Effect of a facemask leak on aerosol delivery from a pMDI-spacer system.

The objective of this investigation was to study the relation between size and position of a mask leak on spacer output and lung dose. An upper-airway model (SAINT model, Erasmus MC) was connected to a breathing simulator. Facemasks with leaks ranging between 0 and 1.5 cm(2) were examined. Leaks were located close to the nose or close to the chin. During simulated breathing, 200 microg budesonide (Pulmicort, AstraZeneca) was delivered to the model via NebuChamber (AstraZeneca) with facemask. Spacer output and lung dose were measured by placing a filter between spacer and facemask or between model and breathing simulator, respectively. Budesonide trapped on the filter was quantified by means of HPLC, and expressed as percentage of the nominal dose. Mean spacer output doses for the nose position were 50, 38, 28, 12, 10, 6, and 0%, and for the chin position were 50, 40, 31, 11, 9, 4, and 0% for leaks of 0, 0.05, 0.1, 0.16, 0.2, 0.3, and larger than 0.4 cm(2), respectively. Mean lung doses for the nose position were 10, 8, 6, 3, 3, 1, 0, 0, 0, and 0%, and for the chin position were 10, 9, 8, 6, 6, 5, 1, 1, 0, and 0% for leaks of 0, 0.05, 0.1, 0.16, 0.2, 0.3, 0.4, 0.5, 1, and 1.5 cm(2). Efficiency of a pMDI-spacer facemask strongly depends on the size of a facemask leak. Spacer output did not depend on the position of the leak. Lung dose was higher for leaks near the chin than for leaks near the nose.

Intramolecular signal transmission in enterobacterial aspartate transcarbamylases II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola.

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropic co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, two trimers of catalytic chains with three dimers of regulatory chains ((c3)2(r2)3), as other enterobacterial ATCases and shows extensive primary structure conservation. In (c3)2(r2)3 ATCases, the association of the catalytic subunits c3 with the regulatory subunits r2 is responsible for the establishment of positive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effectors. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging these blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. herbicola ATCase. A novel structural element involved in allosteric signal recognition and transmission in this family of ATCases was thus identified.

The evolutionary history of carbamoyltransferases: A complex set of paralogous genes was already present in the last universal common ancestor.

Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC alpha and OTC beta) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC beta combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC alpha combination and the other harboring the ATC I/OTC beta combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.

Aspartate transcarbamylase from the hyperthermophilic eubacterium Thermotoga maritima: fused catalytic and regulatory polypeptides form an allosteric enzyme.

In the allosteric aspartate transcarbamylase (ATCase) from the hyperthermophilic eubacterium Thermotoga maritima, the catalytic and regulatory functions, which in class B ATCases are carried out by specialized polypeptides, are combined on a single type of polypeptide assembled in trimers. The ATCases from T. maritima and Treponema denticola present intriguing similarities, suggesting horizontal gene transfer.

Allosteric regulation in a family of enterobacterial aspartate transcarbamylases: intramolecular transmission of regulatory signals in chimeric enzymes.

Several enterobacterial aspartate transcarbamylases (ATCases) exhibit a [2(C3):3(r2)] quaternary structure analogous to that of the Escherichia coli enzyme. Despite their conserved quaternary structures, these enzymes present substantial differences in the co-operativity of substrate binding and in their allosteric regulation by nucleotide effectors. A comparison between different enzymatic species provides an opportunity to expand our understanding of the molecular basis of allostery in ATCase. Chimeric ATCases were constructed by exchanging subdomain regions involved in quaternary structural features, such as the r1-c4 regulatory-catalytic subunit interface analyzed in this study, in order to define the involvement of this interface in the several components of allosteric regulation. The r1-c4 interface was found to constitute an essential element for the recognition and the transmission of the ATP regulatory signal in the Serratia marcescens and the Proteus vulgaris ATCases, as it does in the E. coli ATCase. Besides, the specific amino acid composition of the C-terminal region of the regulatory chain and its interactions with the amino acid residues in the 240s loop of the catalytic chain (r1-c4 interactions) were found to modulate the amplitude of the enzyme's response to ATP. The C-terminal region of the regulatory chain did not appear to participate directly in the regulation of the three native ATCases by CTP. Even when CTP acts as an activator, as in the P. vulgaris and S. marcescens ATCases, its signal follows a route distinct from that of the general activator ATP. Synergistic inhibition by CTP and UTP was found to involve the transmission of a specific UTP signal. This signal appeared different in the various ATCases, involving the C-terminal region of the regulatory chain in the E. coli and S. marcescens ATCases but not in the P. vulgaris ATCase.

Intramolecular transmission of the ATP regulatory signal in Escherichia coli aspartate transcarbamylase: specific involvement of a clustered set of amino acid interactions at an interface between regulatory and catalytic subunits.

Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP. Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites. The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP. These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain. Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding. These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model. This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.

The activation of Escherichia coli aspartate transcarbamylase by ATP. Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains.

The regulatory chain of E. coli aspartate transcarbamylase (E.C. 2.1.3.2) is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.

Prospective study of sentinel headache in aneurysmal subarachnoid haemorrhage.

Retrospective surveys of patients with subarachnoid haemorrhage suggest that minor episodes with sudden headache (warning leaks) may precede rupture of an aneurysm, and that early recognition and surgery might lead to improved outcome. We studied 148 patients with sudden and severe headache (possible sentinel headache) seen by 252 general practitioners in a 5-year period in the Netherlands. Subarachnoid haemorrhage was the cause in 37 patients (25%) (proven aneurysm in 21, negative angiogram in 6, no angiogram done in 6, sudden headache followed by death in 4). 103 patients had headache as the only symptom, 12 of whom proved to have subarachnoid haemorrhage (6 with a ruptured aneurysm). Previous bouts of sudden headache had occurred in only 2. Other serious neurological conditions were diagnosed in 18. In the remaining 93, no underlying cause of headache was found; follow-up over 1 year showed no subsequent subarachnoid haemorrhage or sudden death. In this cohort, acute, severe headache in general practice indicated a serious neurological disorder in 37% (95% CI 29-45%), and subarachnoid haemorrhage in 25% (18-32%). 12% (5-18%) of those with headache as the only symptom. The notion of warning leaks as a less serious variant of subarachnoid haemorrhage is not supported by this study. Early recognition of subarachnoid haemorrhage is important but will probably have only limited impact on the outcome in the general population.

Arginine regulon of Escherichia coli K-12. A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression.

The 12 genes which in E. coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5' non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the repressor, a hexameric protein. In vitro binding experiments with purified repressor (a gift from W. K. Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAb and the bipolar argECBH cluster. A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only. Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves. Symmetrical contacts in the minor groove with A residues have also been identified. Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes. Although the wild-type operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity. Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box. The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect. However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced. Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate Oc type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed. The significance of this lack of correlation is discussed.

Heterotropic interactions in aspartate transcarbamoylase: turning allosteric ATP activation into inhibition as a consequence of a single tyrosine to phenylalanine mutation.

Aspartate transcarbamoylase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allostery. This enzyme shows cooperativity between the catalytic sites, and its activity is feedback inhibited by CTP and activated by ATP. These regulatory processes involve several interfaces between catalytic and regulatory chains as well as between domains within these two types of chains. As far as the regulatory chain is concerned, its two domains are in contact through a hydrophobic interface, in which a tyrosine residue is inserted in a pocket involving two leucine residues of the allosteric domain and a valine and a leucine residue of the zinc domain. To probe the possible implication of this hydrophobic core in the CTP and ATP regulatory effect, the tyrosine was replaced by a phenylalanine through oligonucleotide-directed mutagenesis. Interestingly, the resulting mutant shows a complete inversion of the ATP effect; it is now inhibited by ATP instead of being activated by this nucleotide triphosphate. This mutant remains normally sensitive to the feedback inhibitor CTP. This result shows that the hydrophobic interface between the two domains of the regulatory chain plays an important role in the discrimination between the regulatory signals promoted by the two allosteric effectors.

Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation.

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains.

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals.

The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined. The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals. Three regions are highly conserved. Two of them appear to contain possible Walker-type nt-binding sites [Walker et al., EMBO J. 1 (1982) 945-951] and are therefore candidates for ATP-binding sites. The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

The catalytic site of Escherichia coli aspartate transcarbamylase: interaction between histidine 134 and the carbonyl group of the substrate carbamyl phosphate.

Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme. In the present work, this residue was replaced by an asparagine through site-directed mutagenesis. The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate. In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group. In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3). In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type.

Engineering aspartate transcarbamylase.

Aspartate transcarbamylase from Escherichia coli is one of the most extensively studied regulatory enzymes as a model of cooperativity and allostery. Numerous methods are used to engineer variants of this molecule: random and site-directed mutagenesis, dissociation and reassociation of the catalytic and regulatory subunits and chains, construction of hybrids made from normal and modified subunits or chains, interspecific hybrids and construction of chimeric enzymes. These methods provide detailed information on the regions, domains, interfaces and aminoacid residues which are involved in the mechanism of co-operativity between the catalytic sites, and of regulation by the antagonistic effectors CTP and ATP. These effectors induce the transmission of intramolecular signals whose pathways begin to be delineated.

Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB.

The complete nucleotide sequence of argF is presented, together with that of an operator-constitutive mutant. ArgF is compared with the other gene coding for ornithine carbamoyltransferase (OTCase) in E. coli K-12, argI, and with pyrB, encoding the catalytic monomer of aspartate carbamoyltransferase (ATCase). ArgF and argI appear very closely related having emerged from a relatively recent ancestor gene. The relationship between OTCase and ATCase appears more distant. Nevertheless, the homology observed between the two proteins (mainly in the polar domain) suggests a common origin.

The regulatory region of the divergent argECBH operon in Escherichia coli K-12.

The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed.

Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12.

The argF and argI genes code for similar proteins able to assemble into hybrid isoenzymes and are therefore thought to share a common origin. We show here that the nucleotide sequence of the promoter and operator regions of these two genes are highly homologous. DNA regions preceding the control sites also present significant homologies. The results support the notion of divergent evolution of the two genes from a common ancestor. Like argE and argCBH , argF and argI are controlled by a repressor molecule recognizing a family of similar operator sites. Attenuation appears to play no role in this regulation.