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Random Sequence (random + sequence)
Selected AbstractsActive TEM-1 ,-lactamase mutants with random peptides inserted in three contiguous surface loopsPROTEIN SCIENCE, Issue 10 2006Pascale Mathonet Abstract Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 ,-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two ,-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first ,-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final ,-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified. [source] The relationship of intracanal medicaments to postoperative pain in endodonticsINTERNATIONAL ENDODONTIC JOURNAL, Issue 12 2003E. H. Ehrmann Abstract Aim, To investigate the relationship of postoperative pain to three different medicaments placed in the root canal after a complete biomechanical debridement of the root canal system in patients presenting for emergency relief of pain. Methodology, Two hundred and twenty-three teeth belonging to 221 patients presenting as emergencies to the Royal Dental Hospital of Melbourne were included in the study. Inclusion was limited to patients with a diagnosis of pulp necrosis and acute apical periodontitis. All teeth underwent conventional root canal treatment, which involved the instrumentation to the apices of each canal at the first visit. Canals were instrumented using a stepback technique and hand-files along with irrigants using Milton's (1% sodium hypochlorite) solution followed by 15% EDTAC. The canals were dried and one of the following three medicaments was inserted into the canal in random sequence: Group 1: Ledermix paste (Lederle Pharmaceuticals, Division of Cyanamid, Wolfratshausen, Germany); Group 2: calcium hydroxide paste (Calcipulpe, Septodont, France); and Group 3: no dressing. Before dismissal, the preoperative pain experienced on the previous night was recorded using a visual analogue pain scale. Patients were then instructed to record the degree of pain experienced 4 h after treatment and daily for a further 4 days. Results, The mean score pain for all three groups was between 42 and 48 prior to treatment being commenced. After 4 days, the pain score for Group 2 was 10, for Group 3 was 7 and for Group 1 was 4. Mean preoperative pain level was 44.4 (of a maximum 100) for all groups, and declined by 50% (to 22.1) after 24 h. Patients in Group 1 (Ledermix) experienced significantly less (P = 0.04) postoperative pain than those in the other two groups. There was no significant difference between Group 2 (calcium hydroxide) and Group 3 (no dressing). Conclusion, Under the conditions of this study, painful teeth with acute apical periodontitis that had been dressed with Ledermix paste gave rise to less pain than that experienced by patients who had a dressing of calcium hydroxide or no dressing at all. Ledermix is an effective intracanal medicament for the control of postoperative pain associated with acute apical periodontitis, with a rapid onset of pain reduction. [source] Long-Lasting Resistance to Extinction of Response Reinstatement Induced by Ethanol-Related Stimuli: Role of Genetic Ethanol PreferenceALCOHOLISM, Issue 10 2001Roberto Ciccocioppo Background: The conditioning of ethanol's reinforcing effects with specific environmental stimuli is thought to be a critical factor in long-lasting relapse risk associated with alcoholism. To study the significance of such learning factors in the addictive potential of ethanol, this experiment was designed (1) to characterize the effects of stimuli associated with alcohol availability on the reinstatement of responding at a previously ethanol-paired lever in rats with genetically determined ethanol preference versus nonpreference and (2) to examine the persistence of the motivating effects of these stimuli over time. Methods: Male alcohol-preferring (P) and alcohol-nonpreferring (NP) rats were trained to operantly self-administer ethanol (10% w/v) or water on a fixed-ratio 1 schedule in a 30-min daily session. Ethanol and water sessions were scheduled in random sequence across training days. Ethanol availability was signaled by an olfactory discriminative stimulus (banana extract, S+), and each lever press was paired with brief presentation of the conditioning chamber's house light (CS+). The discriminative stimulus signaling water availability (i.e., nonreward) consisted of anise odor (S,), and lever-responses during water sessions were paired with a brief white noise generation (CS,). The rats then were placed on extinction conditions during which ethanol and water, as well as the corresponding stimuli, were withheld. The effects of noncontingent exposure to the S+ versus S, paired with response-contingent presentation of the CS+ versus CS, on responding at the previously active lever were then determined in 30-min reinstatement sessions. To study the resistance to extinction of the effects of the ethanol-associated stimuli, additional tests were conducted at 3-day intervals for a total of 50 days. Results: The number of ethanol-reinforced responses during self-administration training was significantly greater in P than in NP rats (p < 0.01). After extinction, a significant recovery of responding was observed in both groups of rats under the stimulus conditions associated with ethanol (S+/CS+) but not those associated with water (S,/CS,). However, the response reinstatement was significantly greater in P than NP rats (p < 0.01). In addition, the results revealed a considerable resistance to extinction to the effects of the ethanol-associated stimuli. Throughout the 50-day test period, responding remained significantly above extinction levels in both P and NP rats (p < 0.01), but with an overall greater number of responses in P than NP rats (p < 0.05). Conclusions: The results support the hypothesis that conditioning factors contribute importantly to compulsive ethanol seeking and long-lasting vulnerability to relapse. In addition, the results suggest that genetic predisposition toward heightened ethanol intake extends to greater susceptibility to the motivating effects of ethanol-related environmental stimuli. [source] The frequency spectrum of finite samples from the intermittent silence processJOURNAL OF THE AMERICAN SOCIETY FOR INFORMATION SCIENCE AND TECHNOLOGY, Issue 4 2009Ramon Ferrer-i-Cancho It has been argued that the actual distribution of word frequencies could be reproduced or explained by generating a random sequence of letters and spaces according to the so-called intermittent silence process. The same kind of process could reproduce or explain the counts of other kinds of units from a wide range of disciplines. Taking the linguistic metaphor, we focus on the frequency spectrum, i.e., the number of words with a certain frequency, and the vocabulary size, i.e., the number of different words of text generated by an intermittent silence process. We derive and explain how to calculate accurately and efficiently the expected frequency spectrum and the expected vocabulary size as a function of the text size. [source] Frequencies of hydrophobic and hydrophilic runs and alternations in proteins of known structurePROTEIN SCIENCE, Issue 1 2006Russell Schwartz Abstract Patterns of alternation of hydrophobic and polar residues are a profound aspect of amino acid sequences, but a feature not easily interpreted for soluble proteins. Here we report statistics of hydrophobicity patterns in proteins of known structure in a current protein database as compared with results from earlier, more limited structure sets. Previous studies indicated that long hydrophobic runs, common in membrane proteins, are underrepresented in soluble proteins. Long runs of hydrophobic residues remain significantly underrepresented in soluble proteins, with none longer than 16 residues observed. These long runs most commonly occur as buried , helices, with extended hydrophobic strands less common. Avoiding aggregation of partially folded intermediates during intracellular folding remains a viable explanation for the rarity of long hydrophobic runs in soluble proteins. Comparison between database editions reveals robustness of statistics on aqueous proteins despite an approximately twofold increase in nonredundant sequences. The expanded database does now allow us to explain several deviations of hydrophobicity statistics from models of random sequence in terms of requirements of specific secondary structure elements. Comparison to prior membrane-bound protein sequences, however, shows significant qualitative changes, with the average hydrophobicity and frequency of long runs of hydrophobic residues noticeably increasing between the database editions. These results suggest that the aqueous proteins of solved structure may represent an essentially complete sample of the universe of aqueous sequences, while the membrane proteins of known structure are not yet representative of the universe of membrane-associated proteins, even by relatively simple measures of hydrophobic patterns. [source] Nonspatial intermodal selective attention is mediated by sensory brain areas: Evidence from event-related potentialsPSYCHOPHYSIOLOGY, Issue 5 2001Durk Talsma The present study focuses on the question of whether inter- and intramodal forms of attention are reflected in activation of the same or different brain areas. ERPs were recorded while subjects were presented a random sequence of visual and auditory stimuli. They were instructed to attend to nonspatial attributes of either auditory or visual stimuli and to detect occasional target stimuli within the attended channel. An occipital selection negativity was found for intramodal attention to visual stimuli. Visual intermodal attention was also manifested in a similar negativity. A symmetrical dipole pair in the medial inferior occipital areas could account for the intramodal effects. Dipole pairs for the intermodal attention effect had a slightly more posterior location compared to the dipole pair for the intramodal effect. Auditory intermodal attention was manifested in an early enhanced negativity overlapping with the N1 and P2 components, which was localized using a symmetrical dipole pair in the lateral auditory cortex. The onset of the intramodal attention effect was somewhat later (around 200 ms), and was reflected in a frontal processing negativity. The present results indicate that intra- and intermodal forms of attention were indeed similar for visual stimuli. Auditory data suggest the involvement of multiple brain areas. [source] The relevance of preserving temporary ponds during drought: hydrological and vegetation changes over a 16-year period in the Doņana National Park (south-west Spain)AQUATIC CONSERVATION: MARINE AND FRESHWATER ECOSYSTEMS, Issue 3 2008Laura Serrano Abstract 1.Although the Doņana National Park is given the highest degree of environmental protection in Spain, it is likely that groundwater discharge to several ponds within the Biological Reserve has been damaged by abstraction to a tourist resort located less than 1 km away. 2.Hydrological changes were monitored over 16 years (1 October 1989 to 30 September 2005) by recording the shallow water table of six temporary ponds at 1,8-week intervals, and the duration of pond wet phase (or hydroperiod) during each hydrological cycle. 3.The average rainfall for the study period was 563.2 mm, and included 6 wet, 5 moderate, and 5 dry years in a seemingly random sequence. The average rainy season extended from October until the end of March, while the dry season occupied the rest of the year. 4.The water table generally oscillated following this alternation of rainy and dry seasons, but this fluctuation was minimal during dry years, and even failed to occur at some ponds. 5.Since 1998/99, the average hydroperiod has shortened by 3 months at Charco del Toro pond, and by almost 2 months at Brezo pond, while the rest of the ponds exhibited a reduction of less than 1 month. 6.Vegetation changed in the ponds between May 1990 and 2005. Total plant cover increased (range of increase: 16,65%), and species richness decreased in all ponds (range of species loss: 4,18). 7.The reduction in the hydroperiod probably enhanced the growth of a few woody plants to the detriment of flooding-dependent species as the cover of Pinus pinea increased nearly fourfold at Brezo pond, while that of Scirpus lacustris was halved at Charco del Toro pond. 8.The pumping area for the nearby tourist resort should be relocated, and a specific management strategy should be developed in order to prevent further damage to the ponds. Copyright Š 2007 John Wiley & Sons, Ltd. [source] Lack of agreement between rheumatologists in defining digital ulceration in systemic sclerosisARTHRITIS & RHEUMATISM, Issue 3 2009Ariane L. Herrick Objective To test the intra- and interobserver variability, among clinicians with an interest in systemic sclerosis (SSc), in defining digital ulcers. Methods Thirty-five images of finger lesions, incorporating a wide range of abnormalities at different sites, were duplicated, yielding a data set of 70 images. Physicians with an interest in SSc were invited to take part in the Web-based study, which involved looking through the images in a random sequence. The sequence differed for individual participants and prevented cross-checking with previous images. Participants were asked to grade each image as depicting "ulcer" or "no ulcer," and if "ulcer," then either "inactive" or "active." Images of a range of exemplar lesions were available for reference purposes while participants viewed the test images. Intrarater reliability was assessed using a weighted kappa coefficient with quadratic weights. Interrater reliability was estimated using a multirater weighted kappa coefficient. Results Fifty individuals (most of them rheumatologists) from 15 countries participated in the study. There was a high level of intrarater reliability, with a mean weighted kappa value of 0.81 (95% confidence interval [95% CI] 0.77, 0.84). Interrater reliability was poorer (weighted , = 0.46 [95% CI 0.35, 0.57]). Conclusion The poor interrater reliability suggests that if digital ulceration is to be used as an end point in multicenter clinical trials of SSc, then strict definitions must be developed. The present investigation also demonstrates the feasibility of Web-based studies, for which large numbers of participants can be recruited over a short time frame. [source] Investigation of de novo Totally Random Biosequences, Part ICHEMISTRY & BIODIVERSITY, Issue 8 2006A General Method for in vitro Selection of Folded Domains from a Random Polypeptide Library Displayed on Phage Abstract This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences. [source] Investigation of de novo Totally Random Biosequences, Part IICHEMISTRY & BIODIVERSITY, Issue 8 2006On the Folding Frequency in a Totally Random Library of de novo Proteins Obtained by Phage Display Abstract We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth (,Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 109 clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part,I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of ,Never Born Proteins' are discussed in terms of molecular evolution. [source] Evaluating low level sequence identitiesFEBS JOURNAL, Issue 2 2001AROM homologous?, Are Aspergillus QUTA A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7,18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene136, 49,54]. We tested this hypothesis by a sensitive combination of position-specific log-odds scoring matrix methods. The position-specific log-odds scoring matrices were derived from a large number of 3-dehydroquinate synthase and 5- enolpyruvylshikimate-3-phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position-specific log-odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA,AROM homology even more problematic. [source] Blind identification of sparse Volterra systemsINTERNATIONAL JOURNAL OF ADAPTIVE CONTROL AND SIGNAL PROCESSING, Issue 7 2008Hong-Zhou Tan Abstract This paper is concerned with blind identification for single-input single-output Volterra systems with finite order and memory with the second-order and the third-order statistics. For the full-sized Volterra system (i.e. all its kernels are nonzero) excited by unknown independently and identically distributed stationary random sequences, it is shown that blind identifiability does not hold in the second-order moment (SOM) and the third-order moment (TOM) domain. However, under some sufficient conditions, a class of truncated sparse Volterra systems, where some kernels are restricted to being zero, can be identified blindly and more Volterra parameters can be estimated in TOM than in SOM. Numerical examples illustrate the effectiveness of the proposed methods. Copyright Š 2007 John Wiley & Sons, Ltd. [source] Low free energy cost of very long loop insertions in proteinsPROTEIN SCIENCE, Issue 2 2003Michelle Scalley-Kim Abstract Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 × 108 sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60,80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures. [source] TEM-1 ,-lactamase as a scaffold for protein recognition and assayPROTEIN SCIENCE, Issue 6 2002Daniel Legendre Abstract A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 ,-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to ,-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that ,-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to ,-lactamases binding to streptavidin, ,-lactamase clones binding to horse spleen ferritin and ,-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining ,-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for ,-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme. [source] Cover Picture: ChemBioChem 1/2003CHEMBIOCHEM, Issue 1 2003Sachdev S. Sidhu Dr. The cover picture shows a highly diverse peptide library displayed on cylindrical bacteriophage particles that also contain the cognate DNA. Libraries such as this can be used to identify specific ligands for essentially any protein of interest. Here, phage particles displaying peptides that bind vascular endothelial growth factor are selected from amongst a sea of billions of random sequences. Further information can be found in the Review by Sidhu and co-workers on p. 14 ff. (We thank David Wood and Christian Wiesmann for help in the preparation of the cover picture.) [source] | |||||||||||||