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Results for “Functional characterization”

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PubMed2004

Functional characterization of beta1-integrin-positive epidermal cell populations.

Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide. We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ. Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments. Image analysis was used to determine growth characteristics of the colonies. We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation. The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation. Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different. Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Adult↗

PubMed1996

Subepithelial B cells in the human palatine tonsil. II. Functional characterization.

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike FM B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-mu antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-mu-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-mu antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.

Animals↗

PubMed2004

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector.

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.

Actins↗

PubMed2002

Functional characterization of Delta3,Delta2-enoyl-CoA isomerases from rat liver.

The degradation of unsaturated fatty acids by beta-oxidation involves Delta(3),Delta(2)-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of enoyl-CoAs and the 2,5 --> 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis --> 2-trans isomerization; ECI has a preference for the 3-trans --> 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 --> 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis --> 2-trans and 2,5 --> 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans --> 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 --> 3,5 isomerization.

Animals↗

PubMed2005

Identification, subviral localization, and functional characterization of the pseudorabies virus UL17 protein.

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-DeltaUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-DeltaUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.

Animals↗

PubMed2003

Identification and functional characterization of a Na+-independent large neutral amino acid transporter, LAT1, in human and rabbit cornea.

PURPOSE: The objective of this research was to investigate the presence of an Na(+)-independent, large neutral amino acid transporter, LAT1, on rabbit corneal epithelium and human cornea. METHODS: Freshly excised rabbit corneas were used for transport studies and SIRC (a rabbit corneal cell line) cells for uptake studies. Transport and uptake characteristics of [(3)H]-L-phenylalanine were determined at various concentrations and pH. Inhibition studies were conducted in the presence of other L- and D-amino acids and metabolic inhibitors, such as ouabain and sodium azide, and in the absence of sodium to delineate the mechanism of uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) for large neutral amino acid transporter-1 (LAT1) was performed on total RNA from rabbit cornea, SIRC cells, and human cornea. RESULTS: SIRC uptake of L-Phe was found to be saturable, with K(m) of 73 +/- 9 microM, V(max) of 2.0 +/- 0.1 nanomoles/min per milligram protein, and K(d) of 0.44 +/- 0.6 microL/min per milligram protein. Uptake was independent of pH, energy, and Na(+); inhibited by D-Leu, D-Phe, and an L-system-specific inhibitor 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), but not inhibited by L-Ala and charged amino acids. Transport of L-Phe across rabbit cornea was also saturable (K(m) = 33 +/- 8 microM and V(max) = 0.26 +/- 0.03 nanomoles/min per square centimeter), energy independent, and subject to similar competitive inhibition. LAT1 was identified by RT-PCR in rabbit corneal, SIRC, and human corneal RNA. CONCLUSIONS: A Na(+)-independent, facilitative transport system, LAT1, was identified and functionally characterized on rabbit cornea. LAT1 was also identified on human cornea.

Amino Acids, Cyclic↗

PubMed1998

Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p.

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

Animals↗

PubMedDate not supplied

Functional characterization of sodium-dependent multivitamin transporter in MDCK-MDR1 cells and its utilization as a target for drug delivery.

The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing the MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [3H]biotin uptake and transport was delineated. Transepithelial permeability of the biotin-conjugated prodrug, i.e., biotin-saquinavir, was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the existence of SMVT in MDCK-MDR1 cells. Biotin uptake was Na+, pH, and temperature dependent, but energy independent. Uptake of biotin was found to be saturable with a Km of 13.0 microM, Vmax 21.5 of pmol min-1 (mg of protein)-1, and Kd of 0.12 microL min-1 (mg of protein)-1. Both apical and basal uptake and transepithelial transport of [3H]biotin showed that SMVT localized predominantly on the apical membrane of MDCK-MDR1 cells. [3H]Biotin uptake was inhibited by excess unlabeled biotin and its structural analogues, i.e., desthiolbiotin and valeric acid, and other vitamins such as lipoic acid and pantothenic acid, but not by acetic acid, benzoic acid, biotin methyl ester, and biocytin. Biotin-saquinavir caused lowering of [3H]biotin uptake, which indicates that it is recognized by SMVT. Apical to basal transport of [3H]biotin was also significantly inhibited in the presence of excess biotin or biotin-saquinavir. Transepithelial transport studies of biotin-saquinavir in MDCK-MDR1, wild type MDCK, and Caco-2 cells revealed that permeability of biotin-saquinavir was similar in all three cell lines. A band of SMVT mRNA at 862 bp was identified by RT-PCR. A sodium-dependent multivitamin transporter, SMVT, responsible for biotin uptake and transport, was identified and functionally characterized in MDCK-MDR1 cells. Therefore, the MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of biotin-conjugated prodrugs such as HIV protease inhibitors.

Animals↗

PubMed1992

Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts.

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3+ phenotype and the alpha/beta T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56+. The evaluation of CD45 isoforms showed that both "naive" and "memory" cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.

Adult↗

PubMed1999

Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate.

The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.

Acetonitriles↗

PubMed2003

Molecular cloning, developmental expression, promoter analysis and functional characterization of the mouse CNBP gene.

Striking conservation in various organisms suggests that cellular nucleic acid-binding protein (CNBP) plays a fundamental biological role across different species. However, the regulated expression and physiological properties of the CNBP gene are unknown. In this study, we report the molecular cloning, promoter characterization, developmental expression and functional analysis of the mouse CNBP gene. The gene contains five exons and is localized to chromosome 6 in the region corresponding to band 6 D1-D2. Primer extension assay indicates that the transcription start site is located 230 bp upstream of the initiator Met codon. Our promoter analysis indicates that strong transcription enhancer and silencer regions lie within the 1.6 kb proximal region of the promoter and the upstream -3.0 to -1.6 kb region, respectively. The promoter activity is 10 fold higher in embryonic carcinoma cells than that in fibroblast, as determined by CAT assay. Consistent with its function as a transcription factor, CNBP protein is located in the nucleus of cells. During mouse embryogenesis, CNBP is expressed in the anterior region of the early embryo and in the limb, tail and craniofacial region. Overexpression of CNBP strongly stimulates cell proliferation and increases c-myc promoter activity. Our finds suggest that CNBP may play an important role in cell proliferation and tissue patterning during anterior-posterior axis, craniofacial and limb development by targeting c-Myc.

Animals↗

PubMed2002

Morphological and functional characterization of a rat vaginal smooth muscle sphincter.

The aim of the present study was to gain information about adrenergic-, cholinergic- and non-adrenergic, non-cholinergic (NANC)- transmitter systems/mediators in the rat vagina, and to characterize its smooth muscles functionally. Tissue sections from vagina of Sprague Dawley rats were immunolabelled with antibodies against protein gene product 9.5 (PGP), synaptophysin (Syn), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Circularly cut vaginal smooth muscle preparations from the distal vagina were studied in organ baths. In the paravaginal tissue, a large number of PGP-, NOS-, TH-, VIP-immunoreactive (IR) and few CGRP-IR nerve trunks were observed, giving off branches to the smooth muscle wall. The smooth muscle wall was supplied by a large number of PGP-, Syn-, VAChT-, NPY-, NOS- and TH- IR nerve terminals, whilst only a moderate to few numbers of CGRP-, VIP- and PACAP-IR terminals were identified. Especially the distal part of the vaginal wall, where the circularly running smooth muscle was thickened into a distinct sphincter structure, was very richly innervated, predominantly by PGP- and NOS-IR terminals. Below and within the basal parts of the epithelium in the distal half of the vagina, a large number of PGP- and few NOS- and PACAP-IR varicose terminals were observed. The vaginal arteries were encircled by plexuses of nerve terminals. A large number of these were PGP-, Syn-, VAChT-, NOS-, TH-, NPY- and VIP-IR, and few were CGRP- and PACAP-IR. In isolated preparations of the distal vagina, electrical field stimulation (EFS) caused frequency-dependent contractions, which were reduced by sildenafil, tetrodotoxin (TTX) and phentolamine. In preparations contracted by norepinephrine (NA), EFS produced frequency-dependent relaxations. Pretreatment with the NOS-inhibitor N(G)-nitro-L-arginine, TTX, or the inhibitor of soluble guanylate cyclase, ODQ, abolished the EFS relaxations. In NE precontracted preparations, cumulative addition of sildenafil caused concentration-dependent relaxation. Carbachol contracted the strips concentration-dependently from baseline. It can be concluded that the distal part of the rat vagina forms a distinct smooth muscle sphincter, which is richly innervated by adrenergic, cholinergic and NANC nerves. The present studies suggest that in the rat the L-arginine/NO-system not only plays an important role in the regulation of vaginal smooth muscle tone, but also affects blood flow, and may have sensory functions.

Acetylcholinesterase↗

PubMed2005

Structural and functional characterization of transmembrane segment IV of the NHE1 isoform of the Na+/H+ exchanger.

The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe(161) --> Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD(3)OH:CDCl(3):H(2)O mixture and in Me(2)SO. In CD(3)OH: CDCl(3):H(2)O, TM IV was structured but not as a canonical alpha-helix. Residues Asp(159)-Leu(162) were a series of beta-turns; residues Leu(165)-Pro(168) showed an extended structure, and residues Ile(169)-Phe(176) were helical in character. These three structured regions rotated quite freely with respect to the others. In Me(2)SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe(161) is a pore-lining residue.

Cation Transport Proteins↗

PubMed2006

Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.

Adenylate Cyclase Toxin↗

PubMed1995

Structural and functional characterization of the promoter regions of the NFKB2 gene.

In order to clarify the transcriptional regulation of the NFKB2 gene (lyt-10, NF-kappa Bp100), we have characterized the structure and function of its promoter regions. Based on the nucleotide sequence of cDNA clones and the 5' flanking genomic region of the NFKB2 gene, RT-PCR analysis in a number of human cell lines demonstrated the presence of two alternative noncoding first exons (1a and 1b). Two distinct promoter regions, P1 and P2, were identified upstream of each exon, containing multiple sites of transcription initiation, as shown by RNase protection analysis. Sequence analysis of these regions showed a CAAT box upstream of exon 1a and high G-C content regions within both P1 and P2. Consensus binding sites for transcription factors, including SP1, AP1 and putative NF-kappa B (kappa B sites), were found upstream of each exon. In particular, six kappa B sites were identified, all but one of them capable of binding NF-kappa B complexes in vitro. Transfection in HeLa cells of plasmids containing P1 and P2 sequences linked to a chloramphenicol acetyltransferase reporter gene indicated that both P1 and P2 can act independently as promoters. Co-transfection of NF-kappa B effector plasmids (NF-kappa Bp52 and RelA) with a reporter gene linked to P1 and P2 showed that the NFKB2 promoter regions are regulated by NF-kappa B factors. RelA transactivates the NFKB2 promoter in a dose-dependent manner, whereas NF-kappa Bp52 acts as a repressor, indicating that the NFKB2 gene may be under the control of a negative feedback regulatory circuit.

Base Sequence↗

PubMed2004

Functional characterization of the peptide transporter PEPT2 in primary cultures of human upper airway epithelium.

This study characterizes the expression and function of the peptide transporter hPepT2 (SLC15A2) in differentiated primary cultures of human upper airway lung epithelia obtained from six human donors. Genotype analysis of a SNP in exon 15 of hPepT2 genotypes in six donors revealed an expected distribution of the two main variants present at similar frequency (two AA homozygotes, two BB homozygotes, and two AB heterozygotes). Real-time PCR analysis of the hPepT2 mRNA message revealed no significant differences among genotypes. hPEPT2 was expressed on the apical membrane in all donor specimens, demonstrated by cell surface biotinylation and Western analysis (104 kD). We then compared transepithelial transport of the prototypical substrate (3)H-glycylsarcosine in all donor cultures in the absence and presence of known inhibitors of hPEPT2 to ascertain the phenotype of functionally expressed hPepT2 in the upper airway epithelium. An array of inhibitors included dipeptides, beta-lactam antibiotics, bestatin, and ACE inhibitors. hPEPT2 exhibited saturable Michaelis-Menten-type kinetic parameters for GlySar, corroborating previously reported values for K(T) and J(max). Donor-to-donor variation of transport for different substrates did not correlate with hPepT2 haplotypes in this sample cohort. These findings demonstrate functional hPEPT2 transporter expression in primary cultures of human lung epithelial cells. hPEPT2-mediated transport could serve as a strategy for noninvasive systemic delivery of peptides and peptidomimetics drugs.

Alleles↗

PubMed2000

Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies.

Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to characterize the structural and functional properties and the in vivo expression of the murine GPIb-IX complex (von Willebrand factor receptor). The molecular weights of the subunits are similar to the human homologs: GPIbalpha (150 kd), GPIbbeta (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced shedding of glycocalicin (GC; 130 kd) but only low levels of receptor internalization. The GC concentration in normal mouse plasma was found to be at least 10 times higher than that described for human plasma (approximately 25 microg/mL versus 1-2 microg/mL). Two additional cleavage sites for unidentified platelet-derived proteases were found on GPIbalpha, as demonstrated by the generation of 3 N-terminal fragments during in vitro incubation of washed platelets (GC, 60 kd, 45 kd). Occupancy of GPIbalpha with p0p mAbs or F(ab)(2)-fragments resulted in aggregate formation in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding epitopes of the individual antibodies. GPIb-IX was not detectable immunohistochemically on endothelial cells in the major organs under normal or inflammatory conditions. The authors conclude that the mouse system might become an interesting model for studies on GPIb-IX function and regulation.

Animals↗

PubMed1992

Functional characterization of a variant factor XII (F XII Locarno) in a cross reacting material positive F XII deficient plasma.

The plasma of a healthy woman was found to contain half normal factor XII (FXII) antigen level (0.46 U/ml) without any FXII clotting activity (less than 0.01 U/ml). The variant FXII in this plasma, denoted as FXII Locarno, was partially characterized by immunological and functional studies on the proposita's plasma. FXII Locarno is a single chain molecule with the same size (Mr = 80 kDa) as normal FXII. Isoelectric focusing suggested an excess of negative charge in the variant FXII as compared to normal FXII. In contrast to FXII in normal plasma, FXII Locarno was not proteolytically cleaved upon prolonged incubation of proposita's plasma with dextran sulfate. Adsorption to kaolin was similar for both, abnormal and normal FXII. Incubation of the proposita's plasma with dextran sulfate and exogenous plasma kallikrein showed normal cleavage of FXII Locarno outside of the tentative disulfide loop Cys340-Cys467, but only partial cleavage within this disulfide loop. Furthermore, plasma kallikrein-cleaved abnormal FXII showed neither amidolytic activity nor proteolytic activity against factor XI and plasma prekallikrein. These results suggest a structural alteration of FXII Locarno, affecting the plasma kallikrein cleavage site Arg353-Val354 and thus formation of activated FXII (alpha-FXIIa).

Amides↗