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PubMed2004

A functional analysis of mouse models of cardiac disease through metabolic profiling.

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.

Animals↗
PubMed1988

Graves' disease: phenotypic and functional analysis at the clonal level of the T-cell repertoire in peripheral blood and in thyroid.

We have investigated at the clonal level the repertoire of intrathyroid and peripheral T lymphocytes in three patients with Graves' disease using a high efficiency cloning technique. Clonal efficiencies ranged from 10 to 31% for intrathyroid, and from 19 to 100% for peripheral T cells. In Graves' disease the phenotypic analysis showed similar percentages of CD3+ CD4+ CD8- and CD3+ CD4- CD8+ clones in thyroid infiltrates and peripheral blood. The functional evaluation showed similar or lower proportions of cytolytic clones in thyroid infiltrates with respect to peripheral blood. Furthermore, the proportions of intrathyroid and peripheral T-cell clones capable of releasing interleukin-2 and/or gamma-interferon in response to mitogen stimulation were similar. Finally, 44% of intrathyroid clones were neither cytolytic nor able to release IL-2 and gamma-interferon. These results are strikingly different from those obtained in Hashimoto's thyroiditis, where the large majority of intrathyroid T-cell clones are cytolytic and the proportions of clones able to release gamma-IFN are remarkably increased in thyroid infiltrates when compared to those obtained from peripheral blood. Taken together, these data suggest a different role for T lymphocytes in the pathogenesis of the two major human autoimmune thyroid diseases.

Adult↗
PubMed1999

Functional analysis of conserved cysteines in heparan sulfate N-deacetylase-N-sulfotransferases.

N-Deacetylase-N-sulfotransferases (NDANST) catalyze the two initial modifications of the polysaccharide precursor in the biosynthesis of heparin and heparan sulfate. These modifications are the gating steps in establishing growth factor protein-binding domains of these glycosaminoglycans. We have undertaken a structure-activity analysis of the 841-amino acid Golgi-luminal portion of the rat liver NDANST to localize the two enzymatic functions. Each activity can be assayed in vitro independently of the other when provided with the appropriate substrate, and N-ethylmaleimide treatment selectively inactivates the deacetylase activity. In this study, dithiothreitol treatment of the rat liver NDANST was shown to inactivate the sulfotransferase function, while stimulating deacetylase activity 2-3-fold over the native protein. Site-directed mutagenesis of the eight cysteine (Cys) residues in the rat liver NDANST that are conserved in the mouse mastocytoma protein produced three important findings regarding the localization of each enzymatic function: 1) derivatization of Cys486 with N-ethylmaleimide resulted in total inactivation of the deacetylase activity based on steric hindrance of the active site (this residue was shown not to be involved in enzymatic catalysis), 2) substitution of either Cys159 or Cys486 with alanine resulted in enhanced activity of the deacetylase to the level obtained by dithiothreitol treatment, and 3) alanine substitution of Cys818 or Cys828 completely inactivated the sulfotransferase activity, while substitution of Cys586 or Cys601 resulted in a 90% loss in activity. These findings suggest that the two enzymatic domains within the NDANST localize to different portions of the protein, with two disulfide pairs toward the COOH-terminal half of the protein necessary for the sulfotransferase activity, and Cys residues within the NH2-terminal half influencing or located near the active site of the deacetylase functionality.

Alanine↗
PubMed2005

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosomes deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1.

A magnetosome deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 transposon mutagenesis, and a 5045-bp fragment flanking mini-Tn5 in NM4 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved six putative open reading frames (ORFs); the mini-Tn5 was inserted into ORF4. Functional complementary test indicated that the 5045-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The protein encoded by ORF4 had 25% of identity with the chemotaxis protein CheYIII of Caulobacter crescentus CB15, and the protein encoded by ORF4 contained a conserved signal receiver domain that can receive the signal from the sensor partner of the bacterial two-component systems. It was suggested that the protein encoded by ORF4 may take part in the signal transduction relating to biosynthesis of magnetosomes.

Amino Acid Sequence↗
PubMed2005

Functional analysis of evolutionary conserved clustering of bZIP binding sites in the baculovirus homologous regions (hrs) suggests a cooperativity between host and viral transcription factors.

The genome of the Autographa californica Multinucleocapsid Polyhedrosis Virus (AcMNPV) contains nine interspersed homologous regions (hrs) that function as potent enhancer sequences when linked in cis to either viral or heterologous RNA polymerase II-dependent promoters. Their activity is strongly increased by the binding of the major immediate early viral transregulator IE1 on 28-mer palindromic sites present in hrs. We show that hrs of AcMNPV additionally carry, in the interpalindromic sequences, a large number of cAMP response elements (CRE) and TPA response elements (TRE), known to bind ubiquitous cellular transcription factors of the bZIP family. Moreover, these clusters of CRE and TRE motifs are concentrated in hrs. Analysis of the 25 baculovirus genomes sequenced so far reveals that these motifs are evolutionary conserved in Lepidoptera NPVs, suggesting a functional role in the hr enhancer function. Consistently, EMSA experiments indicate that CRE and on a lesser extent TRE sites specifically bind insect host factors. Moreover, reporter assays reveal that these CRE sites have an additive stimulatory effect on RNAPol II-dependent transcription in Sf9 cells and are potentially able to synergize with the IE1-binding palindrome.

Allosteric Regulation↗
PubMed2004

Functional analysis of the regulatory regions of the matrilin-1 gene in transgenic mice reveals modular arrangement of tissue-specific control elements.

Matrilin-1 is a non-collagenous protein, which functions in the organization of the extracellular matrix by forming collagen-dependent and -independent filamentous networks. It is secreted primarily by chondrocytes in a characteristic spatial, temporal and developmental stage-specific pattern during skeletogenesis. As a first step to define the tissue- and site-specific regulatory regions of the chicken matrilin-1 gene in vivo, we generated transgenic mice harboring various promoter and intronic fragments fused to the LacZ reporter gene. Histological analysis of the transgene expression pattern during ontogenic development revealed specific X-gal staining in most primordial elements of endochondral bones of transgenic mouse lines carrying either the long promoter between -2011 and +67 or the intronic fragment with a short promoter between -338 and +1819. The cartilage-specific activity of the latter transgene, however, was accompanied with variable ectopic expression pattern in neural and other tissues depending on the site of integration. The presence of both promoter upstream and intronic elements was necessary for the high level transgene activity in all chondrogenic tissues and for the extraskeletal transgene expression pattern resembling the most to that of the chicken matrilin-1 gene, e.g. expression in the eye, and lack of expression in the diminishing notochord and nucleus pulposus. The activity of the transgenes was restricted to the columnar proliferating and pre-hypertrophic chondrocytes visualized by BrdU incorporation and distribution of phosphorylated Sox9, respectively. DNA elements between -2011 and -338 also mediated ectopic LacZ expression in cells of neural crest origin. These results suggest that an interplay of modularly arranged cartilage- and neural crest-specific DNA elements control the expression of the matrilin-1 gene. The dispersal of cartilage-specific elements in the promoter upstream and intronic regions shows similarity to the transcriptional regulation of the Col11a2 gene.

Animals↗
PubMed1985

Average, binge and maximum alcohol intake in healthy young men: discriminant function analysis.

Blood samples and data on alcohol consumption, diet, exercise, smoking and drug use were collected from 89 undergraduate men affiliated with Greek houses. This population has previously been identified as one that consumed large amounts of alcohol among university undergraduates. Energy and nutrient intake from foods, exercise and drug use (except caffeine) were not different between those reporting consuming more than or less than 1 oz of alcohol per day. Multiple discriminant analysis using a panel of 35 blood tests correctly classified all subjects according to their self-reported intake of more than or less than 2 oz of alcohol per day; 96% were classified correctly at a cut point of 1.5 oz per day. Correct classification was 96-99% using a measure of binge consumption (quantity-frequency of heavy drinking). When maximum number of drinks per occasion was used in the discriminant function, 90-92% were correctly placed. Discriminant analysis based on a panel of common blood tests appears to be a promising technique to identify young drinkers who consume high amounts of alcohol.

Adolescent↗
PubMed2001

Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification.

The development of efficient DNA sequencing methods has led to the achievement of the DNA sequence of entire genomes from (to date) 55 prokaryotes, 5 eukaryotic organisms and 10 eukaryotic chromosomes. Thus, an enormous amount of DNA sequence data is available and even more will be forthcoming in the near future. Analysis of this overwhelming amount of data requires bioinformatic tools in order to identify genes that encode functional proteins or RNA. This is an important task, considering that even in the well-studied Escherichia coli more than 30% of the identified open reading frames are hypothetical genes. Future challenges of genome sequence analysis will include the understanding of gene regulation and metabolic pathway reconstruction including DNA chip technology, which holds tremendous potential for biomedicine and the biotechnological production of valuable compounds. The overwhelming volume of information often confuses scientists. This review intends to provide a guide to choosing the most efficient way to analyze a new sequence or to collect information on a gene or protein of interest by applying current publicly available databases and Web services. Recently developed tools that allow functional assignment of genes, mainly based on sequence similarity of the deduced amino acid sequence, using the currently available and increasing biological databases will be discussed.

Computational Biology↗
PubMed1994

Functional analysis of mouse Hoxa-7 in Saccharomyces cerevisiae: sequences outside the homeodomain base contact zone influence binding and activation.

The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae. The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems. A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets. The HD of antennapedia is identical to the Hoxa-7 HD. The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied. Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site. We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone). This result is discussed in terms of DNA flexure and two modes of transcription used in S. cerevisiae.

Animals↗
PubMed1989

Expression of calcium-dependent cell adhesion during ocular development: a biochemical, histochemical and functional analysis.

Previous studies of the adhesive properties of embryonic chick neural retina cells indicate a gradual decrease in the expression of calcium-dependent adhesions during retinal histogenesis, a function which has been attributed in part to gp130/4.8, a retinal calcium-dependent adhesion-associated cell surface membrane glycoprotein with a molecular weight of approximately 130 kDa and an isoelectric point of 4.8 (G. B. Grunwald, R. Pratt, and J. Lilien, 1982, J. Cell Sci. 55, 69-83). The experiments described here were done to define the relationship of gp130/4.8 to N-cadherin, another calcium-dependent adhesion molecule found in chick retina, which has a reported molecular weight of 127 kDa and which is recognized by monoclonal antibody NCD-2 (K. Hatta and M. Takeichi, 1986, Nature (London) 320, 447-449). Using two-dimensional gel electrophoresis followed by Western blotting as well as quantitative solid-phase immunoassays, polyspecific antisera recognizing gp130/4.8 were compared with monoclonal antibody NCD-2 for reactivity with proteins of retina and other tissues. The data lead us to conclude that retinal calcium-dependent adhesion proteins gp130/4.8 and N-cadherin are likely to be the same molecule. In order to obtain evidence for a direct correlation of changes in expression of these adhesion proteins with changes in retinal cell adhesivity and related morphogenetic events, parallel studies were carried out with cells from various ocular tissues to examine the functional, biochemical, and immunohistochemical expression of N-cadherin during ocular development. Immunohistochemical mapping of N-cadherin in the developing chick eye reveals three modes of N-cadherin expression which occur simultaneously in different ocular tissues: (1) down-regulation, (2) up-regulation, and (3) steady-state expression. These patterns of expression correlate with changes in the adhesive behavior of cells as well as with discrete stages in the morphogenesis of several ocular tissues. The results suggest that N-cadherin is a versatile cell adhesion protein with a role in both the development of several ocular tissues and the maintenance of specialized structures in the mature eye.

Age Factors↗
PubMed2005

Identification and functional analysis of TopBP1 and its homologs.

The multiple BRCT-domain protein TopBP1 and its yeast homologs have been implicated in many aspects of DNA metabolism, but their molecular functions remain elusive. In this review, we first summarise how the yeast homologs were identified and characterised. We next review the data available from metazoan systems and finally draw parallels with the yeast models. TopBP1 plays important functions in the initiation of DNA replication in all organisms and participates in checkpoint responses both within S phase and following DNA damage. In metazoan systems there is accumulating evidence for additional roles in transcriptional regulation that have not been reported in yeast. Overall, TopBP1 appears to play a key role in integrating different aspects of DNA metabolism, but the mechanistic basis for this remains to be fully explained.

Animals↗
PubMed2003

Functional analysis of the structural basis of homophilic cadherin adhesion.

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.

Binding Sites↗
PubMed1997

Functional analysis of the yeast genome.

The release of the complete genome sequence of the yeast Saccharomyces cerevisiae has ushered in a new phase of genome research in which sequence function will be assigned. The goal is to determine the biological function of each of the > 6,000 open reading frames in the yeast genome. Innovative approaches have been developed that exploit the sequence data and yield information about gene expression levels, protein levels, subcellular localization and gene function for the entire genome.

Fungal Proteins↗
PubMed1993

Human 92 kDa type IV collagenase: functional analysis of fibronectin and carboxyl-end domains.

Two closely related secreted metalloproteases 72 and 92 kDa type IV collagenases (72- and 92T4Cl) consist of several structural domains, the functions of which are poorly understood. Both metalloproteases can bind to gelatin as well as form complexes with specific inhibitors in the proenzyme form. The biologic role of the proenzyme-inhibitor complex formation remained unclear. Here we summarize results demonstrating that the fibronectin-like domain of 92T4Cl mediates gelatin binding of the proenzyme, while the hemopexin like carboxy-terminal domain is essential for the complex formation of the proenzyme with TIMP. The formation of a 92T4Cl proenzyme complex with TIMP prevents dimerization, formation of the novel complex with ClI proenzyme, and activation of the 92T4Cl by stromelysin. Conversely, formation of the covalent 92T4Cl homodimer excludes the formation of a proenzyme-TIMP complex, thus allowing this form of enzyme to enter into the proteolytic cascade of activation. Both components of the 92T4Cl-ClI complex can be activated in a fashion similar to that of free enzymes, yielding a complex active against both gelatin and fibrillar collagen.

Amino Acid Sequence↗
PubMed2004

Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca(2+) release and entry in DT40 B lymphocytes.

We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.

Animals↗
PubMed1997

Mismatch repair in Schizosaccharomyces pombe requires the mutL homologous gene pms1: molecular cloning and functional analysis.

Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic "PMS" genes than to the "MLH" genes, we have designated the S. pombe homologue pms1. Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from > 92% to 80% associated with a low proportion (approximately 50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination.

Adenosine Triphosphatases↗
PubMed2003

The use of functional analysis in evaluating knee kinematics.

The importance of understanding the six-degrees-of-freedom kinematics of the knee during ambulatory activities was examined in the context of the function of total knee arthroplasty. Studies of knee kinematics during walking, stair climbing, and a deep flexion squat indicate that knee kinematics is activity-dependent. A comparative study of patients and healthy subjects during stair climbing indicates the importance of maintaining the function of the posterior cruciate ligament. A second study used walking kinematics derived from patient testing as input to a wear simulator. There was increased wear relative to standard simulator input that was related to the slip velocity at the contact surface. Finally, results from a study of deep flexion indicate that substantial femoral rotation is required during deep flexion activities. The current study shows the importance of studying in vivo knee kinematics for future enhancement in the treatment of the arthritic knee.

Adult↗
PubMed2005

Structural and functional analysis of the interaction of the AAA-peroxins Pex1p and Pex6p.

The AAA-peroxins Pex1p and Pex6p play a critical role in peroxisome biogenesis but their precise function remains to be established. These two peroxins consist of three distinct regions (N, D1, D2), two of which (D1, D2) contain a conserved approximately 230 amino acid cassette, which is common to all ATPases associated with various cellular activities (AAA). Here we show that Pex1p and Pex6p from Saccharomyces cerevisiae do interact in vivo. We assigned their corresponding binding sites and elucidated the importance of ATP-binding and -hydrolysis of Pex1p and Pex6p for their interaction. We show that the interaction of Pex1p and Pex6p involves their first AAA-cassettes and demonstrate that ATP-binding but not ATP-hydrolysis in the second AAA-cassette (D2) of Pex1p is required for the Pex1p-Pex6p interaction. Furthermore, we could prove that the second AAA-cassettes (D2) of both Pex1p and Pex6p were essential for peroxisomal biogenesis and thus probably comprise the overall activity of the proteins.

ATPases Associated with Diverse Cellular Activitie↗