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Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.

Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins. This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6. Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid. Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA. These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp). These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.

Bacillus Phages↗

Factitious and fraudulent fever.

The task of elucidating the etiology of fever of undertermined origin remains a major undertaking. Factitious fever is uncommonly considered of major importance in the differential diagnosis of fever of undetermined origin although it is a readily identifiable, syndrome and one that is easily excluded one it has been considered. Early identification may reduce the necessity for prlonged, expensive and potentially hazardous hospitalizations for such patients. A retrospective study identified 2.2 per cent (11 of 506) of all patients whose fever on their charts was coded as fever of undetermined origin as having factitious fever. These patients either created factitious fever by manipulation of the thermometer or fraudulent fever by self-induced means. A review of the literature yielded an additional 70 cases in which fever was either the sole factitious sign or part of a larger, more complex factitious illness. Patients were typically young, female and often associated with the medical profession. Patients with factitious fever differ from those with the stereotyped Munchausen's syndrome and may be difficult to recognize. Signs leading to the recognition of this syndrome are emphasized. Since the nature of the psychiatric illness may vary from patient to patient, early discovery may facilitate psychiatric intervention as such patients may be more amenable to therapy.

Adolescent↗

Awareness of inner experience: a systems perspective on self-regulatory process in early development.

This brief synopsis of an organizational perspective on early development represents an integration of three major areas of the author's research: that of a detailed observational study of early mother-infant interaction over the first three years of life; that of a continuous neonatal state and caregiving interactional monitoring method over the first two months of life; and that of a 25- to 30-year follow-up on the same infants observed initially. From these data a ground plan is proposed for our thinking about the individual's life span trajectory as a unique construction within a unique context for that individual's interactional and adaptive self-regulatory strategies. Beginning with a review of biological principles, the paradoxical integration of complexity and unity in living systems is traced from the conceptualization of infant state and observations on its regulation through the role of state in the origins of awareness of inner experience, to the consolidation and validation of the self as agent in self-regulation. Central to integration is the recognition process, stemming from one's awareness that another is aware of what one is aware of within oneself. The constructionist perspective on the critical role of specificity in this organizing process is illustrated by reference to the negotiation between caregiver and infant of seven issues of adaptive coordination over the first three years of life. Finally, five propositions are formulated describing such specificity in this organizing process, as one that underlies integration of levels of the living system relating the biological, the developmental, the shaping of the life span trajectory, and the reconstructive therapeutic process.

Awareness↗

Bromodomain: an acetyl-lysine binding domain.

Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyl-lysine binding domains. More recent structural studies of bromodomain/peptide ligand complexes have enriched our understanding of differences in ligand selectivity of bromodomains. These new findings demonstrate that bromodomain/acetyl-lysine recognition can serve as a pivotal mechanism for regulating protein-protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.

Acetylation↗

Glutaminyl-tRNA synthetase: from genetics to molecular recognition.

Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code. They are synthesized by the aminoacyl-tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations. Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Sequence-specific interactions between GlnRS and tRNA(Gln) determine both the accuracy of tRNA selection and the efficiency of aminoacylation. Thus, amino acid recognition is tRNA-dependent. Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA-enzyme complex displays a considerably reduced affinity for glutamine compared to wild-type. This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.

Amino Acyl-tRNA Synthetases↗

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli.

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.

Bacterial Proteins↗

Identification of immune complex antigens in sera of Indian kala-azar patients.

Level of circulating immune complex (IC) in visceral leishmaniasis is much higher than that in control sera. In immunoblot experiment, treatment of kala-azar IC with patient sera showed at least 6 bands of which the band around 55 kDa region was most prominent. The band at 55 kDa is primarily due to the presence of an antigen recognized by its corresponding antibody present in the patient sera. This was confirmed by using radiolabelled antibody from kala-azar patient serum and antipromastigote serum. The heavy chain of IgG originating from IC is also present in the same region which was detected by its recognition with antihuman IgG. The IC gave a band at 55 kDa region with sea-urchin antitubulin. Kala-azar sera also reacted with purified rat brain tubulin. The present results suggest that a tubulin like protein is present at 55 kDa region along with the heavy chain of IgG.

Antigen-Antibody Complex↗

Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently.

The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.

Adenoviruses, Human↗

Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate cross-reactivity to related compounds.

In the context of drug hypersensitivity, our group has recently proposed a new model based on the structural features of drugs (pharmacological interaction with immune receptors; p-i concept) to explain their recognition by T cells. According to this concept, even chemically inert drugs can stimulate T cells because certain drugs interact in a direct way with T-cell receptors (TCR) and possibly major histocompatibility complex molecules without the need for metabolism and covalent binding to a carrier. In this study, we investigated whether mouse T-cell hybridomas transfected with drug-specific human TCR can be used as an alternative to drug-specific T-cell clones (TCC). Indeed, they behaved like TCC and, in accordance with the p-i concept, the TCR recognize their specific drugs in a direct, processing-independent, and dose-dependent way. The presence of antigen-presenting cells was a prerequisite for interleukin-2 production by the TCR-transfected cells. The analysis of cross-reactivity confirmed the fine specificity of the TCR and also showed that TCR transfectants might provide a tool to evaluate the potential of new drugs to cause hypersensitivity due to cross-reactivity. Recombining the alpha- and beta-chains of sulfanilamide- and quinolone-specific TCR abrogated drug reactivity, suggesting that both original alpha- and beta-chains were involved in drug binding. The TCR-transfected hybridoma system showed that the recognition of two important classes of drugs (sulfanilamides and quinolones) by TCR occurred according to the p-i concept and provides an interesting tool to study drug-TCR interactions and their biological consequences and to evaluate the cross-reactivity potential of new drugs of the same class.

Animals↗

Simple bis-thiocarbono-hydrazones as sensitive, selective, colorimetric, and switch-on fluorescent chemosensors for fluoride anions.

Bis-thiocarbono-hydrazones are found to be a class of sensitive, selective, ratiometric, and colorimetric chemosensors for anions such as fluoride (F(-)) or acetate (Ac(-)). The sensitivities, or the binding constants of the sensors with anions, were found to be strongly dependent on the substituents appended on the pi-conjugation framework, the delocalization bridge CH==N, the aromatic moiety, and the hetero atom in the C==X group (X=O, S) of the sensors. Single-crystal structures and (1)H NMR titration analysis shows that the --CH==N-- moiety is a hydrogen-bond donor, and it is proposed that an additional CHF hydrogen bond is formed for the sensors in the presence F(-). A sensor bearing anthracenyl groups is demonstrated as a switch-on fluorescent chemosensor for F(-) and Ac(-). The recognition of F(-) in acetonitrile (MeCN) by a sensor with nitrophenyl substituents is tolerant to MeOH (MeCN/MeOH=10:1, v/v) and water (MeCN/H(2)O=30:1, v/v); at these solvent ratios the absorption intensity of the sensor-F(-) complex solution at maximal absorption wavelength was attenuated to half of the original value in pure MeCN.

Absorption↗

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I.

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.

Adenosine Triphosphatases↗

Efficient detection of three-dimensional structural motifs in biological macromolecules by computer vision techniques.

Macromolecules carrying biological information often consist of independent modules containing recurring structural motifs. Detection of a specific structural motif within a protein (or DNA) aids in elucidating the role played by the protein (DNA element) and the mechanism of its operation. The number of crystallographically known structures at high resolution is increasing very rapidly. Yet, comparison of three-dimensional structures is a laborious time-consuming procedure that typically requires a manual phase. To date, there is no fast automated procedure for structural comparisons. We present an efficient O(n3) worst case time complexity algorithm for achieving such a goal (where n is the number of atoms in the examined structure). The method is truly three-dimensional, sequence-order-independent, and thus insensitive to gaps, insertions, or deletions. This algorithm is based on the geometric hashing paradigm, which was originally developed for object recognition problems in computer vision. It introduces an indexing approach based on transformation invariant representations and is especially geared toward efficient recognition of partial structures in rigid objects belonging to large data bases. This algorithm is suitable for quick scanning of structural data bases and will detect a recurring structural motif that is a priori unknown. The algorithm uses protein (or DNA) structures, atomic labels, and their three-dimensional coordinates. Additional information pertaining to the structure speeds the comparisons. The algorithm is straightforwardly parallelizable, and several versions of it for computer vision applications have been implemented on the massively parallel connection machine. A prototype version of the algorithm has been implemented and applied to the detection of substructures in proteins.

Algorithms↗

Antigen compartmentation and T helper cell tolerance induction.

The process of antigen recognition depends in part on the amount of peptide antigen available and the affinity of the T cell receptor for a particular peptide-major histocompatibility complex (MHC) molecule complex. The availability of self antigen is limited by antigen processing, which is compartmentalized such that peptide antigens presented by MHC class I molecules originate in the cytoplasm, whereas peptide antigens presented by MHC class II molecules are acquired from the endocytic pathway. This segregation of the antigen-processing pathways may limit the diversity of antigens that influence the development and selection of, e.g., CD4-positive, MHC class II-specific T cells. Selection in this case might involve only a subset of self-encoded proteins, specifically those that are plasma membrane bound or secreted. To study these aspects of immune development, we engineered pigeon cytochrome for expression in transgenic mice in two forms: one in which it was expressed as a type II plasma membrane protein, and a second in which it was targeted to the mitochondria after cytoplasmic synthesis. Experiments with these mice clearly show that tolerance is induced in the thymus, irrespective of antigen compartmentation. Using radiation bone marrow chimeras, we further show that cytoplasmic/mitochondrial antigen gains access to the MHC class II pathway by direct presentation. As a result of studying the anatomy of the thymus, we show that the amount of antigen and the affinity of the TCR affect the location and time point of thymocytes under-going apoptosis.

Animals↗

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.

Amino Acid Sequence↗

[Neuropsychological applications of the Rey complex figure and recognition test].

The authors describe the clinical applications of the Rey Complex Figure and Recognition Test and explain how the individual abilities and information-processing characteristics are reflected. Sequential and quantitative evaluation methods are presented. Summarizing the relation between IQ and RCFT they evaluate the differencies in task completion strategies of healthy subjects and subjects with brain damage, suggesting that these differences originate in changes of the information-processing capacity. Application of the RCFT in clinical child psychology is also suggested, primarily in differential diagnostics of children with attention disorder and disruptive disorder. The authors illustrate the protocols of the neuropsychological examination and the role of the RCFT with a case study.

Attention Deficit Disorder with Hyperactivity↗

On the origin of selectivity in recognition by cyclic adenosine 3',5'-monophosphate receptor protein of its specific binding site of the lactose promoter region.

From fluorescence measurements we could analyse the binding of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli to its specific site on a 301 base-pair long DNA fragment containing the control region of the lactose operon. At physiological ionic strength selection of the specific site is strictly dependent on the allosteric effector cAMP, and binding of the cAMP . CRP complex to its specific site is favoured over the non-specific binding by 5 kcal/mol with Kass (specific) = 10(8) M-1 at 37 degrees C.

Binding Sites↗

Switching DNA-binding specificity by unnatural amino acid substitution.

The specificity of protein-nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid-protein complex. We have used cI repressor of bacteriophage lambda as a model system. In the lambda-repressor-DNA complex, the epsilon-NH(2) group (hydrogen bond donor) of lysine-4 of lambda-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site O(L)1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of lambda-repressor from C:G to T:A at position 6 of O(L)1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.

Amino Acid Substitution↗

Patterns of agent interaction scenarios as use case maps.

A use case map (UCM) presents, in general, an abstract description of a complex system and, as such, is a good candidate for representing scenarios of autonomous agents interacting with other autonomous agents. The "gang of four" design patterns are intended for object-oriented software development but at least eight of the patterns illustrate structure, or architecture, that is appropriate for interacting agents, independent of software development. This study presents these particular patterns in the form of UCMs to describe abstract scenarios of agent interaction. Seven of the patterns attempt to balance the decentralized nature of interacting agents with an organized structure that makes for better, cleaner interactions. An example performance analysis is provided for one of the patterns, illustrating the benefit of an early abstraction of complex agent behavior. The original contribution here is a UCM presentation of the causal paths in agent behavior as suggested by software design patterns.

Algorithms↗