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Results for “Functional characterization”

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PubMed2001

Identification and functional characterization of thromboxane A2 receptors in Schwann cells.

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1989

Structural and functional characterization of human immunodeficiency virus tat protein.

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.

Amino Acid Sequence↗

PubMed1984

[Chemical and functional characterization of lymphokines].

Macrophages are heterogeneous with respect to a number of constitutive and inducible functions. In order to study the underlying biological principle, a bone marrow liquid culture system was adopted in which bone marrow cells proliferate and differentiate into macrophages. It was found that maturing macrophages express various constitutive or inducible functions in an ordered sequence. The kinetics of their appearance and disappearance are dependent on the proliferative activity of macrophages. Macrophages in "late" G-1 of the cell cycle express constitutive functions like plasminogen activator production and are inducible by bacterial lipopolysaccharides, Poly I:C and lymphokines to release interferons. The response to lymphokines like migration inhibitory factor (MIF) and chemotactic factors is also transiently expressed during maturation. Using purified MIF, its influence on proliferation, differentiation and activation of macrophages was investigated. The changes induced were monitored following the expression of marker enzymes and of phenotype associated cell surface antigens using monoclonal antibodies. The results showed that functional changes induced by MIF on macrophages are limited and are not related to certain macrophage activating activities (MAF). As determined by flow cytofluorometry, transglutaminase expression and proliferation is consistently down-regulated by MIFs. This together with the shift and the expression of surface antigens indicates that MIFs provide a differentiation signal for a "young" macrophage to become more mature.

Animals↗

PubMed2005

Genetic analysis and functional characterization of polymorphisms in the human concentrative nucleoside transporter, CNT2.

The concentrative nucleoside transporter CNT2 (SPNT1; SLC28A2) plays a role in the absorption and disposition of naturally occurring nucleosides, as well as nucleoside analog drugs. The aim of the present study was to characterize genetic variation in SLC28A2, the gene encoding CNT2, and to functionally analyse non-synonymous variants of CNT2, as a first step towards understanding whether genetic variation in this nucleoside transporter contributes to variation in response to nucleoside analogs. As part of a larger study, DNA samples from an ethnically diverse population (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans and seven Pacific Islanders) were screened and 10 coding region variants of CNT2 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. Six non-synonymous variants were identified, and all were able to transport guanosine. The four common variants (>1% in the sample population) were further characterized with the anti-viral nucleoside analog drug ribavirin. No differences were observed among the four common variants in the uptake kinetics of 3H-ribavirin (Km in microM: 35.6+/-9.27 for CNT2-reference, 40.7+/-6.47 for CNT2-P22L, 31.2+/-15.8 for CNT2-S75R, 26.7+/-6.13 for CNT2-S245T and 49.9+/-14.6 for CNT2-F355S). The variant CNT2-F355S exhibited a change in specificity for the naturally occurring nucleosides, inosine and uridine. All non-synonymous variants of CNT2 took up guanosine, and the four variants examined showed no significant difference in ribavirin kinetics. However, CNT2-F355S (3% allele frequency in the African-American sample) was found to alter specificity for naturally occurring nucleosides, which may have implications for nucleoside homeostasis.

Alleles↗

PubMed1996

Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization.

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.

Animals↗

PubMed2002

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Protein Biosynthesis↗

PubMed1998

Functional characterization of Dictyostelium discoideum mutant myosins equivalent to human familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in beta-myosin heavy chain or other various sarcomeric proteins. To elucidate the functional impact of FHC mutations in myosin heavy chain, we generated Dictyostelium discoideum myosin II mutants equivalent to human FHC mutations by site-directed mutagenesis, and characterized their molecular-basis motor function. The current mutants, i.e. R397Q, F506C, G575R, A699R, K703Q and K703W are equivalent to R403Q, F513C, G584R, G716R, R719Q and R719W FHC mutants respectively. We measured the molecular-basis force and the sliding velocity generated by these myosin mutants. The measurement revealed that the A699R, K703Q and K703W myosins exhibited the lowest level of force with their preserved actin-activated MgATPase activity. F506C mutant showed the least impairment of the motile and enzymatic activities. The motor function of R397Q and G575R myosins were classified as intermediate. These results suggest that ELC binding domain might be important for force production.

Amino Acid Sequence↗

PubMed2003

Biochemical and functional characterization of the Ebola virus VP24 protein: implications for a role in virus assembly and budding.

The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding.

Amino Acid Sequence↗

PubMed1997

Phenotypic and functional characterization of committed and primitive myeloid and lymphoid hematopoietic precursors in human fetal liver.

We studied the phenotypic and functional properties of colony-forming cells (CFCs), primitive long-term culture initiating cells (LTC-ICs) and lymphoid precursors present in human fetal liver (FL) and compared these with their adult bone marrow (BM) counterparts. FL (7-14-week) cells were selected by fluorescence-activated cell sorting based on increasing CD34 antigen expression (34+, 34++, or 34 ), CD38 antigen expression (CD34++/ CD38+, or CD38-), and HLA-DR antigen expression (CD34++/ HLA-DR+ or HLA-DR-). 13 +/- 0.6% of FL CD34-positive cells were 34 . Significantly more FL CD34++/ cells were CD38- (49 +/- 2.4%) and HLA-DR-(72 +/- 6.7%) than BM CD34++ cells (6.8 +/- 0.7% CD38- and 13.3 +/- 3.2% HLA-DR-). FL and BM CFCs were CD34+/++, CD38+, and HLA-DR+. However, significantly more FL CFCs were erythroid (40%) than adult BM CFCs (15%), and FL colonies were larger (8111 +/- 738 cells/CFC) than BM colonies (3466 +/- 272 cells/CFC, p < 0.001). As is seen in adult BM, FL LTC-ICs were CD34++/ CD38-. In contrast to BM LTC-ICs, FL LTC-ICs were almost exclusively CD34++/ HLA-DR+. In addition, a single FL LTC-IC gave rise to >30 CFCs at 5 weeks compared with only 5 +/- 0.9 CFCs per LTC-IC from BM. Finally, we demonstrate that the FL CD34++/ /CD38-/HLA-DR+ population, which contains 3.7% LTC-ICs, also contains primitive lymphoid progenitors capable of differentiating into natural killer (NK) cells. In conclusion, the phenotype of primitive human FL progenitors such as LTC-IC and primitive NK progenitors is CD34++/ /CD38-/HLA-DR+, suggesting that this population may contain FL hematopoietic stem cells. The phenotypic characterization of FL primitive LTC-ICs and NK progenitors will facilitate further studies of the functional properties of these progenitors.

ADP-ribosyl Cyclase↗

PubMed1989

Development and functional characterization of T cell lines from canine Peyer's patches.

We have propagated concanavalin A-stimulated cells from canine Peyer's patches in vitro in the presence of interleukin-2 (IL-2). The cells were characterized as T cells by determination of their phenotype and by functional assays. They are IL-2 dependent and respond to IL-2 of murine, primate and canine origin. The long-term cultured cells provided help for immunoglobulin production by purified autologous B cells and suppressed IgG production by nonseparated autologous peripheral blood mononuclear cells.

Animals↗

PubMed2000

Cloning and functional characterization of the 5'-regulatory region of the human CD86 gene.

The induction of CD86 expression by IFN-gamma on the surface of various antigen presenting cells has been previously reported. In order to understand the mechanisms by which the expression of the CD86 gene is regulated by IFN-gamma at the transcriptional level, we have cloned and characterized the 5'-flanking region of the human CD86 gene. To functionally analyze the upstream regulatory region of the CD86 gene, a series of luciferase reporter gene constructs were prepared and used for transfection of cells from the monocytic line U937 and Raji B cell line. Under basal conditions, functional activity of these constructs was detected in Raji cells, which show high constitutive expression of the CD86 molecule, but not in U937 cells, which show low expression of CD86 in non-activated state. Induction of CD86 expression by stimulation of U937 cells with IFN-gamma revealed the presence of two functional GAS (gamma-interferon activation site) elements. Gel mobility shift assays showed that these two GAS elements specifically bind an IFN-gamma-induced transcriptional complex. The DNA-protein complex was supershifted by antibody to Stat1 alpha (signal transducer and activator of transcription), but not by antibodies to Stat 2, Stat 3 and Sp1, indicating that GAS elements interact with Stat1 alpha. Point mutations in the GAS elements prevented the formation of DNA-protein complex and significantly reduced the responsiveness of the reporter gene to IFN-gamma. These findings suggest that two functional GAS elements within the human CD86 promoter play an important role in the induction of CD86 gene by binding to IFN-gamma-induced Stat1 alpha.

Antigens, CD↗

PubMed1991

Functional characterization of an abnormal factor XII molecule (F XII Bern).

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

Adsorption↗

PubMed1998

Molecular cloning and functional characterization of murine sphingosine kinase.

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

3T3 Cells↗

PubMed1989

Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor gamma/delta.

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) gamma/delta were analyzed. Cell preparations highly enriched for TcR gamma/delta+ cells were obtained by treatment of E rosette-forming lymphocytes with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64-82% TcR gamma/delta+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR gamma/delta+ cell subsets in the peripheral blood. TcR gamma/delta cells were able to form conjugates with the natural killer-sensitive K-562 and with the natural killer-resistant HL-60-R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%-98% of the cells in the TcR gamma/delta+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR gamma/delta+ cells had electron-dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3- or CD3+ granular lymphocyte subsets. Freshly isolated TcR gamma/delta+ cells were unable to lyse K-562 and natural killer-resistant targets, such as HL-60-R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti-CD3 TcR mAb or by lectin. After short-term culture with interleukin 2, TcR gamma/delta+ cells acquired a strong cytolytic activity against K-562 and HL-60-R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti-CD3/TcR mAb.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1995

Functional characterization of the promoter region of the human phospholipid transfer protein gene.

We have identified the functional promoter region of the human phospholipid transfer protein gene. Primer extension analysis indicates multiple sites for transcription initiation. Sequence analysis reveals that the promoter consists of TATA box, high GC region, and several consensus sequences for the potential binding of transcription factors. To assay promoter activity, DNA fragments with various lengths of the 5'-flanking region were fused upstream to the luciferase gene and transfected into HepG2, COS-7, and CHO cells. A minimal promoter of 159 base pairs between -230 and -72 relative to the first transcriptional initiation site is responsible for the full activity. Two motifs, Sp1 and AP-2, are located within this area. It may suggest that the PLTP promoter activity relies primarily on the putative cis-elements in the functional region.

Base Sequence↗

PubMed1996

Synthesis and evaluation of potential N pi and N sigma metal chelation sites within the beta-hydroxy-L-histidine subunit of bleomycin A2: functional characterization of imidazole N pi metal complexation.

The synthesis and evaluation of 4 and 5, fully functionalized deglycobleomycin A2 (2) analogues incorporating an oxazole and a pyrrole in place of the beta-hydroxy-L-histidine imidazole, are detailed. The oxazole agent is only capable of Npi metal complexation through a form related to the N1-H imidazole tautomer of bleomycin A2 (1) while the pyrrole agent may potentially mimic the Nsigma metal complexation capabilities of the imidazole N3-H tautomer. Metal complexes (Fe-II, Fe-III) of 4 and 5 were found to cleave duplex DNA in the presence of O2 (Fe-II) or H2O2 (Fe-III). The oxazole agent 4 which is incapable of Nsigma metal chelation was found to behave analogous to, albeit slightly less effectively than, deglycobleomycin A2 resulting in the characteristic 5'-GC/5'-GT sequence selective cleavage of duplex DNA directly confirming that imidazole/oxazole Npi metal chelation is sufficient for functional reactivity. Importantly, the effective substitution of the oxazole O-1 for the histidine N-1 further illustrates that this group does not require deprotonation upon metal complexation, oxygen activation, or the ensuing oxidation reactions, that the functional bleomycin A2 tautomer is the imidazole N'-H tautomer, and that the imidazole N'-H functionality is not contributing to the polynucleotide recognition through H-bonding to the phosphate backbone or nucleotide bases. In contrast, the pyrrole agent 5 which is incapable of Npi metal chelation, but possesses the capabilities of functioning as a Nsigma metal donor was also found to cleave duplex DNA, but does so in a nonsequence selective fashion with a significantly reduced efficiency and a diminished double to single strand cleavage ratio both only slightly above that of background iron itself. These observations are analogous to those made with 3 which lacks the imidazole altogether and further support the observations that Nsigma coordination, not Npi coordination, of the imidazole is required for the functional activity of bleomycin A2.

Antimetabolites, Antineoplastic↗

PubMed2001

Attention response functions: characterizing brain areas using fMRI activation during parametric variations of attentional load.

We derived attention response functions for different cortical areas by plotting neural activity (measured by fMRI) as a function of attentional load in a visual tracking task. In many parietal and frontal cortical areas, activation increased with load over the entire range of loads tested, suggesting that these areas are directly involved in attentional processes. However, in other areas (FEF and parietal area 7), strong activation was observed even at the lowest attentional load (compared to a passive baseline using identical stimuli), but little or no additional activation was seen with increasing load. These latter areas appear to play a different role, perhaps supporting task-relevant functions that do not vary with load, such as the suppression of eye movements.

Adult↗

PubMed2000

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica).

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.

Amino Acid Sequence↗