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At least 1,261 records · Page 70Linked to original sources
PubMed1989

Dietary restraint in young adolescent girls: a functional analysis.

An investigation of dietary restraint during adolescence was conducted in two phases. First, two school classes of 12- and 14-year-old girls completed a restraint questionnaire. Second, the functional nature of the restraint scores was assessed in 24 girls from each age group using a procedure previously shown to lead to overeating in restrained adults. A wide distribution of restraint scores was found, with restrained individuals present in both age groups. Restraint was found to be functional, predicting higher food consumption in a taste test after imagining eating highly palatable foods. Hunger ratings were elevated by the imagination procedure, although the extent of this increase was similar for both restrained and unrestrained girls. The girls' body weight index was significantly correlated with degree of restraint. The existence and breakdown of restraint at this young age is regarded with concern, particularly in view of the links between dieting and eating disorders.

Adolescent↗
PubMed2004

Functional analysis of late-budding domain activity associated with the PSAP motif within the vesicular stomatitis virus M protein.

A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.

Amino Acid Motifs↗
PubMed1992

A chimeric EGFR/neu receptor in functional analysis of the neu oncoprotein.

As the factor binding to the neu protein has been unknown, it has not been possible to confirm experimentally the proposed growth-factor receptor like functions of the neu protein. To approach this problem we constructed a recombinant receptor which enabled ligand regulation of the neu tyrosine kinase. The hybrid receptor consisted of the extracellular ligand binding, transmembrane and protein kinase C-substrate domains joined to the intracellular tyrosine kinase and carboxyl-terminal domains of the neu protein. Several properties of NIH3T3 cells carrying this construct were tested. We obtained the first experimental evidence that the neu proto-oncogene has mitogenic and transforming activities only in the presence of a ligand stimulating its tyrosine kinase activity. Various cellular and molecular biological parameters indicated that the chimeric receptor behaved very similarly to the EGFR. Also, this chimeric receptor has allowed us to compare the constitutive oncogenic and the ligand-activated non-oncogenic activities of the neu tyrosine kinase. In the future we plan to focus on characterization of possible differences between EGFR and neu signalling in more differentiated cellular backgrounds.

3T3 Cells↗
PubMed1976

Long-term effects of activator (Andresen appliance) treatment. A clinical, biometric, cephalometric roentgenographic and functional analysis.

The purpose of this investigation was to analyse the long-term effects of activator treatment on the dental arches and arch relationships, the dento-facial skeleton and the function of the masticatory system. A follow-up study was performed on 112 patients treated 10-20 years previously with activators as the sole orthodontic appliance. In connection with activator treatment extractions of permanent teeth were performed on 66 patients while 46 patients were treated without tooth extractions. The investigation consisted of four separate portions (Parts I-IV).

Activator Appliances↗
PubMed1991

Functional analysis of the CPS I upstream sequences with a cat assay.

Expression plasmids (pKCPS-CAT) containing carbamyl phosphate synthetase (CPS I) upstream sequences of different lengths were constructed, and the function and characteristics of the sequences were studied with the CAT assay. Results showed that the CPS I upstream sequences exerted highly tissue-specific control on CPS I gene expression, and the -113 approximately -38 bp region relative to the cap site was found to be indispensable for CPS I gene transcription. The -1700 approximately -161 bp region contains sequences which confer an enhancing effect on CPS I gene transcription. Dexamethasone and thioproline (a differentiation inducer) showed enhancing effects on CPS I gene transcription in hepatoma cells. These results would have significance in studies on the gene regulation of CPS I associated with the mechanism of hepatocyte differentiation and carcinogenesis.

Antineoplastic Agents↗
PubMed1997

Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe.

Yeasts are an attractive model for the study of ribosome synthesis. However, our understanding of the relationship between the structure and function of the yeast nucleolus, in which preribosomal particles are synthesized, requires further investigations using microscopic approaches and in situ molecular biology. Combining cryofixation and cryosubstitution of Schizosaccharomyces pombe, we could identify morphologically distinct substructures in the nucleolus similar to the components of nucleoli of higher eukaryotes such as the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC). We complemented this morphological study by performing in situ hybridization and immunocytochemistry at the electron microscopy level. Using a probe complementary to the entire rRNA transcription unit of S. pombe, we detected rDNA at the periphery of the FCs, while immunocytochemistry with antibodies specific for the RNA polymerase I and the gar1 protein provided evidence that transcription and early steps of maturation take place in the DFC that extends throughout the nucleolus. We also present evidence that preribosomal subunits may be exported along tracks to the cytoplasm through all of the pores of the nuclear envelope and not just those in the portion of the envelope close to the nucleolus.

Biological Transport↗
PubMed2000

Expression, purification, and functional analysis of the human serine protease HtrA2.

HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.

Baculoviridae↗
PubMed2005

[Echocardiographic functional analysis of patients with rheumatoid arthritis and collagen diseases].

Cardiac manifestations were observed in patients with rheumatoid arthritis and other collagenoses. Echocardiography is a method of choice to detect pathologies in morphology and function of the heart. Pathophysiologically inflammatory alterations of the endo- as well as perimyocardium can be explained in these patients. In addition, in patients with collagenoses, the coagulation system is activated and the reactivity of the endothelium is reduced. Thus, thrombus formation at the heart valves with consecutive stenosis and/or regurgitations as well as ischemia-induced regional wall motion defects due to reduced vasodilator response of the coronary arteries can be expected. In the literature in patients with rheumatoid arthritis and other collagenoses, pericardial effusion and pulmonary hypertension are most frequently described. The echocardiographic analysis, however, is more complex due to the variability of the patient cohort. Quantification of valve defects and the analysis of wall motion and perfusion at rest and during stress is necessary to detect early changes of the diseases. The prerequisites for successful diagnostic echocardiography in these patients are the knowledge of modern echocardiographic techniques like tissue Doppler and contrast echocardiography and clinical experience with patients with rheumatoid arthritis and other collagenoses. The standardization of the echocardiographic diagnostic procedure becomes more and more important for reproducibility and comparability of the results.

Arthritis, Rheumatoid↗
PubMed1995

[Functional analysis of the CPS I upstream sequences with a CAT assay].

Expression plasmids (pKCPSx-CAT) containing carbamyl phosphate synthetase I (CPS I) upstream sequences of different lengths were constructed, and the function and characteristics of the sequences were studied with the CAT assay. The results showed that the CPS I upstream sequences exerted highly tissue-specific control on CPS I gene expression, and the -142- -38bp region relative to the cap site was found to be indispensable for CPS I gene transcription. The -1700- -161bp region contains sequences which confer an enhancing effect on CPS I gene transcription. Dexamethasone and thioproline (a differentiation inducer) showed enhancing effects on CPS I gene transcription in hepatoma cells. These results would have significance in studies on the gene regulation of CPS I associated with the mechanism of hepatocyte differentiation and carcinogenesis.

3T3 Cells↗
PubMed1997

An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA.

The structure and accessibility of the S. cerevisiae U3A snoRNA was studied in semi-purified U3A snoRNPs using both chemical and enzymatic probes and in vivo using DMS as the probe. The results obtained show that S. cerevisiae U3A snoRNA is composed of a short 5' domain with two stem-loop structures containing the phylogenetically conserved boxes A' and A and a large cruciform 3' domain containing boxes B, C, C' and D. A precise identification of RNA-protein contacts is provided. Protection by proteins in the snoRNP and in vivo are nearly identical and were exclusively found in the 3' domain. There are two distinct protein anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pair and the bases of stem-loop 2 and 4. Box C' is wrapped by the proteins. RNA-protein interactions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP. In accord with this location of the protein binding sites, an in vivo mutational analysis showed that box C' is important for U3A snoRNA accumulation, whereas mutations in the 5' domain have little effect on RNA stability. Our in vivo probing experiments strongly suggest that, in exponentially growing cells, most of the U3A snoRNA molecules are involved in the 10-bp interaction with the 5'-ETS region and in two of the interactions recently proposed with 18S rRNA sequences. Our experimental study leads to a slightly revised version of the model of interaction proposed by J. Hughes. Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis.

Animals↗
PubMed1991

Structural and functional analysis of promoter from gliadin, an endosperm-specific storage protein gene of Triticum aestivum L.

To identify cis-regulatory elements of the gliadin gene, a study of the gliadin gene promoter was conducted by transient expression analysis of plasmid DNAs which were introduced into plant protoplasts by electroporation. The promoter region (-592 bp to +18 bp from the translational start) of this developmentally regulated gene, when fused upstream to the chloramphenicol acetyl transferase (CAT) reporter cassette was unable to direct significant CAT expression in wheat or tobacco suspension cells. Because this monocot gene promoter appeared to be under stringent tissue-specific control, a hybrid promoter approach using a nopaline synthase (nos) promoter was employed. A series of 3' deletions of the gliadin promoter were placed upstream of either a nonfunctional -101 nos or a nearly wild-type -155 nos promoter fused in turn to a CAT reporter gene cassette. Transient expression analysis of these plasmid DNAs in tobacco cells showed that the gliadin fragment could either restore the activity of the non-functional nos promoter (series I) or enhance the activity of the functional nos promoter (series II). The degree of restoration of the promoter function conferred by gliadin fragments of the first series was proportional to the enhancing effect of the same fragments in the second series of constructs. The transcriptional activity of the gliadin (-592 bp to -77 bp) -nos hybrid promoter was reduced by 26% upon 3' deletion of sequences in the region -141 bp to -77 bp, which contains both the TATA and CCAAT boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2002

Structure-function analysis of lipopeptide pheromones from the plant pathogen Ustilago maydis.

Mating of two haploid cells is a prerequisite for the successful infection of corn by the pathogenic fungus Ustilago maydis. Cell-cell recognition is mediated by small lipopeptide pheromones. Genes encoding pheromone precursors as well as pheromone receptors are located in the a mating type locus. Two pheromones are known, the tridecapeptide a1 and the nonapeptide a2, both of which contain an S-prenylated cysteine methyl ester at the C-terminus. It has previously been shown that synthetic pheromones are active in a biological test system. Here, we used the same assay to perform a detailed analysis of synthetic a1 and a2 pheromones. Testing of truncated derivatives of a1 and a2 revealed that in both cases the pheromone function is less sensitive to N-terminal than to C-terminal truncations. Replacement of each amino acid in the a1 pheromone by either alanine or the corresponding D-amino acids revealed that four positions are important for function: the two central glycines (positions 5 and 9), proline at position 7 and tyrosine at position 10. By introducing different naturally occurring as well as synthetic amino acids at position 10, we demonstrate that the presence of an aromatic side chain at this position is necessary for function. We propose a model in which a cis peptide bond at proline 7 favours the formation of a type II' beta turn of the a1 pheromone backbone with glycine 9 in position i+1 (where i refers to the first position of the beta turn). As a result, tyrosine 10, at position i+2 of the turn, would be highly exposed and could be inserted into a structurally well-defined binding pocket of the receptor. The latter may represent an important facet of receptor specificity.

Amino Acid Sequence↗
PubMed2004

Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma.

Although the RhoA and RhoC proteins comprise an important subset of the Rho GTPase family that have been implicated in invasive breast carcinomas, attributing specific functions to these individual members has been difficult. We have used a stable retroviral RNA interference approach to generate invasive breast carcinoma cells (SUM-159 cells) that lack either RhoA or RhoC expression. Analysis of these cells enabled us to deduce that RhoA impedes and RhoC stimulates invasion. Unexpectedly, this analysis also revealed a compensatory relationship between RhoA and RhoC at the level of both their expression and activation, and a reciprocal relationship between RhoA and Rac1 activation.

ADP Ribose Transferases↗
PubMed2005

Identification of the Wnt signaling activator leucine-rich repeat in Flightless interaction protein 2 by a genome-wide functional analysis.

The Wnt signaling pathway acts ubiquitously in metazoans to control various aspects of embryonic development. Wnt ligands bind their receptors Frizzled and low-density lipoprotein receptor-related protein 5/6 and function through Disheveled (Dvl), Axin, adenomatous polyposis coli, glycogen synthase kinase 3beta, and casein kinase (CK) 1 to stabilize beta-catenin and induce lymphocyte enhancer-binding factor (LEF)/T cell factor (TCF)-dependent transcriptional activities. To identify previously unrecognized Wnt signaling modulators, a genome-wide functional screen was performed using large-scale arrayed cDNA collections. From this screen, both known components and previously uncharacterized regulators of this pathway were identified, including beta-catenin, Dvl1, Dvl3, Fbxw-1, Cul1, CK1epsilon, CK1delta, and gamma-catenin. In particular, a previously unrecognized activator, LRRFIP2 (leucine-rich repeat in Flightless interaction protein 2), was found that interacts with Dvl to increase the cellular levels of beta-catenin and activate beta-catenin/LEF/TCF-dependent transcriptional activity. The function of LRRFIP2 is blocked when a dominant negative Dvl (Xdd1) is coexpressed. Expression of LRRFIP2 in Xenopus embryos induced double axis formation and Wnt target gene expression; a dominant negative form of LRRFIP2 suppresses ectopic Wnt signaling in Xenopus embryos and partially inhibits endogenous dorsal axis formation. These data suggest that LRRFIP2 plays an important role in transducing Wnt signals.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Mid-term results of an AGC total knee arthroplasty system survival and function analysis: 2- to 8-year follow-up results.

Between 1993 and 1999, 148 total knee arthroplasties were performed in Dipreca Hospital; 94 were anatomically graduated components (AGC) cam-and-groove posterior-stabilized total knee arthroplasties. All patients underwent implantation with a cemented posterior cruciate-substituting design, with resurfacing of 3 components. Mean age at surgery was 69 years, and patients were followed up for a minimum of 2 years (range, 2-8). The survival analysis at 8 years was 96.6%. Revision for any reason was considered to be the endpoint. Of patients, 3.2% were lost to follow-up. The mean knee pain and function scores were 89 and 64 of 100, respectively. The scores were also analyzed by categories. This knee arthroplasty follow-up analysis shows excellent midterm results.

Aged↗
PubMed2002

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein.

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

Amino Acid Substitution↗
PubMed1987

Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain.

Transcription factor Sp1 is a protein present in mammalian cells that binds to GC box promoter elements and selectively activates mRNA synthesis from genes that contain functional recognition sites. We have isolated a cDNA that encodes the 696 C-terminal amino acid residues of human Sp1. By expression of truncated fragments of Sp1 in E. coli, we have localized the DNA binding activity to the C-terminal 168 amino acid residues. In this region, Sp1 has three contiguous Zn(II) finger motifs, which are believed to be metalloprotein structures that interact with DNA. We have found that purified Sp1 requires Zn(II) for sequence-specific binding to DNA. Thus, it is likely that Sp1 interacts with DNA by binding of the Zn(II) fingers. To facilitate the identification of mutant variants of Sp1 that are defective in DNA binding, we have also devised a bacterial colony assay for detection of Sp1 binding to DNA.

Amino Acid Sequence↗
PubMed1993

Functional analysis of the antigen binding region of an MHC class II molecule.

The MHC class II molecules bind antigenic peptides and present them to T cells. Their ability to carry out these functions depends, in a critical way, on the detailed structure of the membrane-distal alpha 1 and beta 1 domains of these molecules. Using the I-Ak molecule and a series of hen egg lysozyme (HEL) peptide-specific, I-Ak-restricted T cell hybridomas as a model, we have examined the effect of altering essentially all of the polymorphic residues of the murine class II molecule on its ability to present Ag. Our results support the following conclusions: (1) both the location and the structural alteration introduced in a specific amino acid interchange are important in determining the effect the interchange will have on Ag presentation; and (2) changes in amino acids in the floor of the putative Ag binding cleft of the class II molecule can exert a major influence on the presentation of peptides to T cells. By carrying out direct binding experiments between the HEL(46-61) peptide and two mutant I-A molecules that fail to present HEL(46-61) to appropriate T cells, we were able to assess, in a quantitative fashion, the role played by peptide binding in the failure to present Ag. Our results suggest that, in the two cases studied, the failure to bind the HEL(46-61) peptide was not primarily responsible for the failure of the mutant class II molecule to present that peptide. Specifically, an A beta chain mutant that possesses d allelic residues at positions 65-67 in the second PMR of the Ak beta chain actually binds HEL(46-61) at wild type (I-Ak) levels. In contrast, an A alpha chain chimera in which b allelic residues are inserted in the third PMR of the Ak alpha chain, binds HEL(46-61) about three- to four-fold less well than wild type. While this decrease in binding affinity may be partially responsible for the inability of the latter chimeric molecule to present HEL(46-61), it can not be the total explanation because increasing the peptide concn even by an order of magnitude does not restore Ag presentation by APC expressing this chimeric molecule. These results are discussed in terms of the currently accepted model of the class II molecule.

Amino Acid Sequence↗