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At least 865 records · Page 48Linked to original sources
PubMed1998

Expression and functional analysis of a hemopoietic progenitor antigen, NJ-1 (114/A10), in the megakaryocytic lineage.

To analyze the functions of molecules expressed in hemopoietic progenitor cells, we obtained several monoclonal antibodies by immunizing Wistar rats with antigens from a murine immature leukemic cell line, DA-1. Here, we characterize one antibody designated NJ-1, which recognizes a 145-kd molecule, and identify the cDNA encoding the NJ-1 antigen by retrovirus-mediated expression cloning. Sequence analysis of the cDNA reveals that it is identical to a previously reported cDNA encoding a surface molecule of 573 amino acids recognized by monoclonal antibody 114/A10. Our studies show that expression of NJ-1 antigen is upregulated in a murine megakaryoblastic cell line, L8057, when it differentiates into a megakaryocytic lineage in response to 12-O-tetradecanoyl phorbol-13-acetate. Overexpression of NJ-1 antigen in L8057 cells inhibits cell adhesion to fibronectin, suggesting that it may act as a negative regulator of cell adhesion in the megakaryocytic lineage.

Animals↗
PubMed2006

SCORPION2: a database for structure-function analysis of scorpion toxins.

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/.

Animals↗
PubMed1987

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity.

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.

Amino Acid Sequence↗
PubMed2005

Structure-based functional analysis reveals a role for the SM protein Sly1p in retrograde transport to the endoplasmic reticulum.

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.

DNA Mutational Analysis↗
PubMed2002

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function.

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.

Amino Acid Sequence↗
PubMed1994

Toward a functional analysis of self-injury.

This study describes the use of an operant methodology to assess functional relationships between self-injury and specific environmental events. The self-injurious behaviors of nine developmentally disabled subjects were observed during periods of brief, repeated exposure to a series of analogue conditions. Each condition differed along one or more of the following dimensions: (1) play materials (present vs absent), (2) experimenter demands (high vs low), and (3) social attention (absent vs noncontingent vs contingent). Results showed a great deal of both between and within-subject variability. However, in six of the nine subjects, higher levels of self-injury were consistently associated with a specific stimulus condition, suggesting that within-subject variability was a function of distinct features of the social and/or physical environment. These data are discussed in light of previously suggested hypotheses for the motivation of self-injury, with particular emphasis on their implications for the selection of suitable treatments.

Adolescent↗
PubMedDate not supplied

Upper extremity tendon transfers in cerebral palsy: electromyographic and functional analysis.

This study was undertaken to evaluate tendon transfers in a group of children with spastic hemiplegia. Muscle grading, video recordings of function, and electromyographs were performed on 12 patients preoperatively. Postoperative electromyograms were obtained on 16 transferred tendons in eight patients as the children performed assigned tasks. Clinical evaluation demonstrated improvement in all patients at follow-up, which averaged 20 months. Four of the 16 muscles showed changes in their electrical activity postoperatively. Three muscles that fired continuously became phasic, and one muscle changed phase to match its recipient. Pre- and postoperative electromyography with video analysis provided a more complete understanding of the characteristics of tendon function with tendon transfers in cerebral palsy.

Adolescent↗
PubMed2001

Structure-function analysis of CYP27B1 and CYP27A1. Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX).

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1999

Hunting with traps: genome-wide strategies for gene discovery and functional analysis.

With sequence analysis of the human genome well underway, there is an increasingly urgent challenge to understand the fundamental function and interplay of genes that build and maintain an organism. Several approaches will be critical for interpreting gene function, including random cDNA sequencing, expression profiling in different tissues, genetic analysis of human or model organism phenotypes, and creation of transgenic or "knockout" animals. Traditional gene-trapping approaches, in which genes are randomly disrupted with DNA elements inserted throughout the genome, have been used to generate large numbers of mutant organisms for genetic analysis. Recent modifications of gene-trapping methods and their increased use in mammalian systems are likely to result in a wealth of new information on gene function. Various trapping strategies allow genes to be segregated based on criteria like the specific subcellular location of an encoded protein, the tissue expression profile, or responsiveness to specific stimuli. Genome-wide gene-trapping strategies, which integrate gene discovery and expression profiling, can be applied in a massively parallel format to produce living assays for drug discovery.

Genes↗
PubMed2001

Construction of an Escherichia coli knockout strain for functional analysis of tRNA(Asp).

The specific aminoacylation of tRNA is critical for translation of the genetic code. A molecular description of aminoacylation requires knowledge of the relevant three-dimensional structures, biochemical parameters and the structure-function relationship of the synthetase and its substrate tRNA. Extensive structural and biochemical data are available on the aspartic acid system of Escherichia coli, but there is a paucity of cellular functional data. We have developed a system to overcome this deficiency by engineering an E. coli knockout tRNA(Asp) strain, thereby allowing a penetrating analysis of tRNA(Asp) structure and function under conditions that prevail in the cell.

Acylation↗
PubMed2001

Phenotypical and functional analysis of T cells in periodontitis.

To explore aspects of cellular immune responses in the pathogenesis of periodontitis we analyzed phenotype and function of peripheral T cells. Two groups of subjects participated: one group consisted of 10 highly susceptible patients with severe periodontitis (mean age 29 years) and a control group consisted of 10 age, gender and race matched subjects with gingivitis. From all subjects peripheral blood was collected. The results showed that the numbers of CD3+, CD4+ and CD8+ T cells as well as the CD4/CD8 ratio, and the proliferative capacity of T cells, were not different between the two groups of subjects. Also, proportions of naive and memory T cells for both the CD4+ and CD8+ subpopulations were not different. Functional heterogeneity within the CD4+ and CD8+ T cell compartments was determined by intracellular analysis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production. On the basis of these latter analyses among CD4+ and CD8+ cells, T helper (Th) 1 or Th2 function and T cytotoxic (Tc) 1 or Tc2 function, respectively, could be deduced. No significant differences in proportions of CD4+ and CD8+ T cells positive for intracellular IFN-gamma or IL-4 were observed between periodontitis patients and gingivitis controls; however a higher level of intracellular IL-4 in CD8+ T cells was seen in periodontitis patients. This might indicate that there is a shift towards a Tc2 function within the CD8+ T cell subpopulation. The current explorative study suggests that further research into the role of CD8+ T cells in the pathogenesis of periodontitis is warranted.

Adult↗
PubMed1991

Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

Amino Acid Sequence↗
PubMed2004

Computational approaches to structural and functional analysis of plastocyanin and other blue copper proteins.

Computational techniques are becoming increasingly important in structural and functional biology, in particular as tools to aid the interpretation of experimental results and the design of new systems. This review reports on recent studies employing a variety of computational approaches to unravel the microscopic details of the structure-function relationships in plastocyanin and other proteins belonging to the blue copper superfamily. Aspects covered include protein recognition, electron transfer and protein-solvent interaction properties of the blue copper protein family. The relevance of integrating diverse computational approaches to address the analysis of a complex protein system, such as a cupredoxin metalloprotein, is emphasized.

Algorithms↗
PubMed2000

Human thyroid-stimulating hormone: structure-function analysis.

This article provides the reader with an overview of methodological strategies to investigate structure-function relationships of human thyroid-stimulating hormone (hTSH). Various aspects of hTSH production, purification, and characterization described here in more detail are not only relevant to studies on other members of the glycoprotein hormone family, but also applicable to studies of other glycosylated proteins. Knowledge of structure-function relationships of specific hTSH domains is important for a better understanding of the molecular mechanisms of its action. New insights from such studies permit the design of glycoprotein hormone analogs with specific pharmacological properties and potential clinical applications.

Animals↗
PubMed2001

Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus.

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.

Active Transport, Cell Nucleus↗