted dike complex drilled during Legs 137 and 140. Various evidence indicates higher temperatures of hydrothermal alteration in the lower dikes than in the upper dikes, including: the local presence of secondary clinopyroxene in the lower dikes; secondary anorthite and hornblende in the lower dikes vs. mainly actinolite and albite-oligoclase in the upper dikes; generally increasing Al and Ti contents of amphibole downward in the dike section; and greater 18O depletions of the lower dikes (d18O = 3.6-5.0 per mil) compared with the upper dikes. Early high-temperature alteration stages (T = 350°-500°C) resulted in 18O depletions and losses of metals (Cu, Zn) and sulfur from the rocks. Local incorporation of reduced seawater sulfate led to elevated d34S values of sulfide in the rocks (up to 2.5 per mil). Quartz + epidote formed in crosscutting veins at temperatures of 310°-320°C from more evolved fluids (d18O = 1 per mil). Late-stage lower-temperature (~250°C) reactions producing albite, prehnite, and zeolites in the rocks caused slight 18O enrichments, but these were insufficient to offset the 18O depletions caused by earlier higher-temperature reactions. Addition of anhydrite to the rocks during seawater recharge led to increased S contents of rocks that had previously lost S during axial hydrothermal alteration, and to further increases in d34S values of total S in the rocks (up to 12 per mil). Despite the evidence for seawater recharge to near the base of the sheeted dike complex, the paucity of late zeolites in the lower dikes suggests that late-stage, off-axis circulation was mainly restricted to the volcanics and shallowest dikes, or to localized high-permeability zones (faults) at depth.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 Angstrom resolution, have unit-cell parameters a = b = 179.92, c = 185.82 Angstrom and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

A work domain analysis framework for modelling intensive care unit patients

Work domain analysis (WDA) has been applied to a range of complex work domains, but few WDAs have been undertaken in medical contexts. One pioneering effort suggested that clinical abstraction is not based on means-ends relations, whereas another effort downplayed the role of bio-regulatory mechanisms. In this paper it is argued that bio-regulatory mechanisms that govern physiological behaviour must be part of WDA models of patients as the systems at the core of intensive care units. Furthermore it is argued that because the inner functioning of patients is not completely known, clinical abstraction is based on hypothetico-deductive abstract reasoning. This paper presents an alternative modelling framework that conforms to the broader aspirations of WDA. A modified version of the viable systems model is used to represent the patient system as a nested dissipative structure while aspects of the recognition primed decision model are used to represent the information resources available to clinicians in ways that support lsquoif...thenrsquo conceptual relations. These two frameworks come together to form the recursive diagnostic framework, which may provide a more appropriate foundation for information display design in the intensive care unit.

Adoption of a sedation scoring system and sedation guideline in an intensive care unit

Aim. The paper presents a study assessing the rate of adoption of a sedation scoring system and sedation guideline. Background. Clinical practice guidelines including sedation guidelines have been shown to improve patient outcomes by standardizing care. In particular sedation guidelines have been shown to be beneficial for intensive care patients by reducing the duration of ventilation. Despite the acceptance that clinical practice guidelines are beneficial, adoption rates are rarely measured. Adoption data may reveal other factors which contribute to improved outcomes. Therefore, the usefulness of the guideline may be more appropriately assessed by collecting adoption data. Method. A quasi-experimental pre-intervention and postintervention quality improvement design was used. Adoption was operationalized as documentation of sedation score every 4 hours and use of the sedation and analgesic medications suggested in the guideline. Adoption data were collected from patients' charts on a random day of the month; all patients in the intensive care unit on that day were assigned an adoption category. Sedation scoring system adoption data were collected before implementation of a sedation guideline, which was implemented using an intensive information-giving strategy, and guideline adoption data were fed back to bedside nurses. After implementation of the guideline, adoption data were collected for both the sedation scoring system and the guideline. The data were collected in the years 2002-2004. Findings. The sedation scoring system was not used extensively in the pre-intervention phase of the study; however, this improved in the postintervention phase. The findings suggest that the sedation guideline was gradually adopted following implementation in the postintervention phase of the study. Field notes taken during the implementation of the sedation scoring system and the guideline reveal widespread acceptance of both. Conclusion. Measurement of adoption is a complex process. Appropriate operationalization contributes to greater accuracy. Further investigation is warranted to establish the intensity and extent of implementation required to positively affect patient outcomes.

The use of recombinant activated factor VII for refractory bleeding post complex cardiothoracic surgery

We reviewed the outcome following use of recombinant activated factor VII (rVIIa) in patients with major bleeding post cardiothoracic surgery in our unit between January 2002 and July 2004. The unit consists of 16 cardiothoracic intensive care beds in a public metropolitan teaching hospital which serves as a referral centre for heart and lung transplant surgery Patients with refactory bleeding following cardiothoracic surgical procedures who were treated with rVIIa were identified. A total of 12 episodes of rVIIa use were recorded in ten patients, including three episodes with ventricular assist devices, and 5 heart and/or lung transplants. The median dose used was 85 mu g/kg. Chest tube drainage decreased in all patients following administration of rVIIa; median chest tube drainage decreased front 445 ml/h to 171 ml/h (P=0.03). Despite cessation of bleeding, mortality was high, when rVIIa was used after more than 24 hours. In six episodes, despite early rVIIa use (within six hours), continued bleeding necessitated return to theatre, where a surgical source of bleeding was found. In this small retrospective study, rVIIa significantly reduced bleeding that was refractory to standard blood product transfusion. In this series of patients., those that did not respond to rVIla early in the postoperative phase were found to have a surgical source of bleeding.

Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens

Vaccination remains a key tool in the protection and eradication of diseases. However, the development of new safe and effective vaccines is not easy. Various live organism based vaccines currently licensed, exhibit high efficacy; however, this benefit is associated with risk, due to the adverse reactions found with these vaccines. Therefore, in the development of vaccines, the associated risk-benefit issues need to be addressed. Sub-unit proteins offer a much safer alternative; however, their efficacy is low. The use of adjuvanted systems have proven to enhance the immunogenicity of these sub-unit vaccines through protection (i.e. preventing degradation of the antigen in vivo) and enhanced targeting of these antigens to professional antigen-presenting cells. Understanding of the immunological implications of the related disease will enable validation for the design and development of potential adjuvant systems. Novel adjuvant research involves the combination of both pharmaceutical analysis accompanied by detailed immunological investigations, whereby, pharmaceutically designed adjuvants are driven by an increased understanding of mechanisms of adjuvant activity, largely facilitated by description of highly specific innate immune recognition of components usually associated with the presence of invading bacteria or virus. The majority of pharmaceutical based adjuvants currently being investigated are particulate based delivery systems, such as liposome formulations. As an adjuvant, liposomes have been shown to enhance immunity against the associated disease particularly when a cationic lipid is used within the formulation. In addition, the inclusion of components such as immunomodulators, further enhance immunity. Within this review, the use and application of effective adjuvants is investigated, with particular emphasis on liposomal-based systems. The mechanisms of adjuvant activity, analysis of complex immunological characteristics and formulation and delivery of these vaccines are considered.

Broad direction bandwidths for complex motion mechanisms

Growing evidence from psychophysics and single-unit recordings suggests specialised mechanisms in the primate visual system for the detection of complex motion patterns such as expansion and rotation. Here we used a subthreshold summation technique to determine the direction tuning functions of the detecting mechanisms. We measured thresholds for discriminating noise and signal + noise for pairs of superimposed complex motion patterns (signal A and B) carried by random-dot stimuli in a circular 5° field. For expansion, rotation, deformation and translation we found broad tuning functions approximated by cos(d), where d is the difference in dot directions for signal A and B. These data were well described by models in which either: (a) cardinal mechanisms had direction bandwidths (half-widths) of around 60° or (b) the number of mechanisms was increased and their half-width was reduced to about 40°. When d = 180° we found summation to be greater than probability summation for expansion, rotation and translation, consistent with the idea that mechanisms for these stimuli are constructed from subunits responsive to relative motion. For deformation, however, we found sensitivity declined when d = 180°, suggesting antagonistic input from directional subunits in the deformation mechanism. This is a necessary property for a mechanism whose job is to extract the deformation component from the optic flow field. © 2001 Elsevier Science Ltd.