Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in the APC gene are responsible for the dominantly inherited colon cancer syndrome, familial adenomatous polyposis (FAP). We have designed PCR primers which allow amplification by RT-PCR of exons 1-14 of the APC gene in six overlapping segments. The amplicons have been screened for the presence of mutations in patients affected with FAP using heteroduplex analysis. One patient has been identified with an alternatively spliced transcript involving exon 14 and a single base insertion mutation within the same exon.
Mol Cell Probes 1996 Oct
PMID:Mutation detection in exons 1-14 of the adenomatous polyposis coli gene: identification of an alternatively spliced transcript. 891 Aug 93

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.
Mol Biol Evol 1996 Dec
PMID:Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications. 895 77

Desmoid tumours are generally very rare but occur about 100 times more frequently in the colorectal cancer predisposition syndrome familial adenomatous polyposis (MIM 175100), being represented in about 10% of patients. In addition to desmoid disease occurring in familial adenomatous polyposis (FAP) there exist familial infiltrative fibromatosis (MIM 135290) kindreds where there is no evidence of FAP. Previously we have described a kindred with familial infiltrative fibromatosis (FIF) in which desmoid tumours were associated with nonpolyposis colorectal cancer. FAP is caused by mutations in the APC gene and various genotype-phenotype relationships have been defined including reports that colorectal polyposis is less severe with mutations 5' to codon 157 and that the risk of desmoid tumours is high in FAP patients with APC gene mutations between codons 1444 and 1598. There is relatively little information on the phenotype of APC gene mutations 3' to codon 1598; however, one large family has been reported with a mutation at codon 1987 which presents with a highly variable phenotype which includes desmoid disease. We screened our original FIF kindred and three further families with a similar phenotype for mutations in the APC gene. A 4 bp frameshift deletion in codon 1962 was identified in the original FIF kindred and two further apparently unrelated families. Haplotype analysis suggests a common origin for the APC mutation in all three families. Affected individuals had no evidence of congenital hypertrophy of the retinal pigment epithelium. Colorectal polyposis was variable, and most affected patients had either none or a few late onset polyps. These findings demonstrate (i) that FAP and FIF are allelic, and (ii) that APC gene mutations which truncate the APC protein distal to the beta-catenin binding domain are associated with desmoid tumours, absent CHRPE and variable but attenuated polyposis expression.
Hum Mol Genet 1996 Dec
PMID:Familial infiltrative fibromatosis (desmoid tumours) (MIM135290) caused by a recurrent 3' APC gene mutation. 896 44

Activators of the sigma54-holoenzyme catalyze the isomerization of closed complexes between this polymerase and a promotor to open complexes in a reaction that depends upon hydrolysis of a nucleoside triphosphate. The activators normally bind to DNA sites with the properties of transcriptional enhancers and contact the polymerase by means of DNA loop formation. Here, we demonstrate that mutant forms of the activator nitrogen regulatory protein C (NtrC) that lack one helix of the helix-turn-helix (HTH) DNA-binding motif or the entire motif retain residual capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ability to hydrolyze ATP. We show that substitution of alanine for three hydrophilic residues in the second helix of the HTH yields a stable, dimeric form of NtrC defective in DNA-binding. Like mutant forms with deletions of one or both helices, the NtrC3ala protein failed to bind DNA in a sensitive affinity co-electrophoresis assay, indicating that its affinity for a strong enhancer was reduced by at least 5000-fold. (The assay detected enhancer-binding by two mutant forms of NtrC with single amino acid substitutions in the HTH and non-specific DNA-binding by the wild-type protein.) The phosphorylated NtrC3ala protein had normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least tenfold by an oligonucleotide carrying a strong enhancer, the ATPase activity of the phosphorylated NtrC3ala protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC3ala protein activated transcription from the major glnA promoter. In agreement with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templates carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering and facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution.
J Mol Biol 1997 Mar 21
PMID:Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. 909 4

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. Transgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1-0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1-2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent immunopurification with a calcium-dependent mAb directed to the gamma-carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vitro. The higher percentages of Ca(2+)-dependent conformers isolated from the total rhPC by immunopurification correlated well with higher specific activity and lower expression. A rate limitation in gamma-carboxylation of rhPC was clearly identified for the higher expressing animals. Thus, transgenic animals with high expression levels of complex recombinant proteins produced a lower percentage of biologically active protein.
J Mol Recognit
PMID:Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland. 917 18

The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.
J Mol Recognit
PMID:Effect of antibody orientation on immunosorbent performance. 917 36

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.
J Mol Biol 1997 Jul 11
PMID:Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. 923 16

Protein S (PS), which functions as a species-specific anticoagulant cofactor to activated protein C (APC), is a mosaic protein that interacts with the phospholipid membrane via its gamma-carboxyglutamate-rich (Gla) module. This module is followed by the thrombin-sensitive region (TSR), sensitive to thrombin cleavage, four epidermal growth factor (EGF)-like modules and a last region referred to as the sex hormone binding globulin (SHBG) domain. Of these, the TSR and the first EGF-like regions have been shown to be important for the species-specific interaction with APC. Difficulties in crystallising PS have so far hindered its study at the atomic level. Here, we report theoretical models for the Gla and EGF-1 modules of human PS constructed using prothrombin and factor X experimental structures. The TSR was built interactively. Analysis of the model linked with the large body of biochemical literature on PS and related proteins leads to suggestions that (i) the TSR stabilises the calcium-loaded Gla module through hydrophobic and ionic interactions and its conformation depends on the presence of the Gla module; (ii) the TSR does not form a calcium binding site but is protected from thrombin cleavage in the calcium-loaded form owing to short secondary structure elements and close contact with the Gla module; (iii) the PS missense mutations in this region are consistent with the structural data, except in one case which needs further investigation; and (iv) the two PS 'faces' involving regions of residues Arg49-Gln52-Lys97 (TSR-EGF-1) and Thr103-Pro106 (EGF-1) may be involved in species-specific interactions with APC as they are richer in nonconservative substitution when comparing human and bovine protein S. This preliminary model helps to plan future experiments and the resulting data will be used to further validate and optimise the present structure.
J Comput Aided Mol Des 1997 May
PMID:A theoretical model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: from biostructural pathology to species-specific cofactor activity. 926 56

The discovery of thrombin as a Na(+)-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The alllosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na+ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na(+)-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na(+)-dependent regulation discovered for thrombin. Na+ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.
Cell Mol Life Sci 1997 Sep
PMID:Molecular mechanisms of thrombin function. 936 68

Empirical findings have shown that recombinant chimeric proteins may be made more immunogenic if T helper epitopes are incorporated as tandem repeats. In the present study we investigated the mechanisms responsible for the enhanced immunogenicity of fusion proteins composed of the heat-stable enterotoxin of enterotoxigenic E. coli (STa) linked to multiple copies of the ovalbumin323-339 T helper epitope (ova) and a connecting dimer of an Ig-binding region of Staphylococcus aureus protein A (ZZ), which were previously shown to stimulate strong anti-STa titres in mice. We used B cell and macrophage cell lines as APC and IL-2 production by ova-specific T cells as our read-out system. Fusion proteins containing four repeated T helper epitopes were found to be the most immunogenic and resulted in 50-fold higher IL-2 production than constructs with a single T helper epitope. Under limiting APC conditions the construct with four epitopes was the best inducer of IL-2, indicating that this construct was most effectively processed by the APC. Analysis of IL-2R alpha expression by flow cytometry confirmed that four copies gave the highest frequency of activated T cells in culture, indicating a direct correlation between ability to activate T cells and IL-2 production in culture. Also in vivo, the fusion protein with four epitopes exhibited the strongest T cell priming effect. Moreover, both in vitro and in vivo, the ZZ construct was found to serve as an efficient means for targeting of the fusion proteins to B cells, thereby allowing access to the Ig receptor uptake pathway for Ag. The present study provides direct evidence that fusion proteins can be constructed to optimize processing in the individual APC and enhance activation of clonal T cells.
Mol Immunol 1997 Jun
PMID:Tandem repeats of T helper epitopes enhance immunogenicity of fusion proteins by promoting processing and presentation. 939 63


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>