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PubMed2001

Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes.

The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP). The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution. The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A. GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP. Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains. Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP. apo-GK has a much more open conformation than the GK.GMP complex. Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains. The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding. The results suggest that helix 3 plays an important role in domain movement. Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open. Therefore, the LID domain movement may facilitate the binding of MgATP. The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK.

Acetylation↗

PubMed2001

The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

Amino Acid Motifs↗

PubMed2001

Extending the accuracy limits of prediction for side-chain conformations.

Current techniques for the prediction of side-chain conformations on a fixed backbone have an accuracy limit of about 1.0-1.5 A rmsd for core residues. We have carried out a detailed and systematic analysis of the factors that influence the prediction of side-chain conformation and, on this basis, have succeeded in extending the limits of side-chain prediction for core residues to about 0.7 A rmsd from native, and 94 % and 89 % of chi(1) and chi(1+2 ) dihedral angles correctly predicted to within 20 degrees of native, respectively. These results are obtained using a force-field that accounts for only van der Waals interactions and torsional potentials. Prediction accuracy is strongly dependent on the rotamer library used. That is, a complete and detailed rotamer library is essential. The greatest accuracy was obtained with an extensive rotamer library, containing over 7560 members, in which bond lengths and bond angles were taken from the database rather than simply assuming idealized values. Perhaps the most surprising finding is that the combinatorial problem normally associated with the prediction of the side-chain conformation does not appear to be important. This conclusion is based on the fact that the prediction of the conformation of a single side-chain with all others fixed in their native conformations is only slightly more accurate than the simultaneous prediction of all side-chain dihedral angles.

Computational Biology↗

PubMed2001

Intestinal and hemodynamic impairment following mesenteric ischemia/reperfusion.

BACKGROUND: Clinical intestinal ischemia/reperfusion (I/R) injury results in local and systemic dysfunction. A rat model of transient mesenteric occlusion has been used to study this phenomenon. However, a systematic analysis of the rat model with respect to intestinal permeability and hemodynamics has not been carried out. MATERIALS AND METHODS: In anesthetized rats, the superior mesenteric artery was occluded for 60 min, followed by reperfusion for 4 h. Intestinal impairment was evaluated via histological examination and by measuring ex vivo apparent permeability coefficients (Papp) of mannitol (0.18 kDa), inulin (5 kDa), and dextran (70 kDa). Hemodynamic effects of intestinal I/R were determined by monitoring mean arterial pressure (MAP) and heart rate (HR) via a catheter placed in the femoral artery. RESULTS: The animal model was associated with increased ex vivo Papp for mannitol and inulin. Although I/R injury was accompanied by significant histological disruption, there was no observable alteration in dextran permeability, suggesting that the loss in normal barrier function was limited to low-molecular-weight compounds. Hemodynamic measurements indicated that reperfusion induced a precipitous and sustained fall in MAP. HR values fell sharply following reperfusion but gradually increased and eventually "overshot" to values greater than baseline. CONCLUSIONS: Our findings demonstrate the selective loss of barrier function of the small bowel following intestinal I/R. Furthermore, these results also illustrate the importance of selecting appropriate permeability markers for the evaluation of intestinal damage. In light of the significant hemodynamic disruption accompanying the animal model, our investigation also points toward the need for developing therapeutic strategies that mitigate the local and systemic effects of intestinal I/R injury.

Animals↗

PubMed1999

Optimal stoichiometric designs of ATP-producing systems as determined by an evolutionary algorithm.

The design of metabolic pathways is thought to be the result of an optimization process such that the structure of contemporary metabolic routes maximizes a particular objective function. Recently, it has been shown that some essential stoichiometric properties of glycolysis can be explained on the basis of the requirement for a high ATP production rate. Because the number of stoichiometrically feasible designs increases strongly with the number of reactions involved, a systematic analysis of all the possibilities turns out to be inaccessible beyond a certain system size. We present, therefore, an alternative approach to compute in a more efficient way the optimal design of glycolysis interacting with an external ATP-consuming reaction. The algorithm is based on the laws of evolution by natural selection, and may be viewed as a particular version of evolutionary algorithms. The following conclusions are derived: (a) evolutionary algorithms are very useful search strategies in determining optimal stoichiometries of metabolic pathways. (b) Essential topological features of the glycolytic network may be explained on the basis of flux optimization. (c) There is a strong interrelation between the optimal stoichiometries and the thermodynamic and kinetic properties of the participating reactions. (d) Some subsequences of reactions in optimal pathways are strongly conserved at variation of system parameters, which may be understood by applying principles of metabolic control analysis.

Adenosine Triphosphatases↗

PubMed2001

A yeast expression vector and leucine selection in Escherichia coli to aid in the identification of novel genes.

The complete sequencing of the Saccharomyces cerevisiae genome provides a powerful tool for studying and elucidating essential cellular processes. To aid in the application of this resource to investigations into the molecular mechanisms of endoplasmic reticulum-associated protein degradation, a simple procedure was designed to generate a unique 2-microm LEU2-selectable yeast expression vector. Putative genes easily inserted into this vector are under control of the ADH1 promoter and transcription terminator sequences. Furthermore, a LEU2 selection in both yeast and Escherichia coli was used to allow the isolation of underrepresented plasmid from a pool of multiple vectors. Together, these advances in technology will be useful in the systematic analysis of novel yeast gene function.

3-Isopropylmalate Dehydrogenase↗

PubMedDate not supplied

School physical education: effect of the Child and Adolescent Trial for Cardiovascular Health.

BACKGROUND: Physical inactivity is a risk behavior for cardiovascular and other diseases. Schools can promote public health objectives by increasing physical activity among youth. METHODS: The Child and Adolescent Trial for Cardiovascular Health (CATCH) was a multicenter, randomized trial to test the effectiveness of a cardiovascular health promotion program in 96 public schools in four states. A major component of CATCH was an innovative, health-related physical education (P+) program. For 2.5 years, randomly assigned schools received a standardized PE intervention, including curriculum, staff development, and follow-up. RESULTS: Systematic analysis of 2,096 PE lessons indicated students engaged in more moderate-to-vigorous physical activity (MVPA) in intervention than in control schools (P = 0.002). MVPA during lessons in intervention schools increased from 37.4% at baseline to 51.9%, thereby meeting the established Year 2000 objective of 50%. Intervention children reported 12 more min of daily vigorous physical activity (P = 0.003) and ran 18.6 yards more than control children on a 9-min run test of fitness (P = 0.21). CONCLUSIONS: The implementation of a standardized curriculum and staff development program increased children's MVPA in existing school PE classes in four geographic and ethnically diverse communities. CATCH PE provides a tested model for improving physical education in American schools.

Adolescent↗

PubMed2002

Expression of FLAG fusion proteins in insect cells: application to the multi-subunit transcription/DNA repair factor TFIIH.

The multi-subunit transcription/DNA repair factor TFIIH was used as a model system to show that the expression of FLAG fusion proteins in insect cells constitutes a versatile tool for both structural and functional investigations. In the present study, we have constructed recombinant baculoviruses expressing the four core TFIIH subunits fused at their N-terminus to the FLAG peptide. Using these recombinant viruses we have established protocols based on anti-FLAG immunoaffinity chromatography that allow the systematic analysis of pairwise interaction within multiprotein complexes and have developed a double tag strategy (FLAG and hexahistidine tags) for the identification and purification of stable TFIIH subcomplexes. A simple purification procedure was developed that leads to the isolation of recombinant TFIIH containing the full set of subunits. The purified recombinant TFIIH was shown to be active in a transcription assay and to be structurally homologous to the endogenous complex by electron microscopy and image analysis.

Animals↗

PubMed2000

The influence of the radiofrequency excitation and conversion pulses on the lineshapes and intensities of the triple-quantum MAS NMR spectra of I = 3/2 nuclei.

A rigorous examination of the various multiple-quantum magic angle spinning sequences is carried out with reference to sensitivity enhancement in the isotropic dimension and the lineshapes of the corresponding MAS peaks in the anisotropic dimension. An echo efficiency parameter is defined here, which is shown to be an indicator of the performance aspects of the various sequences. This can be used in the design of further new experiments in this field. A consequence of such a systematic analysis has been the combination of a spin-lock pulse for excitation of multiple-quantum coherences and an amplitude-modulated pulse for their conversion to observable single-quantum coherences. This approach has resulted in an improved performance over other sequences with respect to both the anisotropic lineshapes and the isotropic intensities.

Anisotropy↗

PubMed1999

Mechanism of interferon action: functional characterization of positive and negative regulatory domains that modulate transcriptional activation of the human RNA-dependent protein kinase Pkr promoter.

The PKR protein kinase is an important regulator of viral mRNA translation. A approximately 50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). The Pkr promoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bp IFN-stimulated response element (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5' flanking region of the human Pkr gene to define how the novel KCS element acts to affect basal as well as IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) revealed that nuclear proteins bound selectively to the KCS element in a manner that was not dependent upon either IFN treatment or protein binding at the adjacent ISRE element. KCS protein binding activity in vitro correlated with activation of transcription in vivo in transient transfection assays. Competitionsupershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Sp1. In addition to the positive regulatory domain containing the KCSISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned approximately 400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affected Pkr transcription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within the Pkr promoter that function through the KCS element to affect basalIFN-inducible transcription of Pkr.

Base Sequence↗

PubMed1999

Proteolytic processing in infectious bursal disease virus: identification of the polyprotein cleavage sites by site-directed mutagenesis.

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of an immune depressive disease that affects domesticated and wild avian species. The expression strategy of IBDV includes the synthesis of a 110-kDa polyprotein containing the capsid precursor polypeptides. The polyprotein is autocatalitically processed rendering three polypeptides: NH2-VPX-VP4-VP3-COOH. We have carried out a systematic analysis, using a series of plasmids encoding polyproteins containing either deletions or single amino acid substitutions, to identify the processing sites. The results obtained showed the existence of two sites, 511LAA513 and 754MAA756, that are essential for the processing of the VPX-VP4 and VP4-VP3 precursors, respectively. These sequences are highly conserved among IBDV strains form serotypes 1 and 2. A secondary VPX-VP4 processing site was detected in a 19-amino acid stretch located upstream of the 511LAA513 site. Analyses using versions of the 754MAA756 VP4-VP3 processing site containing conservative and nonconservative amino acid substitutions demonstrated that the specificity of the cleavage is dictated by the conserved AA dipeptide.

Amino Acid Sequence↗

PubMed1987

Metal regulatory elements of the mouse metallothionein-I gene.

The promoter of the mouse metallothionein-I (mMT-I) gene contains multiple metal regulatory elements (MREs) which allow transcription of the gene to be induced by heavy metals. Insertion into the promoter of the TK gene of two or more synthetic MREs enables that gene to respond to heavy metals. We tested each of the MREs from the mMT-I promoter in this assay, and found four to be functional. We have commenced a systematic analysis of single nucleotide changes within an MRE, to determine the contribution of each nucleotide. The MRE core sequence in which single nucleotide changes can abolish function is TGCRCNCG; certain changes outside this sequence have lesser effects. MREs can act cooperatively with distal promoter elements of the TK gene, but in the presence of just a TATA-box they function as heavy-metal dependent promoter elements. Experiments to determine the effect of spacing suggest a range of at least 90 bp for interaction of two MREs, but the range for efficient stimulation of transcription from a TATA-box appears to be shorter. Stimulation of MT gene transcription by heavy metals is probably mediated by heavy metal-activated regulatory proteins binding cooperatively to the multiple MREs.

Animals↗

PubMed2001

Cellular receptors and hantavirus pathogenesis.

Hantaviruses cause two potentially lethal diseases, HPS and HFRS, and both diseases result in defects in vascular permeability and platelet function. Human beta 3 integrins confer cellular susceptibility to HPS- and HFRS-causing hantaviruses, a fact directly linking platelets, endothelial cells, and hantavirus diseases to the use of [figure: see text] cellular receptors that maintain capillary integrity and regulate platelet function. The role of vitronectin, PAI-1, uPAR, and complement cascades in hantavirus pathogenesis are unstudied but may contribute to specific disease syndromes effected by hantaviruses. The divergence of hantavirus surface glycoproteins and common beta 3-integrin usage provides further insight into the interaction of hantaviruses with cells. G1 and G2 glycoprotein variation is likely to contribute to additional interactions that determine pathogenic responses to individual viruses. beta 3-integrin usage also suggests that common elements exist on G1 or the more highly conserved G2 surface glycoprotein, which mediate viral attachment to integrins. Although there is currently no data defining the virion attachment protein, the development of antibodies that recognize the hantavirus attachment protein and block integrin interactions is of interest since it is likely to provide an additional point for therapeutic intervention and vaccine development. There are a plethora of effects that could be elicited by hantavirus regulation of cellular beta 3 integrins and their ligands that are consistent with hantavirus diseases. Since beta 3 integrins are critical adhesive receptors on platelets and endothelial cells and regulate both vascular permeability and platelet activation and adhesion, the use of these receptors by hantaviruses is likely to be fundamental to hantavirus pathogenesis. The lack of an animal model for hantavirus pathogenesis has prevented a systematic analysis of immune and cellular responses to hantavirus infections, and it impedes our ability to study protective or therapeutic approaches to hantavirus diseases. However, recent findings suggest that human beta 3 integrins within transgenic mice may provide animal models of hantavirus pathogenesis and have the potential to radically alter the ability to investigate hantavirus disease.

Animals↗

PubMed1995

A near-upstream element in a plant polyadenylation signal consists of more than six nucleotides.

A plant polyadenylation signal consists of three distinct components: a far-upstream element (FUE) that can control utilization of several polyadenylation sites, one or more near-upstream elements (NUEs) that control utilization of each site in a transcription unit, and polyadenylation site (CSs) themselves. NUEs have previously been suggested to be related to the mammalian polyadenylation signal AAUAAA. However, many plant genes do not contain AAUAAA-like motifs near their polyadenylation sites. To better understand the nature of NUEs, we conducted a systematic analysis of the NUE for one polyadenylation site (site 1) in the pea rbcS-E9 gene; this NUE lacks an AAUAAA motif. Linker substitution studies showed that the NUE for site 1 in this gene resides in the sequence AAAUGGAAA. Single-nucleotide substitutions in this domain had modest effects on the functioning of this NUE. Replacement of part of this sequence with the sequence AAUAAA increased the efficiency of this NUE. However, alteration of nucleotides immediately 3' of the AAUAAA reversed this effect. Our results indicate that the NUE for site 1 consists of as many as 9 nucleotides, that these 9 bases do not include an element that is intolerant of single base changes, that the sequence AAUAAA can function as a NUE for site 1, and that sequences flanking AAUAAA can affect the efficiency of functioning as a NUE.

Base Sequence↗

PubMed1989

Stereo and eye movement.

We describe a method to solve stereo correspondence using controlled eye (or camera) movements. Eye movements supply additional image frames and monocular depth estimate, which can be used to constrain stereo matching. Because the eye movements are small, traditional stereo techniques of stereo with multiple frame will not work. We develop an alternative approach using a systematic analysis to define a probability distribution for the errors. Our matching strategy then matches the most probable points first, thereby reducing the ambiguity for the remaining matches. We demonstrate this algorithms with several examples.

Computer Simulation↗

PubMed1995

Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells.

The Rhesus (Rh) blood group system shows complex polymorphisms in the human. Some of the heterogeneity may be generated by alternative RNA splicing. For a systematic analysis of Rh-related mRNA isoforms expressed in reticulocytes, we isolated mRNA, which was then reverse transcribed and amplified by the polymerase chain reaction (PCR) to give Rh-related cDNAs of two segments of 704 bp and 975 bp. The PCR amplification of the 5'-region yielded a single PCR product, whereas a complex electrophoretic pattern of PCR bands was derived from the 3'-region. A highly reproducible ladder of multiple additional bands migrated below the PCR products corresponding to the full-size cDNAs for RhPI and RhPII and encoding two different Rh polypeptides. Eleven and five truncated isoforms of the RhPI and RhPII cDNAs, respectively, were identified in the PCR products. These isoforms appear to be generated by combinatorial splicing of six RhPI and three RhPII exons. Our results suggest that the Rh-related polypeptides consist of a mixture of RhPI and RhPII polypeptide isoforms differing at the C terminus. Multiple RNA splicing pathways are thus operative in the two Rh-related genes even within a single cell lineage of human erythroid cells.

Alternative Splicing↗

PubMed1993

Presentation of the rifle project risk factors and life expectancy. The RIFLE Research Group.

Plans are presented for conducting an epidemiological observational project which will pool the data from nine Italian field studies covering over 70,000 men and women aged 20 to 69 years, belonging to 52 population samples distributed in 13 different regions of the country. The main objective is to produce multivariate models for the prediction of all-cause mortality, life expectancy and selected causes of death as a function of some baseline characteristics. The entry examinations of the population samples were performed between 1978 and 1987 with the measurements of risk factors and other personal characteristics mainly related to cardiovascular diseases and other chronic conditions. They include a large number of anthropometric, social, biochemical, biophysical, clinical, nutritional and behavioural measurements, although only a limited subset of them is common to all the studies. The collection of data on life status, mortality and causes of death has already been completed for 45 out of the 52 samples, although for lengths of follow-up varying from 4 to 12 years. The systematic analysis of this data bank will be started in 1993.

Adult↗

PubMed1981

Presence of a smooth muscle system in aortic valve leaflets.

The location and the spatial arrangement of smooth muscle cells in aortic valves have been assessed by a systematic analysis of serial semithin sections of plastic embedded porcine and human aortic leaflets, combined with an electron microscope study. The investigation showed that smooth muscle cells, either single and arranged in thin bundles, and other cell types such as myofibroblasts are constantly present in the aortic valve leaflets. In addition, it was possible to devise a model of the three dimensional, specific organization of the smooth muscle bundles which can be interpreted as an intrinsic muscle system of the leaflets. As the muscular elements might play an active role in the normal functioning of the valve, their presence should be taken into account in designing (bio)prosthetic leaflets and in the evaluation of valve pathology.

Animals↗