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PubMed2000

Cloning of the rat ADAMTS-1 gene and its down regulation in endothelial cells in cirrhotic rats.

AIMS: This study was undertaken in order to identify genes which are regulated during the process of liver fibrosis. METHODS: The differential display method and RNA from rat endothelial cells before and after induction of cirrhosis were used. RESULTS: A 496 bp fragment, which was down regulated in liver endothelial cells from a cirrhotic animal, was cloned. The cloned fragment showed a 95% homology with the newly cloned mouse ADAMTS-1 gene (a disintegrin and metalloproteinase with thrombospondin motifs), which is implicated in inflammation. The fragment was found to span the 3' of exon 6, the whole exon 7 and the 5' of exon 8. Sequencing of the entire coding region of the rat gene showed a 94% homology at the nucleic acid level and 96% homology at the amino acid level. The sequences responsible for the function of the protein were conserved. Northern blot analysis, using the cloned fragment as a probe, confirmed the finding that the gene was down-regulated in endothelial cells derived from livers of cirrhotic animals. In situ PCR analysis localised the ADAMTS-1 gene in the liver endothelial cells from normal animals. CONCLUSIONS: Regulation of the expression of genes which belong to the metalloproteinase family in liver endothelial cells might be important in the development of liver cirrhosis.

ADAM Proteins↗
PubMed2005

A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk.

The identification of common variants that contribute to the genesis of human inherited disorders remains a significant challenge. Hirschsprung disease (HSCR) is a multifactorial, non-mendelian disorder in which rare high-penetrance coding sequence mutations in the receptor tyrosine kinase RET contribute to risk in combination with mutations at other genes. We have used family-based association studies to identify a disease interval, and integrated this with comparative and functional genomic analysis to prioritize conserved and functional elements within which mutations can be sought. We now show that a common non-coding RET variant within a conserved enhancer-like sequence in intron 1 is significantly associated with HSCR susceptibility and makes a 20-fold greater contribution to risk than rare alleles do. This mutation reduces in vitro enhancer activity markedly, has low penetrance, has different genetic effects in males and females, and explains several features of the complex inheritance pattern of HSCR. Thus, common low-penetrance variants, identified by association studies, can underlie both common and rare diseases.

Animals↗
PubMed2005

The macro domain is an ADP-ribose binding module.

The ADP-ribosylation of proteins is an important post-translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.

Adenosine Diphosphate↗
PubMed1996

Cloning, sequencing and expression of rat liver pyruvate carboxylase.

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated M(r) of 129848. This is flanked by a 5' untranslated region of 66 bp and a 3' untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249-10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

Amino Acid Sequence↗
PubMed2000

Using the CATH domain database to assign structures and functions to the genome sequences.

The CATH database of protein structures contains approximately 18000 domains organized according to their (C)lass, (A)rchitecture, (T)opology and (H)omologous superfamily. Relationships between evolutionary related structures (homologues) within the database have been used to test the sensitivity of various sequence search methods in order to identify relatives in Genbank and other sequence databases. Subsequent application of the most sensitive and efficient algorithms, gapped blast and the profile based method, Position Specific Iterated Basic Local Alignment Tool (PSI-BLAST), could be used to assign structural data to between 22 and 36 % of microbial genomes in order to improve functional annotation and enhance understanding of biological mechanism. However, on a cautionary note, an analysis of functional conservation within fold groups and homologous superfamilies in the CATH database, revealed that whilst function was conserved in nearly 55% of enzyme families, function had diverged considerably, in some highly populated families. In these families, functional properties should be inherited far more cautiously and the probable effects of substitutions in key functional residues carefully assessed.

Algorithms↗
PubMed2001

A multivariable model for predicting the need for blood transfusion in patients undergoing first-time elective coronary bypass graft surgery.

BACKGROUND: The incidence of blood transfusion in coronary artery bypass graft (CABG) surgery remains high. Preoperative identification of those at high risk for requiring blood will allow for the cost-effective use of some blood conservation modalities. Multivariable analysis techniques were used in this study to develop a prediction rule for such a purpose. STUDY DESIGN AND METHODS: Data were prospectively collected for all patients undergoing elective first-time CABG surgery from January 1997 to September 1998 at a tertiary-care teaching hospital (n = 1007). The prediction rule was developed on the first two-thirds of the sample by using logistic regression methods to examine the relationship of patient demographics, comorbidities, and preoperative Hb with perioperative blood transfusion. The remaining one-third of the sample was used to validate the rule. RESULTS: The transfusion rate was 29.4 percent. The prediction rule included preoperative Hb (g/dL, OR 0.928, p<0.0001), weight (kg, OR 0.938, p<0.0001), age (years, OR 1.037, p<0.01), and sex (male/female, OR 0.493, p<0.01); receiver operating characteristic = 0.86. When externally validated, the rule had a sensitivity of 82.1 percent and a specificity of 63.6 percent (at a selected probability cutoff). CONCLUSION: A simple and valid prediction rule is developed for predicting the risk of blood transfusion in patients undergoing first-time elective CABG surgery.

Aged↗
PubMed2000

Axillary recurrence following conservative surgery and radiotherapy in early breast cancer.

At the institute, since the late 1980s, there has been a uniform treatment protocol for the management of the regional lymph nodes in patients referred for radiotherapy following breast-conserving surgery. An analysis of 2,277 consecutive patients referred for radiotherapy between 1989 and 1992, with particular reference to regional lymph node management, has been undertaken. Axillary surgery alone was used in 517 patients (23%); 1,191 (52%) patients had no axillary surgery but had radiotherapy to the axilla, and infraclavicular and supraclavicular fossae by a single anterior field, delivering 40 Gy in 15 daily fractions over 3 weeks; and 474 patients (21%) had axillary surgery followed by radiotherapy. Ninety-five patients (4%) underwent no axillary treatment. There was a total of 155 axillary recurrences with a median follow-up of 5.9 years, giving an actuarial nodal control rate of 94% at 5 years (95% confidence interval (CI) 93.1-95.1). The overall survival at 5 years was 86% (95% CI 84.6-87.5). There was a trend towards improved axillary control with surgery alone compared with radiotherapy alone (4.5% versus 5.9% actuarial axillary failure rate at 5 years). An extremely low incidence of brachial plexus neuropathy secondary to radiotherapy was reported. The multidisciplinary treatment protocol used gave a high rate of regional node control, with minimal recorded morbidity.

Adult↗
PubMed1991

Phase-locking effects in a system of nonlinear oscillators.

Models for the excitation of collective modes in a system of nonlinear classical oscillators, initially out of phase, are discussed. The oscillators may be coupled in a dissipative or conservative manner. The analysis is based on the results of recent studies dealing with the problem of the free excitation of a coherent pulse, analogous to "superradiance" in two-level quantum systems. Several physical examples from the realms of electrodynamics and acoustics are discussed. The processes discussed here may be thought of as chaos-order transitions, provided that "chaos" is understood not as a stochastic nature of an individual oscillator but as the absence of a coherent component in their collective field.

Journal Article↗
PubMed2002

Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone.

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. We have previously documented the adaptive evolution of the Drosophila CenH3 (Cid) in comparisons of Drosophila melanogaster and Drosophila simulans, a divergence of approximately 2.5 million years. We have proposed that rapidly changing centromeric DNA may be driving CenH3's altered DNA-binding specificity. Here, we compare Cid sequences from a phylogenetically broader group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. Our analysis also reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres.

Amino Acid Sequence↗
PubMed2005

The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila.

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different "strengths." Here we use a combination of Bcd-site cluster analysis and evolutionary conservation to predict Bcd-dependent CRMs. We tested 14 predicted CRMs by in vivo reporter gene assays; 11 show Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, we do not detect a strong correlation between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, we find that binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Kruppel, which may limit the posterior extent of activation by the Bcd gradient. We propose that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene.

Animals↗
PubMed2006

New insights into myosin evolution and classification.

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

Animals↗
PubMed2002

Network of coupled promoting motions in enzyme catalysis.

A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.

Animals↗
PubMed2006

Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides.

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-beta-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.

Adenosine Triphosphate↗
PubMed1989

Evolutionary conserved modules associated with zinc fingers in Xenopus laevis.

Many DNA-binding proteins that are involved in the differential regulation of gene expression are composed of multiple discrete modules. Association of the homeobox-encoded helix-turn-helix DNA-binding motif with conserved modules, such as the paired box or the POU domain, has led to the definition of structurally and functionally related subfamilies of regulatory proteins. The zinc finger, which is the second major nucleic acid-binding motif characterized to date, defines large multigene families in higher eukaryotes; we have isolated more than 100 Xenopus finger protein-encoding cDNAs and in this study we show that at least 10 of these clones share extensive sequence homologies in a region of more than 200 amino acids in the N-terminal nonfinger portion of the predicted proteins, which is connected to variable finger clusters. We refer to this element as a finger-associated boxes (FAX) domain. Cross-hybridization with human genomic DNA indicates that the finger-associated boxes domain is evolutionary conserved. Northern blot analysis shows that the corresponding genes are differentially expressed in the course of early Xenopus embryogenesis.

Amino Acid Sequence↗
PubMed1991

Expression of GATA-binding proteins during embryonic development in Xenopus laevis.

Proteins that recognize the core sequence GATA are important regulators of hematopoietic-specific gene transcription. We have characterized cDNAs encoding the Xenopus laevis homologues of three related transcription factors, designated GATA-1, -2, and -3. Comparative sequence analysis reveals strong conservation of the zinc-finger DNA-binding domain among all vertebrate GATA-binding proteins. GATA-2 and GATA-3 polypeptides are homologous throughout their entire sequences, whereas GATA-1 sequence is conserved only in the region responsible for DNA binding. In Xenopus, RNAs encoding GATA-binding proteins are expressed in both larval and adult erythroid cells. GATA-1, -2, and -3 RNAs are first detectable in early gastrula (Nieuwkoop developmental stage 11). This is earlier than the appearance of the early larval alpha T1 globin RNA (stage 15), beta T1 globin RNA (stage 26), or blood island formation (stage 30). The expression of GATA-1, -2, and -3 in early development may signal an early commitment of mesoderm to form hematopoietic tissue.

Amino Acid Sequence↗
PubMed1996

Selection of linkers for a catalytic single-chain antibody using phage display technology.

Phage display has been evaluated as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. Preliminary experiments with a conventional linker failed to yield a functional single-chain version of a catalytic antibody with chorismate mutase activity. A random linker library was therefore constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire ( approximately 5 x 10(6) different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. There are apparently many viable solutions to the problem of linking individual VH and VL domains, but subtle differences in sequence dramatically influence the production, stability, and recognition properties of the scFv. The success of these experiments suggests that phage display will be generally useful for identifying peptide sequences for covalently linking any two protein domains.

Amino Acid Sequence↗
PubMed1998

Yeast and viral RNA 5' triphosphatases comprise a new nucleoside triphosphatase family.

Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, the hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium-dependent and has a turnover number of 1 s-1. Here we show that purified recombinant Cet1p possesses a robust ATPase activity (Km = 2.8 microM; Vmax = 25 s-1) in the presence of manganese. Cobalt is also an effective cofactor, but magnesium, calcium, copper, and zinc are not. Cet1p displays broad specificity in converting ribonucleoside triphosphates and deoxynucleoside triphosphates to their respective diphosphates. The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs. Mutational analysis establishes that conserved glutamate and arginine side chains within these motifs are essential for the RNA triphosphatase and ATPase activities of Cet1p in vitro and for Cet1p function in vivo. These findings are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel family of phosphohydrolase enzymes with a common active site.

Acid Anhydride Hydrolases↗
PubMed1999

A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding.

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

Alanine↗