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PubMed1998

Determining the evolutionary potential of a gene.

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base-substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta-galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.

Amino Acid Sequence↗
PubMed2006

Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin.

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.

Amino Acid Sequence↗
PubMed2026

Deep DNA and protein level feature integration for robust clinical variant interpretation using probabilistic gradient boosting.

A major challenge in clinical genomics is to classify genetic variations correctly, since it directly affects disease diagnosis and personal care. The existing methods tend to be based on the combination of different factors, such as protein structure, population frequencies, phenotypic annotations, and sequence conservation. Nevertheless, these methods often cannot be used to achieve the necessary interpretability, quantify uncertainty, and address rare cases. This paper presents a probabilistic gradient boosting model on variant pathogenicity prediction. The suggested framework applies biological characteristics at both level of DNA and protein levels while also scaling the level of uncertainty in clinical decision making. Our machine learning aims to solve the issues of variant interpretation by managing the features and through probability-based pathogenicity prediction. The framework formulation is aimed at generalizing over various datasets and minimizing overfitting. At the same time, it can ensure reasonable performance to facilitate clinical experiments. The model has also been tested on three standard datasets and demonstrated to be more predictive of the pathogenic effect of variants, in comparison with a variety of existing tools. The probabilistic gradient boosting model proposed had ROC AUC values of 0.9293, 0.9610, and 0.9646 on ClinVar variants, GRCh37, and GRCh38 human genome respectively. Furthermore, the dataset was ensured to include both exonic and intronic variants, and Variants of Uncertain Significance were also taken into consideration for Performance Testing. Through this it also aims to provide better clinical significance which will lead to a good interpretable tool for priority of variants for a large variety of disease conditions.

ClinVar↗
PubMed1996

The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most-characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm x 8 nm) were deduced from electron-microscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) alpha H gamma alpha L delta beta. Its two agonist/competitive-antagonist-binding sites have been localized by photolabelling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane-spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand-gated ion channels. The binding site for regulatory non-competitive antagonists has been localized by photolabelling and site-directed-mutagenesis studies within this ion channel. Therefore a three-dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/ competitive-antagonist-binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.

Acetylcholine↗
PubMed2003

Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice.

S100B, the EF-hand Ca(++)-binding protein with gliotrophic and neurotrophic properties implicated in the pathogenesis of Alzheimer's disease, is coined as a glial marker, despite its documented presence in rodent brain neurons. We have generated a transgenic mouse whose EGFP reporter, controlled by the -1,669/+3,106 sequence of the murine S100B gene, allows the direct microscopic observation of most S100B-expressing cells in the central nervous system (CNS). From embryonic day 13 onward, EGFP expression was targeted to selected neuroepithelial, glial, and neuronal cells, indicating that cell-specific expression of S100B is regulated at the transcriptional level during development. In adult mice, the highest level of EGFP expression was found in ependymocytes; astrocytes; and spinal, medullar, pontine, and deep cerebellar S100B neurons. Our results, thus, agree with earlier reports suggesting that S100B is not a CNS glial-specific marker. In addition, we detected EGFP and S100B in forebrain neurons previously thought not to express S100B in the mouse, including neurons of primary motor and somatosensory neocortical areas, the ventral pallidum and prerubral field. Another interesting finding was the selected EGFP targeting to neonatal S100B oligodendrocytes and adult NG2 progenitors as opposed to mature S100B oligodendrocytes. This finding suggests that, except for oligodendrocytes at the last stage of myelin maturation, the -1,669/+3,106 sequence of the S100B gene is a useful reagent for driving expression of transgenes in most S100B-expressing cells of mouse brain.

Animals↗
PubMed2006

Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage.

Active deep-sea hydrothermal vents are areas of intense mixing and severe thermal and chemical gradients, fostering a biotope rich in novel hyperthermophilic microorganisms and metabolic pathways. The goal of this study was to identify the earliest archaeal colonizers of nascent hydrothermal chimneys, organisms that may be previously uncharacterized as they are quickly replaced by a more stable climax community. During expeditions in 2001 and 2002 to the hydrothermal vents of the East Pacific Rise (EPR) (9 degrees 50'N, 104 degrees 17'W), we removed actively venting chimneys and in their place deployed mineral chambers and sampling units that promoted the growth of new, natural hydrothermal chimneys and allowed their collection within hours of formation. These samples were compared with those collected from established hydrothermal chimneys from EPR and Guaymas Basin vent sites. Using molecular and phylogenetic analysis of the 16S rDNA, we show here that at high temperatures, early colonization of a natural chimney is dominated by members of the archaeal genus Ignicoccus and its symbiont, Nanoarchaeum. We have identified 19 unique sequences closely related to the nanoarchaeal group, and five archaeal sequences that group closely with Ignicoccus. These organisms were found to colonize a natural, high temperature protochimney and vent-like mineral assemblages deployed over high temperature outflows within 92 h. When compared phylogenetically, several of these colonizing organisms form a unique clade independent of those found in mature chimneys and low-temperature mineral chamber samples. As a model ecosystem, the identification of pioneering consortia in deep-sea hydrothermal vents may help advance the understanding of how early microbial life forms gained a foothold in hydrothermal systems on early Earth and potentially on other planetary bodies.

Base Sequence↗
PubMed2000

Deep coagulation of dermal collagen with repetitive Er:YAG laser irradiation.

BACKGROUND AND OBJECTIVE: Er:YAG lasers are known to effectively ablate human skin with minimal thermal damage to subjacent dermal tissue. We have investigated whether deep coagulation of dermal collagen, similar to that observed with the CO(2) laser, could be achieved with repetitive Er:YAG laser exposures. STUDY DESIGN/MATERIALS AND METHODS: Skin on the back of a Sprague-Dawley rat in vivo was irradiated with sequences of 1-10 Er:YAG laser pulses at a repetition rate of 10 or 33 Hz and single-pulse fluences from 0.8 to 1.4 J/cm(2). The resulting lesions were biopsied within 1 hour after laser exposure, and the histologic sections were examined by using optical microscopy. RESULTS: The depth of dermal collagen denaturation increases dramatically when 3-10 low-fluence Er:YAG laser pulses are stacked at a repetition rate of 10 or 33 Hz. CONCLUSION: Coagulation of dermal collagen deeper than 200 microm below the epidermal-dermal junction is feasible by using the appropriate settings of a repetitive Er:YAG laser.

Animals↗
PubMed2000

"Paleogenomics": looking in the past to the future.

The complete sequence of the human and other vertebrate and nonvertebrate genomes provide a wealth of information on the organization, relationships and evolution of the metazoans. Soon the fine structure of our innermost biological identity will be unveiled and what has so far remained deep and secret will shine like an unearthed treasure and shape and fuel our future quests. A key treasure, for many molecular scientists interested in molecular evolution and development would be the knowledge of the genome of the ancestral precursor of all metazoans. In the absence of fossil DNA, this knowledge will forever remain a yearning for dreamy molecular biologists. And yet, will not the power of deduction and reconstitution of information gained through man's sophisticated technologies one day recreate a "virtual" metazoan ancestor?

Animals↗
PubMed2004

MRI of the fetal posterior fossa.

MRI is a useful tool to complement US for imaging of the fetal posterior fossa (PF). In France, the discovery of a PF malformation in the fetus frequently leads to termination of pregnancy (80% in a personal series). However, despite improved accuracy in the diagnosis of PF abnormalities, prognosis remains uncertain. The first objective of this review is to document the normal MRI landmarks of the developing fetal PF. Because of their thinness, the visibility of the cerebellar fissures is dramatically delayed on MRI compared to macroscopic data. An important landmark is identification of the primary fissure of the vermis, normally seen at around 25-26 weeks' gestation (WG) on the sagittal slice, separating the larger posterior lobe from the anterior lobe (volume ratio around 2:1). The prepyramidal and secondary fissures are usually only identifiable after 32 WG and the hemispheric fissures are difficult to see until the end of pregnancy. Considering the signal changes, high signal on T2-weighted (T2-W) sequences is seen from 25 WG in the posterior part of the brain stem (tegmentum and ascending sensory tracts) related to myelination. The low signal intensities seen within the cerebellum on T2-W images correspond to high cellularity of grey matter (deep nuclei), as there is no myelination within the white matter before 38 WG. The second objective is to highlight the signs highly predictive of a poor neurological prognosis. Lack of pontine curvature or vermian agenesis without a PF cyst (small volume of PF) is greatly associated with poor neurological status. The third objective is to propose a diagnostic strategy in difficult cases where prognosis is important, e.g. the Dandy Walker continuum. Analysis of the cerebellum is often impossible if a PF cyst is present (whatever its nature) as the mass effect usually blurs the foliation and even impairs evaluation of the normal ratio between the posterior and anterior lobes of the vermis. Isolated cerebellar hypoplasias raise the question of prognosis and genetic counselling. Such uncertainties require an amniocentesis and a careful search for other anomalies (cerebral and extracerebral). Unilateral abnormalities of a cerebellar hemisphere can be associated with good neurological status if they are isolated. The final objective is to discuss other rare PF fetal abnormalities, such as vascular malformations and tumours.

Cranial Fossa, Posterior↗
PubMed1994

Cortical and thalamic cellular correlates of electroencephalographic burst-suppression.

This experimental study on anesthetized cats used intracellular recordings of cortical, thalamocortical and reticular thalamic neurons (n = 54), as well as multi-site extracellular recordings (n = 36), to investigate the cellular correlates of EEG burst-suppression patterns, defined as alternating wave bursts and periods of electrical silence. Burst-suppression was elicited by the administration of the same or other anesthetic agents upon the background of an already synchronized EEG activity. About 95% of cortical cells entered burst-suppression, in close time-relation with EEG activity, displaying sequences of phasic depolarizing events associated with bursts of EEG waves and an electrical silence of the neuronal membrane during flat EEG epochs. The membrane potential (Vm) hyperpolarized by approximately 10 mV prior to any EEG change and the slow rhythms reflecting deep stages of anesthesia progressively disorganized with transition to burst-suppression. During flat EEG epochs, the apparent input resistance (tested through short hyperpolarizing current pulses) decreased (range 12-60%) and neuronal responsiveness to orthodromic volleys (tested by thalamic and cortical evoked excitatory postsynaptic potentials) was dramatically reduced. It is proposed that the decreased input resistance is mainly due to an increase in K+ conductances. At variance with cortical neurons, only 60-70% of thalamic cells ceased firing before overt EEG burst-suppression and were completely silent during flat periods of EEG activity. The remaining 30-40% of thalamic cells discharged rhythmic (1-4 Hz) spike bursts during periods of EEG silence. This rhythm, within the frequency range of delta waves, is generated in thalamic cells by the interplay between two of their intrinsic currents at critical levels of Vm hyperpolarization. However, with the deepening of burst-suppression, when silent EEG periods became longer than 30 sec, thalamic cells also ceased firing. The assumption that full-blown burst-suppression is achieved through virtually complete disconnection in brain circuits implicated in the genesis of the EEG is corroborated by the revival of normal cellular and EEG activities after volleys setting into action thalamic and cortical networks.

Animals↗
PubMed2004

Polymorphic corneal amyloidosis: a disorder due to a novel mutation in the transforming growth factor beta-induced (BIGH3) gene.

PURPOSE: To characterize the clinicopathologic phenotype as well as the molecular genetic basis of an autosomal dominant form of corneal amyloidosis. DESIGN: Clinicopathologic and molecular genetic study of a family with a form of corneal amyloidosis. PARTICIPANTS: Forty-nine individuals from one family were studied. METHODS: The medical records of affected family members were reviewed, and corneal tissue from those who had undergone penetrating keratoplasty (PK) was examined. Several family members were examined clinically, and corneas were photographed. Deoxyribonucleic acid from blood or buccal swabs was extracted from each consenting family member to determine the status of their transforming growth factor beta-induced (TGFBI) gene. The coding region of the TGFBI gene was analyzed for mutations in the proband's DNA, and compared with the nucleotide sequences of normal individuals. This was performed by amplifying and sequencing all exons of the TGFBI gene. In all other family members, only exons 4, 8, 11, and 12 of the gene were amplified, sequenced, and analyzed for mutations. MAIN OUTCOME MEASURES: Clinicopathologic manifestations in relation to mutational status of the TGFBI gene. RESULTS: Slit-lamp biomicroscopy revealed bilateral multiple polymorphic, polygonal, refractile, chipped ice-appearing gray and white opacities. There were also occasional deep filamentous lines that did not form a distinct lattice pattern. Corneal tissue of affected individuals who underwent PK contained widespread deposits of amyloid within the corneal stroma, particularly in the deep central stroma. Twelve members of the family were found to have a heterozygous single mutation in the TGFBI gene leading to a predicted amino acid substitution of aspartic acid for alanine (A546D). Nine of these individuals had ophthalmologist-documented corneal disease. The remaining 3, who were 11, 14, and 15 years old, were asymptomatic. In addition, 4 inconsequential polymorphisms with the nucleotide changes 387 G/A (R129R), 981 G/A (V327V), 1416 T/C (L472L), and 1620 C/T (F540F) were found. CONCLUSION: A distinct, progressive form of corneal amyloidosis with an autosomal dominant mode of inheritance is characterized clinically by the presence of refractile polymorphic corneal opacities. We have designated this entity, which is caused by an A546D mutation in the TGFBI gene, polymorphic corneal amyloidosis.

Adolescent↗
PubMed2006

Unravelling the phylogeny of Lejeuneaceae (Jungermanniopsida): evidence for four main lineages.

With about 1000 species in approximately 90 genera, Lejeuneaceae are the largest family of liverworts and make up a large and important part of cryptogamic diversity in the humid tropics. Maximum parsimony, Maximum likelihood and Bayesian analyses of a dataset including four markers (rbcL, psbA, trnL-trnF region of cp DNA, nrITS region) of 134 accessions resulted in similar topologies that support the presence of four main lineages within Lejeuneaceae. Model-based analyses support a division of Lejeuneaceae into two lineages corresponding to the subfamilies Ptychanthoideae and Lejeuneoideae. The latter lineage splits into the tribes Lejeuneeae, Brachiolejeuneeae and the genus Symbiezidium. In contrast, the Maximum parsimony analysis resolves Brachiolejeuneeae and Symbiezidium in serial sister relationships to the remainder of Lejeuneaceae. Sporophyte characters support a split into two subfamilies as seen in the model-based analyses. Some deep nodes remain unresolved, possibly indicating a series of initial diversifications which occurred over a short time period.

Base Sequence↗
PubMed1997

Simultaneous intrusion and retraction using a three-piece base arch.

Flared incisors and deep overbite are challenging to treat orthodontically. This paper describes the use of a three-piece base arch and Class I elastics to correct deep overbite while simultaneously closing spaces. An analysis of the biomechanics and a discussion of the appliance design are presented to help understand how the incisor axial inclination can be corrected and controlled during orthodontic therapy. A clinical example illustrates the treatment sequence.

Adult↗
PubMed1999

Diversity of Holocene life forms in fossil glacier ice.

Studies of biotic remains of polar ice caps have been limited to morphological identification of plant pollen and spores. By using sensitive molecular techniques, we now demonstrate a much greater range of detectable organisms; from 2000- and 4000-year-old ice-core samples, we obtained and characterized 120 clones that represent at least 57 distinct taxa and reveal a diversity of fungi, plants, algae, and protists. The organisms derive from distant sources as well as from the local arctic environment. Our results suggest that additional taxa may soon be readily identified, providing a plank for future studies of deep ice cores and yielding valuable information about ancient communities and their change over time.

Base Sequence↗
PubMed1992

Determination of plasma (1-->3)-beta-D-glucan: a new diagnostic aid to deep mycosis.

We present additional evidence that plasma from patients with deep-seated mycoses contains (1-->3)-beta-D-glucan. Digestion of such samples with endo-(1-->3)-beta-D-glucanase completely abolished the ability of the plasma to activate factor-G, a horseshoe crab coagulation enzyme that is extremely sensitive to this polysaccharide. Measurement of plasma (1-->3)-beta-D-glucan is a promising method for the diagnosis of deep-seated mycoses and for monitoring the response of these infections to antifungal therapy.

Amino Acid Sequence↗
PubMed1998

Ultrastructural analyses of the Caenorhabditis elegans rol-6 (su1006) mutant, which produces abnormal cuticle collagen.

Roller mutants of Caenorhabditis elegans rotate around their long axis and move in circular paths. Isolation and sequence of the rol-6 gene of C. elegans have shown that it encodes a cuticle collagen. In this paper, we describe the morphological alterations seen in the cuticle of the right roller mutant rol-6 (su1006) at the ultrastructural level. Deep-etched replica analyses showed that the honeycomb elements, fibers organized in a pentagonal fashion above the fishbone fibrous layer, completely fill the intermediate layer, which is observed to be largely empty spaces in the wild-type strain. The honeycomb fibers appear to connect the cortical and basal regions of the mutant cuticle. These fibers are likely to be involved in generating the helical twist of the mutant animals. Deep-etched replicas also revealed a delicate network of filaments on the nematode surface.

Animals↗
PubMed2006

Cloning and expression analysis of an ovine PAP-like protein cDNA, a gene differentially expressed in scrapie.

In a previous study, the mRNA level of a pancreatitis-associated protein (PAP)-like protein was found to be elevated in the ileal Peyer's patch of lambs during the early phase of scrapie infection. Here, we report the isolation of the ovine PAP-like protein cDNA which encodes a putative 178 amino acid protein with a signal peptide and a C-lectin binding domain. Comparisons of REG/PAP proteins between various species showed that the deduced amino acid sequences were conserved. The overall amino acid identity between the ovine PAP-like protein and bovine, human and rat REG/PAP proteins varied from 23% to 85%. In Northern blot analysis the expression of the ovine PAP-like protein mRNA was restricted to the ileal and jejunal Peyer's patches. The cellular expression of the PAP-like protein mRNA in the ovine intestine was further characterized by in situ hybridization. PAP-like protein mRNA was detected in cells of the epithelial lining in most crypts and in some intestinal villi in the ileum and jejunum while in the colon and rectum, the PAP-like protein mRNA expression was only detected in the deep portion of a few crypts. The data provided will offer the possibility to search for a link between this PAP-like protein and early events in the development of scrapie.

Animals↗
PubMed1999

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough.

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27 degrees 32.7'N, 126 degrees 58.5'E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 microm in diameter. Growth was observed at temperatures between 50 degrees and 93 degrees C, with an optimum at 85 degrees C. The pH range for growth is 5.0-9.0, with an optimum around 7.0. Strain RG-20 requires 1%-4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 microg/ ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si'cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349).

DNA, Archaeal↗