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At least 397 records · Page 22Linked to original sources
PubMed2006

[Influence of rotatory malposition of femoral implant in failure of unicompartimental medial knee prosthesis].

PURPOSE OF THE STUDY: Rotatory malposition of the femoral component of a unicompartmental knee arthroplasty (UKA) is a key element of mechanical failure despite proper alignment and position of the tibial implant. The purpose of this study was to describe a method for measuring femoral implant rotation on the anteroposterior x-ray using an original geometric model. MATERIAL AND METHODS: 276 medial UKA (227 non-cemented Uni Goeland, Depuy; Uni, AMP) and 49 cemented (Miller-Galante, Zimmer) were reviewed and analyzed at a mean 11 years (range 7 - 15) using the Knee Society Score. Mean objective and functional scores were 43 and 47 points preoperatively. Postoperative alignment and position of the femorotibial contact point were determined. The frontal and sagittal position of the tibial plateau was noted by the angle formed with the mechanical axis. Angles alpha and B of the femoral implant formed between the mechanical axis and the greater condylar axis and the inferior condylar line passing through the apex of the condylar convexity respectively were measured on the AP view. Frontal rotation (angle B) of the femoral implant reflected the orientation of the distal condylar cut. The frontal measurement (angle alpha) was validated using simple geometric formulas: knowing angles alpha and B and the size of the implant and the displacement of the point of contact was measured together with the true rotation of the femoral implant or anteroposterior divergence, function of the difference alpha-B which is its frontal projection and reflects the orientation of the posterior condylar cut. RESULTS: Mean alignment was 3 degrees varus. At last follow-up, outcome was satisfactory in 81.2% of the knees (224 UKA). Mean objective and functional scores were 90 and 87 points. Angles alpha and B were parallel and orthogonal to the mechanical axis or off by less than 4 degrees external rotation (+6 degrees to -4 degrees). The mean difference alpha-B (frontal projection of the femoral implant transverse rotation) was 1 degrees. The point of contact was 4 mm or less from the middle of the tibia. Failures were observed in 18.8% of knees (52 UKA): 4 for diverse reasons and 48 for mechanical failure, including 3 with polyethylene wear without loosening (two by eccentric point of contact and neutral position and one by inverted misalignments). 45 UKA (16.3%) presented loosening of the tibial plateau alone with rotatory femoral malposition: mean a angle off 13% from the mechanical axis (+16 degrees to -9 degrees ), mean B angle off 8 degrees (+12 degrees to -8 degrees), mean alpha-B difference 5 degrees , femoral implant in square position on polyethylene with a peripheral point of contact > or 5 mm from the middle of the tibial plateau. The tibial implant exhibited mean varus of 1.5 degrees. DISCUSSION: The main cause of failure was rotatory malposition of the condylar implant often associating frontal with transversal rotation. This increased mediolateral translation of the point of contact during knee motion causing abrasion and excessive pressure on the medial portion of the plateau. Frontal malrotation externalizes the posterior cut on the condyle tilted by the varus position, without correcting the varus of the mechanical axis in flexion. To avoid such prosthetic malrotations, three corrections must be made before making the bone cuts: cancel the external rotation of the condyles in flexion by positioning the distal cut guide in extension; re-establish the mechanical axis by reducing the internal tilt of the condyles resulting from the varus position both in extension (distal cut) and flexion (posterior cut). Improved instrumentations and reproducible techniques are needed to re-establish the 3D anatomic orientation of the femoral component, a key element for longer life of unicompartmental prostheses. This method for measuring the rotation of the femoral implant and the displacement of the point of contact using a geometric model applied to the AP view is useful for better understanding UKA failures.

Adult↗
PubMed1992

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.

Animals↗
PubMed2004

The AZFa gene DBY (DDX3Y) is widely transcribed but the protein is limited to the male germ cells by translation control.

We explored the function of the human DEAD-box Y RNA helicase DBY (DDX3Y) gene located in the (AZFa) region on the human Y chromosome (Yq11.21). Deletion of this Y interval is known to be a major cause for the occurrence of a severe testicular pathology, the Sertoli-cell-only (SCO) syndrome. DBY has a structural homologue on the short arm of the X chromosome DBX (DDX3X) (Xp11.4). We found widespread transcription of both genes in each tissue analyzed, although predominantly in testis tissue. However, translation of DBY was detected only in the male germ line, whereas DBX protein was expressed in all tissues analyzed. In testis tissue sections, DBY protein was found predominantly in spermatogonia, whereas DBX protein was expressed after meiosis in spermatids. We conclude that although both RNA helicases are structurally very similar, they have diverged functionally to fulfill different roles in the RNA metabolism of human spermatogenesis, and that deletion of the DBY gene is the most likely cause of the severe testicular pathology observed in men with AZFa deletions.

Chromosomes, Human, Y↗
PubMed1996

Sequence divergence associated with species-specific splicing of the nonmuscle beta-tropomyosin alternative exon.

Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.

Alternative Splicing↗
PubMed2003

A novel Mg(2+)-dependent O-methyltransferase in the phenylpropanoid metabolism of Mesembryanthemum crystallinum.

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg(2+)-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg(2+)-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.

Acyl Coenzyme A↗
PubMed2026

Comparative proteomics reveals distinct functions and localization of invasive Entamoeba histolytica and non-invasive Entamoeba moshkovskii proteins.

BACKGROUND: Entamoeba histolytica is a pathogenic protozoan accountable for amoebiasis, while Entamoeba moshkovskii is considered non-invasive. Despite morphological similarity, the molecular mechanisms underlying their different pathogenicity remain largely undefined. METHODS: Trophozoite proteins from axenic cultures of E. histolytica and E. moshkovskii were separated and identified using GeLC-MS/MS, and classified using Gene Ontology. Selected and differentially expressed proteins were validated by peptide-specific antibody production, ELISA, and immunofluorescence to determine cellular localization. RESULTS AND DISCUSSION: A total of 1,077 and 1,201 proteins were identified from E. histolytica and E. moshkovskii, respectively. The 801 of Entamoeba common proteins included kinases, GTPase-activating proteins, and heat shock proteins, reflecting conserved cellular processes. E. histolytica-unique proteins involved in nitrogen compound metabolism, vesicle-mediated transport, and catalytic activities, whereas E. moshkovskii proteins were related to lipid metabolism and environmental resilience. Subcellular localization revealed species-specific distribution of MmpL and AIG1-family proteins, suggesting potential roles in pathogenicity and host-immune response. A large proportion of hypothetical proteins was identified, highlighting gaps and opportunities for future study. CONCLUSIONS: Our study highlights conserved and divergent functions and cellular locations of Entamoeba species-specific proteins as insights for distinct pathogenicity and adaptation. MmpL and AIG1 proteins were proposed as potential targets for further diagnostic and therapeutic development.

Proteomics↗
PubMed1998

Uniqueness and redundancy in GABA production.

GABA is present in organisms belonging to at least four of the five kingdoms. It acts as a neurotransmitter, a paracrine signaling molecule, a metabolic intermediate, or a trophic factor. In mammals, GABA synthesis depends on two forms of the enzyme glutamic acid decarboxylase--GAD65 and GAD67--that may serve distinctive functions within GABA-producing cells. The two GADs derive from two genes, which are differentially regulated, though nearly every GABA-producing cell contains both forms of GAD. GAD67 predominates early in development and after neuronal injury, consistent with a possible role in producing GABA for trophic use. In the embryo, GAD67 transcripts also undergo alternative splicing, which gives rise to truncated forms. In the mature neuron, GAD67 is present in both terminals and the cell body, where it may subserve a nonsynaptic, intracellular GABA pool. In contrast, GAD65 is usually expressed later in development and is primarily localized to nerve terminals. GAD65 enzymatic activity is more subject to regulation by cofactor binding and neuronal activity, consistent with its involvement in the production of synaptic GABA. Thus, while both GAD67 and GAD65 mediate the synthesis of GABA, their unique distributions and expression patterns suggest divergent functional roles.

Animals↗
PubMed2001

Metabolic enzymes and phenotypic expression among human locomotor muscles.

Percutaneous biopsies were taken from the right vastus lateralis (VL), tibialis anterior (TA), soleus (Sol), and lateral gastrocnemius (LG) muscles of eight recreationally active adult males. Approximately 60 fibers in each sample were analyzed for their type (I, IIa, or IIx), cross-sectional area (CSA), and succinic dehydrogenase (SDH), alpha glycerol phosphate dehydrogenase (GPDH) and calcium-activated actomyosin adenosine triphosphatase (qATPase) activities. This was done to test the hypothesis that metabolic enzyme activities are more reflective of the functional diversity among human locomotor muscles than fiber type composition. The results showed that enzymatic characteristics differed more or less than expected between muscles of the same or different fiber type. For example, the relative CSA occupied by fast fibers was only about 50% greater in the mixed (LG and VL) than in the slow (Sol and TA) muscles (57 vs. 38%). At the same time, average fiber SDH activity and fiber type specific SDH:qATPase*%CSA, both used as estimates of fatigue resistance, were greater in Sol and LG than in TA and VL. As a result, the two slow muscles and the two mixed muscles had different values, and a mixed muscle (LG) had higher values than a slow muscle (TA). The findings suggest that differences in enzymatic profile, more than fiber type composition, afford human locomotor muscles the capacity to perform their purportedly divergent functional tasks.

Energy Metabolism↗
PubMed1999

The ethological analysis of imitation.

Theorists and experimental researchers have long debated whether animals are able to imitate. A variety of definitions of imitation have been proposed to describe this complex form of social learning. Experimental research on imitation has often been hampered by either a too loose 'anthropomorphic' approach or by too narrow 'behaviourist' definitions. At present neither associative nor cognitive theories are able to offer an exhaustive explanation of imitation in animals. An ethological approach to imitation offers a different perspective. By integrating questions on function, mechanism, development and evolution one can identify possible directions for future research. At present, however, we are still far from developing a comprehensive theory of imitation. A functional approach to imitation shows that, despite some evidence for imitative learning in food processing in apes, such learning has not been shown to be involved in the social transmission of either tool-use skills or communicative signals. Recently developed procedures offer possible ways of clarifying the role of imitation in tool use and visual communication. The role of imitation in explorative play in apes is also investigated and the available data suggest that copying during play might represent a behavioural homologue of human imitation. It is proposed that the ability to copy the behaviour of a companion is under a strong genetic influence in many social species. Many important factors have not been examined experimentally, e.g. the effect of the demonstrator, the influence of attention and memory and the ability to generalize. The potential importance of reinforcement raises the possibility that copying abilities serving divergent functions might be partly under the control of different mechanisms.

Animals↗
PubMed2001

Divergence of volatile anesthetic effects in inhibitory neurotransmitter receptors.

BACKGROUND: The mechanism of volatile anesthetic (VA) action is unknown. Inhibitory receptors for the neurotransmitters gamma-aminobutyric acid (GABA) or glycine are typically positively modulated by VAs and may be important targets for their action. The existence of a GABA receptor subtype (p), which is uniquely inhibited by VAs, suggested a chimeric receptor approach to identify portions of these proteins that may be necessary for anesthetic effects. METHODS: A silent mutation resulting in the addition of a unique restriction enzyme recognition site was introduced in GABA receptor type A alpha2, glycine alpha1, and p subunit cDNAs. Chimeras were constructed by rejoining restriction digest fragments and were expressed in Xenopus oocytes. Modulation of submaximal agonist-evoked peak currents by the VAs chloroform, enflurane, halothane, or isoflurane was measured using two-electrode voltage clamp. RESULTS: Four chimeras were constructed and designated glyrho, rhogly, alpha2rho, and rhoalpha2. Glyrho formed glycine-gated receptors with currents that were enhanced by chloroform or halothane but were inhibited by enflurane or isoflurane. Chimeras rhogly and rhoalpha2 each formed GABA-gated receptors with currents that were inhibited by chloroform or halothane but enhanced by enflurane or isoflurane. CONCLUSIONS: These data show, for the first time, functional divergence of VA action on a single protein target. The VAs in this study fall into two distinct groups with respect to their effects on these receptors. This grouping parallels the chemistry of these compounds. Our results support the involvement of multiple protein domains in the mechanism of VA modulation of GABA and glycine receptors.

Amino Acid Sequence↗
PubMed2026

Rewiring Cellular Context as A Central Mechanism Governing Cancer Stem Cell Survival: Insights from ESC Comparisons.

Cancer stem cells (CSCs) drive tumor initiation, metastasis, and therapy resistance, yet their remarkable persistence remains poorly understood. While CSCs share stemness attributes with embryonic stem cells (ESCs), including self-renewal, transcriptional plasticity, and permissive chromatin, they exhibit a fundamentally divergent regulatory logic that prioritizes survival over developmental fidelity. ESCs maintain globally open chromatin that supports transcriptional hyperactivity but predisposes them to apoptosis under genotoxic stress, whereas CSCs maintain dynamically inducible, permissive chromatin at survival loci while repressing differentiation programs, enabling adaptive stress responses. We advance the hypothesis that CSC persistence emerges not from any single factor, but from the integrative rewiring of signaling cascades (Wnt/β-catenin, Notch, Hedgehog, PI3K/AKT/mTOR), stress-responsive transcription factors (HIFs, NF-κB, STAT3), and core pluripotency networks (OCT4, SOX2, NANOG) within a survival-centric context, reinforced by dynamic chromatin remodeling, inducible super-enhancer landscapes, and microenvironmental cues (hypoxia, inflammation, matrix stiffness). Within this framework, the E2F family serves as a key contextual integrator: in ESCs, constitutive E2F activity triggers p53-mediated apoptosis upon DNA damage, preserving genomic integrity; in CSCs, deregulated E2F activity redirects transcription toward DNA repair, antioxidant defenses, and anti-apoptotic programs. This functional divergence underscores that phenotypic outcome is determined by the broader cellular and epigenetic landscape rather than any single factor. We conclude that CSC persistence is an emergent property of this integrated, survival-centric program, fundamentally distinct from the developmental imperative of ESCs. Effective therapeutic strategies must therefore move beyond targeting individual pathways to dismantle the interconnected regulatory networks that define the CSC survival context, offering a more robust approach to overcome therapy resistance and prevent tumor relapse.

Cancer Stem Cells (CSCs)↗
PubMed1999

Contrasting calcium dependencies of SAPK and ERK activations by glutamate in cultured striatal neurons.

Stress-activated protein kinase (SAPK) and extracellular signal-regulated kinase (ERK), both members of the mitogen-activated protein kinase (MAPK) family, may in some circumstances serve opposing functions with respect to cell survival. However, SAPK and ERK can also be coordinately activated in neurons in response to glutamate stimulation of NMDA receptors. To explore the mechanisms of these MAPK activations, we compared the ionic mechanisms mediating SAPK and ERK activations by glutamate. In primary cultures of striatal neurons, glutamatergic activation of ERK and one of its transcription factor targets, CREB, showed a calcium dependence typical of NMDA receptor-mediated responses. In contrast, extracellular calcium was not required for glutamatergic, NMDA receptor-mediated activation of SAPK and phosphorylation of its substrate, c-Jun. Increasing extracellular calcium enhanced ERK activation but reversed SAPK activation, further distinguishing the calcium dependencies of these two NMDA receptor-mediated effects. Finally, reducing extracellular sodium prevented the glutamatergic activation of SAPK but only partially blocked that of ERK. These contrasting ionic dependencies suggest a mechanism by which NMDA receptor activation may, under distinct conditions, differentially regulate neuronal MAPKs and their divergent functions.

Animals↗
PubMed2004

p53-regulated transcriptional program associated with genotoxic stress-induced apoptosis.

By using a genome-wide approach, we sought the identification of p53-regulated genes involved in cellular apoptosis. To this end, we assessed the transcriptional response of HCT116 colorectal cancer cells during apoptosis induced by the anticancer drug 5-fluorouracil as the function of p53 status, and we identified 230 potential targets that are regulated by p53. Previously identified p53 targets known to be involved in growth arrest and apoptosis were observed to be induced, thus validating the approach. Strikingly, we found that p53 regulates gene expression primarily through transcriptional repression (n = 189) rather than activation (n = 41), and selective blockade of p53-dependent gene repression resulted in the reduction in 5-fluorouracil-induced apoptosis. Reporter and chromatin immunoprecipitation assays demonstrated that p53 can suppress the promoter activities of three further studied candidate genes PLK, PTTG1, and CHEK1 but would only bind directly to PTTG1 and CHEK1 promoters, revealing that p53 can repress gene expression through both direct and indirect mechanisms. Moreover, RNA(i)-mediated knockdown of PLK and PTTG1 expression was sufficient to induce apoptosis, suggesting that repression of novel anti-apoptotic genes by p53 might contribute to a significant portion of the p53-dependent apoptosis. Our data support the divergent functions of p53 in regulating gene expression that play both synergistic and pleiotropic roles in p53-associated apoptosis.

Apoptosis↗
PubMed2002

Molecular cloning and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus.

The DMRT genes constitute a family of genes, which possess a common motif called the DM domain. DMRT1 is considered to be involved in sex determination and/or sex differentiation, but not much information exists about the function of the other gene family members. We cloned DMRT genes of two important model fish species, the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus. Based on sequence similarity and genomic structure with known DMRT genes, the gene from the medaka was identified as OlaDMRT4, and those from the platyfish as XmaDMRT2 and XmaDMRT4. OlaDMRT4 was assigned to the linkage group 18 (LG18) of the medaka by linkage analysis and fluorescence in situ hybridization. The earlier cloned medaka DMRT1, 2 and 3 genes form a cluster on LG9. Therefore, OlaDMRT4 does not belong to the DMRT gene cluster. In adult medaka fish, OlaDMRT4 is expressed in the brain, eyes, gill, kidney, as well as testis and ovary. During development, OlaDMRT4 exists as maternal transcripts, and is expressed until early larval stages. This pattern of expression differs from the other known medaka DMRT genes. Surprisingly it is also not the same as its putative tilapia ortholog (DMO). These differences in expression suggest that DMRT4 might fulfill divergent functions in different species.

Animals↗
PubMed2002

XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.

3T3 Cells↗
PubMed2003

Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome.

Zinc-finger-containing proteins can be classified into evolutionary and functionally divergent protein families that share one or more domains in which a zinc ion is tetrahedrally coordinated by cysteines and histidines. The zinc finger domain defines one of the largest protein superfamilies in mammalian genomes;46 different conserved zinc finger domains are listed in InterPro (http://www.ebi.ac.uk/InterPro). Zinc finger proteins can bind to DNA, RNA, other proteins, or lipids as a modular domain in combination with other conserved structures. Owing to this combinatorial diversity, different members of zinc finger superfamilies contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover. Accordingly, mutations of zinc finger genes lead to aberrations in a broad spectrum of biological processes such as development, differentiation, apoptosis, and immunological responses. This study provides the first comprehensive classification of zinc finger proteins in a mammalian transcriptome. Specific detailed analysis of the SP/Krüppel-like factors and the E3 ubiquitin-ligase RING-H2 families illustrates the importance of such an analysis for a more comprehensive functional classification of large protein families. We describe the characterization of a new family of C2H2 zinc-finger-containing proteins and a new conserved domain characteristic of this family, the identification and characterization of Sp8, a new member of the Sp family of transcriptional regulators, and the identification of five new RING-H2 proteins.

Alternative Splicing↗
PubMed2005

On the quality of tree-based protein classification.

MOTIVATION: Phylogenetic analysis of protein sequences is widely used in protein function classification and delineation of subfamilies within larger families. In addition, the recent increase in the number of protein sequence entries with controlled vocabulary terms describing function (e.g. the Gene Ontology) suggests that it may be possible to overlay these terms onto phylogenetic trees to automatically locate functional divergence events in protein family evolution. Phylogenetic analysis of large datasets requires fast algorithms; and even 'fast', approximate distance matrix-based phylogenetic algorithms are slow on large datasets since they involve calculating maximum likelihood estimates of pairwise evolutionary distances. There have been many attempts to classify protein sequences on the family and subfamily level without reconstructing phylogenetic trees, but using hierarchical clustering with simpler distance measures, which also produce trees or dendrograms. How can these trees be compared in their ability to accurately classify protein sequences? RESULTS: Given a 'reference classification' or 'group membership labels' for a set of related protein sequences as well as a tree describing their relationships (e.g. a phylogenetic tree), we propose a method for dividing the tree into monophyletic or paraphyletic groups so as to optimize the correspondence between the reference groups and the tree-derived groups. We call the achieved optimal correspondence the 'accuracy of a tree-based classification (TBC)', which measures the ability of a tree to separate proteins of similar function into monophyletic or paraphyletic groups. We apply this measure to compare classical NJ and UPGMA phylogenetic trees with the trees obtained from hierarchical clustering using different protein similarity measures. Our preliminary analysis on a set of expert-curated protein families and alignments suggests that there is no uniformly superior algorithm, and that simple protein similarity measures combined with hierarchical clustering produce trees with reasonable and often the most accurate TBC. We used our measure to help us to design TIPS, a tree-building algorithm, based on agglomerative clustering with a similarity measure derived from profile scoring. TIPS is comparable with phylogenetic algorithms in terms of classification accuracy and is much faster on large protein families. Due to its time scalability and acceptable accuracy, TIPS is being used in the large-scale PANTHER protein classification project. The trees produced by different algorithms for different protein families can be viewed at http://panther.appliedbiosystems.com/pub/tree_quality/trees.jsp. For every tree and every level of classification granularity we provide the optimal TBC along with the reference classification. AVAILABILITY: The script that evaluates the accuracy of TBC is available at http://panther.appliedbiosystems.com/pub/tree_quality/index.jsp

Algorithms↗
PubMed1992

Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

Alphavirus↗