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B Knudsen

Publications and source records attributed to B Knudsen.

At least 19 recordsLinked to original sources
PubMed2004

Evolutionary rate variation and RNA secondary structure prediction.

Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type of approach. Determining these rates can be hard to do reliably without a large and accurate initial alignment, which ideally also has structural annotation. Hence, one must often apply rates extracted from other RNA families with trusted alignments and structures. Here, we investigate this problem by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions. In addition we obtained an alignment of the 5' HIV-1 region that is more consistent with the structure than that currently in the database. We added randomized noise to the original values of the rates to investigate the stability of predictions to rate matrix deviations. We find that changes within a fairly large range still produce reliable predictions and conclude that using rates from a limited set of RNA sequences is valid over a broader range of sequences.

Algorithms↗
PubMed2001

A likelihood ratio test for evolutionary rate shifts and functional divergence among proteins.

Changes in protein function can lead to changes in the selection acting on specific residues. This can often be detected as evolutionary rate changes at the sites in question. A maximum-likelihood method for detecting evolutionary rate shifts at specific protein positions is presented. The method determines significance values of the rate differences to give a sound statistical foundation for the conclusions drawn from the analyses. A statistical test for detecting slowly evolving sites is also described. The methods are applied to a set of Myc proteins for the identification of both conserved sites and those with changing evolutionary rates. Those positions with conserved and changing rates are related to the structures and functions of their proteins. The results are compared with an earlier Bayesian method, thereby highlighting the advantages of the new likelihood ratio tests.

Amino Acid Sequence↗
PubMed2001

SRPDB (Signal Recognition Particle Database).

Signal recognition particle (SRP) is a stable cytoplasmic ribonucleoprotein complex that serves to translocate secretory proteins across membranes during translation. The SRP Database (SRPDB) provides compilations of SRP components, ordered alphabetically and phylogenetically. Alignments emphasize phylogenetically-supported base pairs in SRP RNA and conserved residues in the proteins. Data are provided in various formats including a column arrangement for improved access and simplified computational usability. Included are motifs for identification of new sequences, SRP RNA secondary structure diagrams, 3-D models and links to high-resolution structures. This release includes 11 new SRP RNA sequences (total of 129), two protein SRP9 sequences (total of seven), two protein SRP14 sequences (total of 10), two protein SRP19 sequences (total of 16), 10 new SRP54 (ffh) sequences (total of 66), two protein SRP68 sequences (total of seven) and two protein SRP72 sequences (total of nine). Seven sequences of the SRP receptor alpha-subunit and its FtsY homolog (total of 51) are new. Also considered are ss-subunit of SRP receptor, Flhf, Hbsu, CaM kinase II and cpSRP43. Access to SRPDB is at http://psyche.uthct. edu/dbs/SRPDB/SRPDB.html and the European mirror http://www.medkem. gu.se/dbs/SRPDB/SRPDB.html

Databases, Factual↗
PubMed2001

tmRDB (tmRNA database).

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and accessible on the World Wide Web at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. Mirror sites are located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Institute of Biological Sciences, Aarhus, Denmark (http://www.bioinf.au. dk/tmRDB/). The tmRDB provides information and citation links about tmRNA, a molecule that combines functions of tRNA and mRNA in trans-translation. tmRNA is likely to be present in all bacteria and has been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana. This release adds 26 new sequences and corresponding predicted tmRNA-encoded tag peptides for a total of 86 tmRNAs, ordered alphabetically and phylogenetically. Secondary structures and three-dimensional models in PDB format for representative molecules are being made available. tmRNA alignments prove individual base pairs and are generated manually assisted by computational tools. The alignments with their corresponding structural annotation can be obtained in various formats, including a new column format designed to improve and simplify computational usability of the data.

Databases, Factual↗
PubMed2001

Semi-automated update and cleanup of structural RNA alignment databases.

UNLABELLED: We have developed a series of programs which assist in maintenance of structural RNA databases. A main program BLASTs the RNA database against GenBank and automatically extends and realigns the sequences to include the entire range of the RNA query sequences. After manual update of the database, other programs can examine base pair consistency and phylogenetic support. The output can be applied iteratively to refine the structural alignment of the RNA database. Using these tools, the number of potential misannotations per sequence was reduced from 20 to 3 in the Signal Recognition Particle RNA database. AVAILABILITY: A quick-server and programs are available at http://www.bioinf.au.dk/rnadbtool/

Base Sequence↗
PubMed2001

Characterisation of the gastrointestinal bacterial community in pigs fed fermented liquid feed and dry feed: composition and fermentation capacity (phenotypic fingerprint).

Feeding pigs with fermented liquid feed (FLF) has been shown to reduce the number of enteropathogens such as Salmonella and Brachyospira hyodysenteriae as well as coliform bacteria in general in the gastrointestinal tract (GIT). Also the commensal bacterial populations have been shown to respond to the use of FLF, e.g. the total counts of anaerobes, including lactic acid bacteria are reduced. In the present work we demonstrate that the capacity to ferment a series of substrates (mainly low-molecular weight carbohydrates) is reduced in caecum, colon and faeces of pigs fed FLF compared to pigs fed dry feed. This reduction could be due to the fact that these substrates are partially depleted by fermentation in the liquid feed prior to entering the animal. Therefore nutrient availability may be limited in the large intestine of pigs fed FLF, which may again affect bacterial enzyme synthesis and growth and thus the possibility for pathogenic and zoonotic bacteria to establish.

Animal Feed↗
PubMed2000

Statistical alignment: computational properties, homology testing and goodness-of-fit.

The model of insertions and deletions in biological sequences, first formulated by Thorne, Kishino, and Felsenstein in 1991 (the TKF91 model), provides a basis for performing alignment within a statistical framework. Here we investigate this model.Firstly, we show how to accelerate the statistical alignment algorithms several orders of magnitude. The main innovations are to confine likelihood calculations to a band close to the similarity based alignment, to get good initial guesses of the evolutionary parameters and to apply an efficient numerical optimisation algorithm for finding the maximum likelihood estimate. In addition, the recursions originally presented by Thorne, Kishino and Felsenstein can be simplified. Two proteins, about 1500 amino acids long, can be analysed with this method in less than five seconds on a fast desktop computer, which makes this method practical for actual data analysis.Secondly, we propose a new homology test based on this model, where homology means that an ancestor to a sequence pair can be found finitely far back in time. This test has statistical advantages relative to the traditional shuffle test for proteins.Finally, we describe a goodness-of-fit test, that allows testing the proposed insertion-deletion (indel) process inherent to this model and find that real sequences (here globins) probably experience indels longer than one, contrary to what is assumed by the model.

Algorithms↗
PubMed2000

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling.

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

Animals↗
PubMed1999

E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens.

OBJECTIVES: Approximately 30% of clinically localized prostate adenocarcinomas treated by radical prostatectomy (RP) will recur within 10 years. To prevent recurrence, new adjuvant therapies are in development that seek to treat high-risk patients after surgery. To identify patients as candidates for these treatments, improved biomarkers for predicting prognosis are needed. Reduced expression of E-cadherin has been proposed as a new marker for predicting prognosis in prostate adenocarcinoma. Since few studies have examined the relation between risk factors for disease progression and E-cadherin expression using routinely processed RP specimens, we used RP specimens to correlate downregulation of E-cadherin and pathologic stage at RP. METHODS: Primary adenocarcinomas (n = 76) from formalin-fixed and paraffin-embedded RP specimens were evaluated by immunohistochemistry against E-cadherin (HECD-1) using heat-induced epitope retrieval and automated staining (BioTek Solutions). Normal appearing prostate epithelium was used as an internal control for each specimen. Staining was scored as an estimate of the percentage of tumor cells in each specimen that showed strong plasma membrane staining. RESULTS: Specimens were divided into three categories with respect to Gleason score: intermediate (score 5 to 6, n = 31), intermediate to high (score 7, n = 25), and high (score 8 to 9, n = 20). For pathologic stage, specimens were divided into three categories: low stage/organ confined (pT2, n = 30), intermediate stage/limited extraprostatic extension (pT3a, n = 25), and high stage/seminal vesicle-pelvic lymph node metastases (pT3b-any pTN1, n = 21). In univariate analysis, reduced levels of E-cadherin correlated with advanced Gleason score (P = 0.003) and advanced pathologic stage (P = 0.008). In multivariate analysis, E-cadherin, preoperative prostate-specific antigen, and Gleason score all contributed independently to the prediction of high-stage disease (P<0.0001). Ten pelvic lymph node metastases from this same patient cohort were stained for E-cadherin. All were positive and 9 of 10 were moderately to strongly positive. CONCLUSIONS: Since essentially all patients in the high-stage category have a very high likelihood of disease recurrence, we conclude that the study of E-cadherin in a prospective manner as a potential biomarker of disease progression in patients with clinically organ-confined prostate cancer who undergo RP is warranted. Additionally, our finding that most metastatic tumor cells in pelvic lymph nodes express E-cadherin supports the notion that the establishment of the distant colonization and growth of metastatic tumor cells may be facilitated by expression or re-expression of previously downregulated E-cadherin. This would strongly suggest that irreversible genetic inactivation through mutation or allelic loss at 16q2.3 is probably not the mechanism of E-cadherin downregulation in most abnormally expressing primary prostate carcinomas.

Adenocarcinoma↗
PubMed1999

RNA secondary structure prediction using stochastic context-free grammars and evolutionary history.

MOTIVATION: Many computerized methods for RNA secondary structure prediction have been developed. Few of these methods, however, employ an evolutionary model, thus relevant information is often left out from the structure determination. This paper introduces a method which incorporates evolutionary history into RNA secondary structure prediction. The method reported here is based on stochastic context-free grammars (SCFGs) to give a prior probability distribution of structures. RESULTS: The phylogenetic tree relating the sequences can be found by maximum likelihood (ML) estimation from the model introduced here. The tree is shown to reveal information about the structure, due to mutation patterns. The inclusion of a prior distribution of RNA structures ensures good structure predictions even for a small number of related sequences. Prediction is carried out using maximum a posteriori estimation (MAP) estimation in a Bayesian approach. For small sequence sets, the method performs very well compared to current automated methods.

Algorithms↗
PubMed1999

Advances in non-peptide glucagon receptor antagonists.

This review deals with glucagon receptor antagonism as a possible treatm ent of Type 2 diabetes. The role of glucagon in animal models has been studied by glucagon antibodies as model antagonists. Depending upon the animal model studied, selective glucagon deficiency produced by immunone utralisation suggests that glucagon plays a modest (rats) to substantial (rabbits) role in the maintenance of euglycaemia and is an important dia betogenic factor. These data strongly suggest that glucagon antagonism may be a beneficial and safe therapeutic approach for the treatment of T ype 2 diabetes. Further, the progress on non-peptide glucagon receptor antagonists is reviewed with special focus on the different classes of g lucagon receptor antagonists published, namely quinoxalines /pyrrolo[1,2 -a]quinoxalines, mercaptobenzimidazoles, 2-pyridyl-3,5-diarylpyrroles, q uinoline hydrazones, 4-phenylpyridines, and alkylidene hydrazides.

Animals↗
PubMed1998

Neuronal excitatory properties of human immunodeficiency virus type 1 Tat protein.

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

Animals↗
PubMed1997

Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium.

Prostate-specific membrane antigen (PSMA), initially defined by monoclonal antibody (mAb) 7E11, is a now well-characterized type 2 integral membrane glycoprotein expressed in a highly restricted manner by prostate epithelial cells. 7E11 has been shown to bind an intracellular epitope of PSMA that, in viable cells, is not available for binding. Herein, we report the initial characterization of the first four reported IgG mAbs that bind the external domain of PSMA. Competitive binding studies indicate these antibodies define two distinct, noncompeting epitopes on the extracellular domain of PSMA. In contrast to 7E11, these mAbs bind to viable LNCaP cells in vitro. In addition, they show strong immunohistochemical reactivity to tissue sections of prostate epithelia, including prostate cancer. These mAbs were also strongly reactive with vascular endothelium within a wide variety of carcinomas (including lung, colon, breast, and others) but not with normal vascular endothelium. These antibodies should prove useful for in vivo targeting to prostate cancer, as well as to the vascular compartment of a wide variety of carcinomas.

Animals↗
PubMed1996

Proline-rich sequences mediate the interaction of the Arg protein tyrosine kinase with Crk.

Arg is a ubiquitously expressed member of the Abelson family of nonreceptor protein-tyrosine kinases. Defining the Arg sequences that mediate its interaction with other proteins is essential to elucidating its role in cellular signaling. In this report we demonstrate that Arg associates with c-Crk, an adaptor protein composed of an SH2 domain and two SH3 domains, and examine the molecular mechanism of the interaction. In vitro experiments revealed that three proline-rich sequences with distinct specificities for SH3 domains are located in the Arg C-terminal domain, just C-terminal to the kinase domain, and that two of these sequences bind to the Crk N-terminal SH3 domain. These two sequences conform to the PxLPxK/R motif that has been observed in other proteins that bind the Crk N-terminal SH3 domain. The interaction of Arg with c-Crk in living cells was confirmed by the detection of coimmunoprecipitation in coinfected Sf9 cells. In addition, increased phosphorylation of c-Crk was observed in cotransfected COS cells, indicating that Crk is an Arg substrate. The site of c-Crk phosphorylation by Arg was identified as tyrosine 221, a residue whose modification has been shown to result in an intramolecular SH2 interaction and a folded conformation. These experiments extend the known Arg protein interacting motifs to include SH3 binding sites and suggest that Arg may function as an effector as well as a regulator of Crk activity.

Animals↗
PubMed1996

Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic.

Tat is an 86- to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca2+]i), and affected neuronal excitability. Tat31-61 but not Tat48-85 dose dependently increased cytotoxicity and levels of [Ca2+]i in cultured human fetal brain cells. Similarly, Tat31-61 but not Tat48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca2+]i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors.

Animals↗
PubMed1995

Cellular proteins binding to the first Src homology 3 (SH3) domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways.

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

BACKGROUND: Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS: In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Amino Acid Sequence↗