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Results for “Functional characterization”

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PubMed2003

Functional characterization of a new human Ad4BP/SF-1 variation, G146A.

Ad4BP/SF-1 plays key roles at all levels of the hypothalamic-pituitary-steroidogenic organ axis and its functional disruption causes endocrine disorders of these organs. However, only three human subjects with Ad4BP/SF-1 mutations have been reported to date, suggesting limited clinical significance as a cause of inborn adrenal or sexual abnormalities. We report the first functional characterization of a new variation found in the hinge region of human Ad4BP/SF-1, G146A. Resulting from a single nucleotide shift (GGG-->GCG), G146A bears slightly diminished transactivation activity evidenced by both adrenal specific cyp11A promoter and ovary specific cyp19 promoter II. The variation does not affect protein expression or stability, exhibiting no dominant negative effect. G146A has a normal interaction pattern with standard co-regulators and subnuclear distribution pattern, and can be considered as a nonsynonymous single nucleotide polymorphism, since it occurs in normals and patients with adrenal diseases. In normal Japanese the allele C frequency is 8%, while in a preliminary population of patients with adrenal diseases it is elevated to 30%; suggesting the G146A variation might be of clinical importance.

Adrenal Gland Diseases↗

PubMed2002

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151.

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.

Bacterial Proteins↗

PubMed2003

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster.

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.

Amino Acid Sequence↗

PubMed1993

Biochemical and functional characterization of aminopeptidase N expressed by human melanoma cells.

A cell surface protein expressed on melanoma cells, but not on normal melanocytes, was biochemically and functionally characterized. Microsequencing of the M(r) 143,000 affinity-purified protein revealed amino acid sequence identity to aminopeptidase N (EC 3.4.11.2). In situ expression, indirect immunofluorescence, and Western blotting demonstrated that aminopeptidase N is tightly associated with extracellular matrix components. A specific polyclonal antiserum and the competitive inhibitors of aminopeptidase N, bestatin and amastatin, inhibited invasion of an aminopeptidase N-expressing metastatic melanoma cell line through the reconstituted basement membrane Matrigel in a dose-dependent manner. In vitro digestion of Matrigel with affinity-purified aminopeptidase N revealed an enzyme-sensitive M(r) 160,000 protein. These experiments suggest a role for aminopeptidase N in melanoma invasion of basement membranes.

Amino Acid Sequence↗

PubMed2001

Phenotypic and functional characterization of rhesus monkey decidual lymphocytes: rhesus decidual large granular lymphocytes express CD56 and have cytolytic activity.

In this study, we carried out a phenotypic and functional characterization of lymphocytes isolated from the uterine endometrium of the pregnant rhesus monkey. A majority (80%) of these cells were CD56(bright+), CD3- had typical large granular lymphocyte/uterine natural killer (NK) cell morphology and contained numerous cytoplasmic granules. Flow cytometric evaluation showed that rhesus decidual CD56(bright+) cells shared other phenotypic features of human uterine NK cells, including low levels of CD45RA and CD62L expression. A majority of the rhesus uterine CD56(bright+) cells expressed low levels of CD 16 but were CD2-. In contrast, most rhesus CD16+ peripheral blood cells were CD56-. In addition to the primary population of CD56(bright+) cells, a minor subset of smaller and less granular CD56(intermediate+) decidual lymphocytes was identified, the majority of which were CD16-, CD2(+). Decidual CD56+ cells did not express monocyte/macrophage markers, including CD14, CD64 and CD68. Decidual lymphocytes effectively lysed K562, Raji and particularly 721.221 targets in cytotoxicity assays. Together, these results suggest that as in human pregnancy, rhesus decidual CD56(bright+) cells represent a distinct lymphocyte subset that belongs to the NK cell lineage.

Animals↗

PubMed2000

Functional characterization of two low density lipoprotein receptor gene mutations by fluorescence flow cytometric assessment of receptor activity in stimulated human T-lymphocytes.

We report a functional characterization of the W23X and W66G low density lipoprotein (LDL) receptor gene mutations. The authors used two-color fluorescence flow cytometry to measure LDL receptor activity in stimulated T-lymphocytes, prepared from patients heterozygous for the W23X or W66G mutation, and compared the results with measurements of LDL receptor activity in stimulated T-lymphocytes prepared from unrelated healthy control subjects. It was found that the W23X mutation significantly reduced LDL receptor expression and LDL binding and internalization, and that the W66G mutation significantly reduced LDL receptor expression and LDL binding. LDL internalization in patients heterozygous for the W66G mutation was not significantly reduced. The data support the concepts that the W23X mutation prevents production of LDL receptors (class I) and that the W66G mutation produces LDL receptors unable to recycle normally in cells (class V).

Adult↗

PubMed1997

Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotropic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used for heterologous expression of the rabbit intestinal peptide transporter PepT1 and its functional characterization. PepT1 mediates the electrogenic transmembrane transport of di- and tripeptides and peptido-mimetics such as beta-lactam antibiotics and ACE-inhibitors. Functional expression of PepT1 was determined in different recombinant clones by flux studies employing the radiolabeled dipeptide 3H-(D)-Phe-(L)-Ala. One clone (GS-PepT1) displayed high level functional expression that was pH dependent and saturable with an app. K0.6 of 1.17 +/- 0.18 mM. Inhibition of 3H-(D)-Phe-(L)-Ala uptake into GS-PepT1 by selected dipeptides, tripeptides and peptidomimetics including beta-lactam antibiotics and ACE-inhibitors revealed the same substrate specifity as reported for PepT1 when expressed in mammalian cells or Xenopus laevis oocytes. Pichia cells expressing PepT1 will provide an excellent tool for in vitro bioavailability studies for peptides and peptidomimetics. Moreover, to our knowledge, this is the first demonstration of functional expression of a mammalian membrane transport protein using P. pastoris.

Animals↗

PubMed1981

A new preparation of modified immune serum globulin (human) suitable for intravenous administration. II. Functional characterization.

The commercial immune serum globulin (ISG) product is suitable for use in passive immunization only by intramuscular administration; intravenous use results in vasomotor side reactions. We report the functional characterization of a chemically modified ISG (MISG) that is essentially free of nonspecific complement-binding activity and contains no new antigenic determinants. The half-life values for MISG given intravenously to rabbits, guinea pigs, and humans are shown not to be different from that of ISG. The MISG we describe is shown to fulfill the in vitro and in vivo criteria of stability, safety, function, and half-life, making it suitable for human intravenous administration.

Animals↗

PubMed2001

Cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme gene. Regulatory role of Sp1 and AP-2.

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.

Amino Acid Sequence↗

PubMed1995

Transcriptional regulation of the gene encoding the human C-type lectin leukocyte receptor AIM/CD69 and functional characterization of its tumor necrosis factor-alpha-responsive elements.

The human activation antigen CD69 is a member of the C-type animal lectin superfamily that functions as a signal-transmitting receptor. Although the expression of CD69 can be induced in vitro on cells of most hematopoietic lineages with a wide variety of stimuli, in vivo it is mainly expressed by T-lymphocytes located in the inflammatory infiltrates of several human diseases. To elucidate the mechanisms that regulate the constitutive and inducible expression of CD69 by leukocytes, we isolated the promoter region of the CD69 gene and carried out its functional characterization. Sequence analysis of the 5'-flanking region of the CD69 gene revealed the presence of a potential TATA element 30 base pairs upstream of the major transcription initiation site and several putative binding sequences for inducible transcription factors (NF-kappa B, Egr-1, AP-1), which might mediate the inducible expression of this gene. Transient expression of CD69 promoter-based reporter gene constructs in K562 cells indicated that the proximal promoter region spanning positions -78 to +16 contained the cis-acting sequences necessary for basal and phorbol 12-myristate 13-acetate-inducible transcription of the CD69 gene. Removal of the upstream sequences located between positions -78 and -38 resulted in decreased promoter strength and abolished the response to phorbol 12-myristate 13-acetate. We also found that tumor necrosis factor-alpha (TNF-alpha) is capable of inducing the surface expression of the CD69 molecule as well as the promoter activity of fusion plasmids that contain 5'-flanking sequences of the CD69 gene, suggesting that this cytokine may regulate in vivo the expression of CD69.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs.

BACKGROUND: The complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions. RESULTS: The experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes. CONCLUSIONS: By combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.

Animals↗

PubMed1982

Functional characterization of the cells in chronic neutrophilic leukemia.

Light and electron microscopy of neutrophils from chronic neutrophilic leukemia (CNL) did not reveal differences from normal mature neutrophils. However, functional characterization of CNL cells showed marked differences when compared to normal cells. CNL neutrophils were much less viable in suboptimal conditions. Their survival was further reduced by autologous serum and was corrected by normal human serm. CNL cells showed very active phagocytosis, but their bactericidal activity was reduced in suboptimal conditions. The total content of lysozyme and beta-glucuronidase was lower in CNL cells compared to normal neutrophils, but the release of these enzymes from stimulated cells was much higher than normal. This observation is compatible with a marked lysosomal lability. Cells from the patients' peripheral blood and bone marrow showed excessive growth in CFU-C assays. Marked susceptibility of CNL cells to cytotoxic activity of cold agglutinins, SLE sera, and CSFs was observed and may signify qualitative and/or quantitative differences in the membrane structure of CNL neutrophils, as compared to normal cells.

Aged↗

PubMed1992

Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD45 (CD45RO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis.

The biochemical and functional characterization, and the regulation of plasma membrane expression of the leucocyte tyrosine phosphatase CD45, have been investigated in neutrophils from healthy donors and patients undergoing haemodialysis. CD45 proteins of 180 kD and 130-150 kD were precipitated from neutrophils from both healthy subjects and haemodialysed patients. Prolonged storing, as well as trypsin treatment of samples containing the 180-kD CD45 protein, generated the 130-150-kD polypeptides. The 130-150-kD CD45 polypeptides carried extracellular CD45 epitopes, including the sialic acid-related UCHL1 epitope (CD45RO). Furthermore, these trypsin-generated CD45 polypeptides did not possess phosphatase activity, which could be detected on the 180-kD protein. A remarkable quantitative increase of cell surface expression of the neutrophil CD45 components was detected both after in vitro neutrophil activation and after dialysis treatment with neutropenic membranes. The CD45 biochemical pattern did not qualitatively change upon either in vitro or in vivo dialysis-induced neutrophil activation. The upregulated expression of CD45 on neutrophils from dialysed patients correlated with the neutropenic effect induced by the different dialyser membranes. Maximal upregulation of CD45 expression was observed after 15 min of dialysis with neutropenic membranes, and normal expression levels were restored after 1 h. By contrast, increase of CD45 plasma membrane expression induced in vitro by treatment of normal neutrophils with the degranulatory agents fMLP or Ca2+ ionophore was maintained. These results demonstrate that neutrophil cell surface expression of the 180-kD CD45 protein is upregulated during the in vivo haemodialysis process, and suggest that a proteolytic activity could regulate the enzymatic activity of CD45 by degranulation of its cytoplasmic phosphatase domains.

Antigens, CD↗

PubMed2005

Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Amino Acid Motifs↗

PubMed1993

Functional characterization of the 5'-regulatory region of the human thrombomodulin gene.

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.

Base Sequence↗

PubMed1999

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis.

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.

Animals↗

PubMed1996

Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.

Adenosine Triphosphate↗

PubMed2002

Functional characterization of calcium channels localized on the growth cones of cultured rat dorsal root ganglion cells.

Ca2+ channels on growth cones of cultured rat dorsal root ganglion (DRG) neurons were functionally characterized with an optical method using Fura-2. An increase in intracellular calcium concentration ([Ca2+](i)) of the growth cone was induced in response to electrical stimulation to the DRG cell body. The ([Ca2+](i))-increase was partly inhibited by either of omega - conotoxin GVIA (omega - CgTx, 3 microM) or omega - agatoxin IVA (omega - aga IVA, 300 nM) and completely blocked by both present at the same time, but was not affected by nicardipine (30 microM). The omega - CgTx - as well as omega - aga IVA - sensitive Ca2+ channels were immunologically localized on the growth cones using field emission scanning electron microscopy. It is concluded that the omega - CgTx - as well as omea - aga IVA - sensitive Ca2+ channels are involved in the ([Ca2+](i))-increase in the growth cones of the cultured DRG neurons, leading to glutamate release before synapse formation.

Animals↗