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At least 1,369 records · Page 76Linked to original sources
PubMed2003

Did the loss of sigma factors initiate pseudogene accumulation in M. leprae?

Pseudogenes are non-functional regions in the genome that have arisen as a consequence of accumulating mutations that either result in the premature termination of proteins during protein synthesis or the disruption of transcription. There have been various discussions of the origins of pseudogenes and the models for their formation, but there has been little input on how pseudogenes could have accumulated in an organism. In this brief communication, I propose a two-step model for the accretion of pseudogenes in the Mycobacterium leprae genome, triggered by the loss of different sets of sigma factors at different time points during the course of evolution.

DNA-Directed RNA Polymerases↗
PubMed1999

A ribozyme that lacks cytidine.

The RNA-world hypothesis proposes that, before the advent of DNA and protein, life was based on RNA, with RNA serving as both the repository of genetic information and the chief agent of catalytic function. An argument against an RNA world is that the components of RNA lack the chemical diversity necessary to sustain life. Unlike proteins, which contain 20 different amino-acid subunits, nucleic acids are composed of only four subunits which have very similar chemical properties. Yet RNA is capable of a broad range of catalytic functions. Here we show that even three nucleic-acid subunits are sufficient to provide a substantial increase in the catalytic rate. Starting from a molecule that contained roughly equal proportions of all four nucleosides, we used in vitro evolution to obtain an RNA ligase ribozyme that lacks cytidine. This ribozyme folds into a defined structure and has a catalytic rate that is about 10(5)-fold faster than the uncatalysed rate of template-directed RNA ligation.

Base Sequence↗
PubMedDate not supplied

New directions in comparative physiology and biochemistry: mechanisms, adaptations, and evolution.

Historically, the discipline of comparative physiology and biochemistry has had two major goals: (1) elucidation of mechanisms and their adaptative significance, and (2) understanding of the evolution of mechanisms and adaptations. In general, the first goal has dominated the field. In a mechanistic/adaptational approach, the diversity of organisms is an experimental parameter in the investigation. Lineage-specific characteristics reveal both how physiological systems work and how different kinds of animals are adapted to different kinds of environments. We believe that this approach is far from outdated, in part because many animal groups have been investigated superficially if at all, and in part because the incorporation of fundamentally new technologies into our discipline permits us to address previously intractable questions about even intensively studied animal groups. In evolutionary physiology and biochemistry, the diversity of lineage-specific physiological systems and how they came to be is the subject of investigation. Early attempts to employ the evolutionary approach were not only few in number, they were unsatisfying in outcome because neither phylogenetic nor mechanistic/adaptational knowledge was adequate to serve as a firm foundation. We agree with earlier authors that new and more sophisticated applications of this approach, together with progress in understanding both animal phylogeny and mechanisms/adaptations, all promise to allow us at last to fulfill our second historic goal. In our view, an integration of the two approaches seems to present the most productive trajectory into the next century.

Adaptation, Physiological↗
PubMed1999

Universal modular glottiscope system: the evolution of a century of design and technique for direct laryngoscopy.

Since Kirstein introduced formal direct examination (autoscopy) of the glottis in 1895, a great number of laryngoscopes have been produced to view the vocal folds; however, none have had universal appeal. The primary goals for the designs have been to optimize exposure and to facilitate instrumentation of the glottis. An analysis of more than 50 laryngoscopes was done to assess key design characteristics that would ideally be present in a laryngoscope for optimally viewing the musculomembranous vocal folds. The Pro/Engineer and Pro/Mechanica computer programs were used to model the universal modular glottiscope. This new laryngoscope comprises a plurality of specially designed examining tubes that are bivalved proximally to improve utilization of hand instrumentation, and form a single tube distally to achieve internal distention of the supraglottal tissues. The distal lumen has an arcuate isosceles-triangular conformation to optimally expose the glottis. The base of the tube is detachable for the difficult intubation or the placement of bronchoscopes. The examining tubes vary in size and dimension to accommodate the diversity of human anatomy. The tubes are easily attachable to and detachable from an ergodynamically designed universal handle that can be joined to fulcrum laryngoscope holders or suspension gallows. The universal modular glottiscope evolved from the selective integration of optimal 20th-century design modifications of Kirstein's original autoscope. This new laryngoscope is ideally suited for phonomicrosurgery as well as for difficult intubation.

History, 20th Century↗
PubMed2006

Antagonistic coevolution with parasites increases the cost of host deleterious mutations.

The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations.

Directed Molecular Evolution↗
PubMed2006

Duplication, divergence and formation of novel protein topologies.

The rearrangement or permutation of protein substructures is an important mode of divergence. Recent work explored one possible underlying mechanism called permutation-by-duplication, which produces special forms of motif rearrangements called circular permutations. Permutation-by-duplication, involving gene duplication, fusion and truncation, can produce fully functional intermediate proteins and thus represents a feasible mechanism of protein evolution. In spite of this, circular permutations are relatively rare and we discuss possible reasons for their existence.

Amino Acid Motifs↗
PubMed2006

Miniaturising the laboratory in emulsion droplets.

Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (>10(10) per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.

Cell-Free System↗
PubMed2004

SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription.

Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged epsilon stem-loop on the pregenomic RNA, and the epsilon-templated synthesis of the 5' terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV epsilon (Depsilon) RNA. Employing the SELEX method on a constrained library of Depsilon upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired Depsilon represents an exceptional rather than a prototypic avian HBV epsilon signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.

Animals↗
PubMed2006

[Primary sarcomas of the breast].

Primary sarcomas of the breast are extremely rare with less than 1% of all malignant tumours of the breast reported in literature. At our Institution 1315 malignant tumours of the breast were diagnosed between 1999-2004; nine of them corresponded to primary sarcomas: angiosarcoma (3), leiomyosarcoma (1), low-grade fibromyxoid sarcoma (1), dematofibrosarcoma protuberans (1), liposarcoma (1), osteosarcoma (1), malignant peripheral nerve sheath tumour (1). Histopathological specimens stained with routine techniques and immunoperoxidase were reviewed; proliferation index and p53 over-expression were also determined. Patients' clinical reports were also reviewed to determine prognosis (favorable and unfavorable). The incidence observed (0.7%) is similar to those already published by others authors. Proliferation index was correlated with type of evolution, being an unfavourable prognosis factor when it was equal or major to 30%. Most of the tumours (67%) showed p53 (mayor or equal to 20% of nuclear staining) over-expression but this did not show a direct relationship with the evolution of each neoplasm.

Adult↗
PubMed2005

Altering protein specificity: techniques and applications.

Protein engineering constitutes a powerful tool for generating novel proteins that serve as catalysts to induce selective chemical and biological transformations that would not otherwise be possible. Protocols that are commonly employed for altering the substrate specificity and selectivity profiles by mutating known enzymes include rational and random methods as well as techniques that entail evolution, selection and screening. Proteins identified by these techniques play important roles in a variety of industrial and medicinal applications and in the study of protein structure-function relationships. Herein we present a critical overview of methods for creating new functional proteins having altered specificity profiles and some practical case studies in which these techniques have been applied to solving problems in synthetic and medicinal chemistry and to elucidating enzyme function and biological pathways.

Biotechnology↗
PubMed2002

Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus.

The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation. This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.

Adaptation, Physiological↗
PubMed2006

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.

Alcohol Dehydrogenase↗
PubMed2003

Precise oculomotor correlates of visuospatial mental rotation and circular motion imagery.

Visual imagery is a basic form of cognition central to activities such as problem solving or creative thinking. Phenomena such as mental rotation, in which mental images undergo spatial transformations, and motion imagery, in which we imagine objects in motion, are very elusive. For example, although several aspects of visual imagery and mental rotation have been reconstructed through mental chronometry, their instantaneous evolution has never been directly observed. We paired mental chronometry to eye movement recording in subjects performing a visuospatial mental rotation task and an instructed circular motion imagery task. In both tasks, sequences of spontaneous saccades formed curved trajectories with a regular spatio-temporal evolution. In the visuospatial mental rotation task, saccadic amplitude decreased progressively within each sequence, resulting in an average gaze rotation with a bell-shaped asymmetrical angular velocity profile whose peak and mean increased with the amount of the to-be-performed rotation, as in reaching movements. In the second task, the average gaze rotation reproduced faithfully the to-be-imagined constant-velocity circular motion, thus excluding important distortions in the oculomotor performance. These findings show for the first time the instantaneous spatio-temporal evolution of mental rotation and motion imagery. Moreover, the fact that visuospatial mental rotation is modeled as a reaching act suggests that reaching pertains to the realm of visuospatial thinking, rather than being restricted to the motor domain. This approach based on eye movement recording can be profitably coupled to methods such as event-related potentials, transcranial magnetic stimulation, or functional magnetic resonance to study the precise neuronal dynamics associated with an ongoing mental activity.

Adult↗
PubMed1998

Serial radiography in the infant shaken impact syndrome.

Certain CT and/or MRI abnormalities have been used medicolegally to time intracranial injuries from the infant shaken impact syndrome (ISIS). For example, parenchymal hypodensities on CT scans are said to arise only after 6-48 h have elapsed postinjury, and the presence of chronic or mixed subdural hematomas suggests injury that occured 1-4 weeks prior. However, these statements are based largely upon inference from data obtained in other conditions such as ischemic anoxic injury and chronic subdural hemorrhage in adults. Direct evidence about the evolution of intracranial injuries in infants with ISIS is sparse, and the radiographic changes following ISIS have never been systematically studied on serial imaging studies. One hundred-seventeen serial CT and MRI scans obtained from 33 infants with ISIS were reviewed retrospectively. The exact scan dates and times were obtained directly from the scans. Acute subdural hemorrhage was the most common intracranial abnormality and was present in 27 (81%) of the 33 infants. Other intracranial abnormalities included chronic subdural collections, subarachnoid hemorrhage, epidural hematomas, parenchymal hypodensities, edema and contusions, and atrophy and encephalomalacia. In 15 of the 33 infants, the injury could be timed with reasonable certainty, and the evolution of the radiographic changes followed over time. Six of the 15 infants had evidence of prior cranial trauma such as chronic subdural collections (5 infants) or mild atrophy (1 infant). Of the remaining 9 infants, parenchymal abnormalities such as hypodensities, edema and contusion appeared in virtually all of the initial scans performed approximately 3 h following the report of injury. One 'chronic' subdural collection was absent on the first scan performed 2.75 h postinjury, but appeared on a second scan performed 17 h later, suggesting that some 'chronic' subdural fluid collections may arise much sooner than previously thought. These findings challenge some of the current dogma about the timing of radiographic changes following abuse and are important in timing the alleged abuse for legal purposes.

Battered Child Syndrome↗
PubMed1999

The power of evolution: accessing the synthetic potential of P450s.

Cytochromes P450 can catalyse hydroxylation reactions that are of considerable potential synthetic value, but a number of practical difficulties have hitherto prevented their use for this purpose. Recent advances, including intelligently designed laboratory evolution experiments, promise to overcome these obstacles, and to add P450s to the enzymatic armoury of the chemist.

Animals↗