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PubMed2005

Insights on a new PDI-like family: structural and functional analysis of a protein disulfide oxidoreductase from the bacterium Aquifex aeolicus.

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (PDI)-like proteins. Members of this family are characterized by a molecular mass of about 26kDa and by two Trx folds, each comprising a CXXC active site motif. We report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO). Functional studies have revealed the high catalytic efficiency of this enzyme in reducing, oxidizing and isomerizing disulfide bridges. Site-directed mutagenesis experiments have suggested that its two active sites have similar functional properties, i.e. that each of them imparts partial activity to the enzyme. This similarity was confirmed by the analysis of the enzyme crystal structure, which points to similar geometrical parameters and solvent accessibilities for the two active sites. The results demonstrated that AaPDO is the most PDI-like of all prokaryotic proteins so far known. Thus, further experimental studies on this enzyme are likely to provide important information on the eukaryotic homologue.

Amino Acid Sequence↗
PubMed1993

Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression.

In eukaryotes, RNA polymerase I transcription is controlled by DNA elements located within the spacers that separate the tandemly arranged rRNA genes. Unlike rRNA coding sequences, the intergenic spacers evolve rapidly and have little sequence similarity even among closely related species. Nonetheless, the arrangement of functional elements, such as spacer promoters and enhancers, is thought to be highly conserved. Here, we identify spacer promoters in the plant Arabidopsis thaliana, thereby demonstrating their existence in both the plant and animal kingdoms. We also use an Arabidopsis transient expression system to perform transcriptional analysis of the ribosomal gene promoter. Spacer promoters share sequence similarity with the gene promoter from -91 to +22 relative to the transcription start site, +1. Deletion analysis shows that sequences required for RNA polymerase I transcription reside within these boundaries. Spacer sequences upstream of the gene promoter have only a small positive effect on transcription in transfected protoplasts but can increase transcription from a Xenopus ribosomal gene promoter in injected frog oocytes. This trans-kingdom enhancer effect further suggests that the functional elements within eukaryotic ribosomal genes are highly conserved.

Animals↗
PubMed2002

Functional analysis of the Escherichia coli molybdopterin cofactor biosynthesis protein MoeA by site-directed mutagenesis.

Five moeA mutants were generated by replacing some conserved amino acids of MoeA by site-directed mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Mo-molybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterin-derived dephospho FormA as compared to bacteria transformed with wild-type moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

Blotting, Western↗
PubMed2005

Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas.

Pituitary tumor-transforming gene-1 (PTTG-1) is a novel protooncogene overexpressed in numerous cancer cell lines and cancers. In this study we elucidate the expression of PTTG-1 in uterine leiomyomas and its functional role in the development of this disease. By comparing 23 pairs of leiomyomas and matched pairs of myometria, we found that the expression of PTTG-1 is significantly elevated in leiomyoma. The expression of PTTG-1 is independent of the menstrual cycle and is not affected by ovarian hormones. In contrast, basic fibroblast growth factor (bFGF) time- and dose-dependently stimulates PTTG-1 expression, which results in increasing cell proliferation. Forced expression of PTTG-1 by transient transfection stimulates bFGF and VEGF expression as well as changes the expression pattern of cell cycle proteins. Western blotting analysis demonstrates that the expressions of PTTG-1, bFGF, and the cell proliferation marker, proliferating cell nuclear antigen, are positively correlated with each other, which supports the hypothesis that the positive feedback loop between PTTG-1 and bFGF increases leiomyoma cell proliferation. In summary, we have shown for the first time that PTTG-1 is up-regulated in human uterine leiomyomas and that the positive feedback loop between PTTG-1 and bFGF may be pivotal in the growth of leiomyoma cells.

Base Sequence↗
PubMed2004

Functional analysis of the NPDC-1 gene.

Mouse NPDC-1 (Neural Proliferation Differentiation and Control-1) is specifically expressed in neural cells when they stop dividing and start to differentiate. The NPDC-1 protein has been shown to interact with the E2F1 transcription factor, D-type cyclins and Cdk2. Immunocytochemical studies and subcellular fractionation of rat brains disclosed a partial colocalization of NPDC-1 with synaptic vesicle proteins, suggesting additional functional interactions. Here, we report the characterization of the mouse and human genes that were found to display very similar structures. We mapped the human gene to chromosome 9q34.3. No obvious pathological defect has been previously linked to this region. In order to gain further insights into its function(s), we generated null mice for the NPDC-1 gene. We did not detect any macroscopic phenotypical defect. Analysis of the upstream sequence of the mouse NPDC-1 gene delineated two regions involved in its negative and positive transcriptional regulation. Evidence for the regulation of NPDC-1 by Krox family transcription factors is presented.

Animals↗
PubMed1998

Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis.

The Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-kappa B. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.

Arabidopsis↗
PubMed1987

A functional analysis of phonological knowledge and generalization learning in misarticulating children.

It has been suggested that a child's productive phonological knowledge may be one factor that potentially accounts for individual differences in generalization learning observed among phonologically disordered children (Dinnsen & Elbert, 1984; Elbert, Dinnsen, & Powell, 1984). This paper evaluates the hypothesis that productive phonological knowledge influences generalization. Three related studies involving 6 functionally misarticulating children were conducted. In the first study, a description of each child's phonological system was developed using procedures of standard generative analysis. Based upon these descriptions, each child's productive phonological knowledge of his or her own sound system was determined and then ranked on a continuum ranging from "most" to "least" knowledge relative to the adult target. The second study implemented an experimental treatment program based upon each child's productive phonological knowledge, with treatment sounds selected directly from each child's continuum of knowledge. The third study reassessed each child's productive phonological knowledge following treatment. The results of these three studies indicated that a child's productive phonological knowledge of the sound system influenced the overall amount of generalization learning. However, the extent of generalization learning was associated with the point on the knowledge continuum at which treatment was initiated. These findings are discussed with reference to individual differences in generalization learning.

Articulation Disorders↗
PubMed2002

Characterization and in vivo functional analysis of splice variants of cypher.

Previously, we reported two splice variants of Cypher, a striated muscle-specific PDZLIM domain protein, Cypher1 and Cypher2. We have now characterized four additional splice isoforms, two of which are novel. The six isoforms can be divided into skeletal or cardiac specific classes, based on the inclusion of skeletal or cardiac specific domains. Short and long isoforms share an N-terminal PDZ domain, but the three C-terminal LIM domains are unique to long isoforms. By RNA and protein analysis, we have demonstrated that Cypher isoforms are developmentally regulated in both skeletal and cardiac muscle. We have previously shown that knockout of Cypher is neonatal lethal. To investigate the function of splice variants in vivo, we have performed a rescue experiment of the Cypher null mutant by replacing the endogenous Cypher gene with cDNAs encoding either a short or long skeletal muscle isoform. In contrast to Cypher null mice, a percentage of mice that express only a short or a long skeletal muscle-specific isoform can survive to at least 1 year of age. Although surviving mice exhibit muscle pathology, these results suggest that either isoform is sufficient to rescue the lethality associated with the absence of Cypher.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4+, Lyt-2.2- phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-gamma transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.

Animals↗
PubMed2005

Entamoeba histolytica: structural and functional analysis of the Ehadh112 gene promoter.

The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.

Animals↗
PubMed2002

Characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation.

A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.

Animals↗
PubMed1996

Structure-function analysis of human alpha1,3-fucosyltransferase. Amino acids involved in acceptor substrate specificity.

A series of molecular biology experiments were carried out to identify the catalytic domain of two human alpha1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino acids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62-361 (containing amino acids 62-361) and FucT V76-374 (containing amino acids 76-374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an alpha1,4-fucosyltransferase, whereas FucT V is an alpha1,3-fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results obtained from domain swapping experiments demonstrated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme's substrate specificity. However, swapping a region near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both alpha1,3- and alpha1,4-fucosyltransferase activity. This region contains 8 of the amino acid sequence differences that occur between the two proteins.

Amino Acid Sequence↗
PubMed2000

Functional analysis of the pro-apoptotic factor Bax using hammerhead ribozymes.

A pro-apoptotic protein Bax is a Bcl-2 family member and forms homodimers and also heterodimerizes with death antagonists, Bcl-2 and Bcl-XL. To elucidate the detail of function of Bax in cells, we constructed a hammerhead ribozyme targeted to the Bax mRNA. The level of Bax protein in Hela-K cells expressing Bax-ribozyme was decreased compared with that of wild type Hela-K cells. Therefore, the Bax-ribozyme should be useful for the future investigations of the details of apoptosis pathway.

Apoptosis↗
PubMed1987

Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II.

The calcium-calmodulin-dependent protein kinase II is a major component of brain synaptic junctions and has been proposed to play a variety of important roles in brain function. A complementary DNA representing a portion of the smaller 50-kilodalton subunit of the rat brain enzyme has been cloned and sequenced. The calmodulin-binding region has been identified and a synthetic analog prepared that binds calmodulin with high affinity in the presence of calcium. Like the 50-kilodalton kinase polypeptide, the concentration of the messenger RNA varies both neuroanatomically and during postnatal development of the brain. The broad tissue and species cross-reactivity of the complementary DNA suggests that the 50-kilodalton subunit found in rat brain is evolutionarily conserved and is the product of a single gene.

Age Factors↗
PubMed2003

Structural and functional analysis of mutations along the crystallographic dimer interface of the yeast TATA binding protein.

The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF(II)145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.

Adenosine Triphosphatases↗
PubMed1977

The psychobiological model and multiple discriminant function analysis of high-calibre oarsmen.

Candidates for New Zealand rowing teams (N = 181) were given a battery of physiological, performance, anthropometric and psychological tests in order to test the psychobiological model for prediction of athletic success. A series of stepwise multiple discriminant function analyses were conducted on both separate sub-sets of variables and their combinations. The expectation that the model would not differentiate between the three age categories of oarsmen (Juniors, Colt and Senior) except for age-related factors was upheld. A more specific test of the model was an examination of the accuracy of discrimination within each age group between those who were selected for New Zealand teams and those who were not selected. The results indicated that such differentiation was best when the biological and psychological variables were used in concert. The major discriminators between selected and nonselected oarsmen on the psychobiological functions were certain anthropometric and psychological variables. Further support for the notion of the existence of a rowing stereotype was provided by testing the Senior discriminant function on the other two groups. It was concluded that the results were in accordance with the multidisciplinary psychobiological model.

Adolescent↗
PubMed1994

A functional analysis and the potential clinical significance of 7 categories of sperm morphology.

Sperm morphology is a critical factor in the evaluation of infertile men. Patients often exhibit a morphology profile with consistent increases of specific abnormal categories of sperm morphology. We analyzed the functional ability of 7 categories of sperm morphology using assays that measure sperm viability longevity, deoxyribonucleic acid packing, acrosomal status, and the stimulation of the acrosome reaction with human cumulus/oocyte complexes and calcium ionophore A23187. Each morphology category was separately analyzed initially and at 3, 6, 12, 18 and 24 hours of in vitro culture. The data indicate that all categories of abnormal sperm morphology exhibit various functional debilities. However, tapered sperm most closely approached normal sperm function. All abnormal categories possessed subpopulations of sperm that functioned normally to the limits of these assays. These data indicate that increases in any of the categories of abnormal sperm morphology may lead to decreased fertility due to diminished functional ability indicated by the assays performed in this study.

Acrosome↗