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Results for “Functional characterization”

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PubMed1993

Phenotypical and functional characterization of Fc gamma receptor I (CD64)-negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity.

Fc gamma receptor I-positive (CD64+) and Fc gamma receptor I-negative (CD64-) monocytes were prepared from highly purified (elutriation-derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64+ and CD64- monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64- monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64- cells were found to be esterase-positive, and 85% were positive for the the CD64- cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64- monocytes show significantly higher expression of CD45RA and Fc gamma receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64- monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64+ monocytes. CD64- monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-gamma and also higher proliferation in responding autologous T cells than PPD-pretreated CD64+ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon-gamma release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64- monocytes than of irradiated CD64+ monocytes. Furthermore, when CD64- and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-alpha release from CD64- than from CD64+ monocytes, indicating a higher anti-viral capacity of this subset. CD64- monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64- monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-alpha.

Antigen-Presenting Cells↗

PubMed1991

Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions.

The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII.

Amino Acid Sequence↗

PubMed2003

Glucose transporter GLUT12-functional characterization in Xenopus laevis oocytes.

We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.

Animals↗

PubMed2002

Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome.

The extracellular Ca(2+)-sensing receptor (CaSR) plays an essential role in extracellular Ca(2+) homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca(2+) and ERK activity, showed a dramatic reduction in the EC(50) for extracellular Ca(2+) compared with the wild-type and a loss-of-function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.

Bartter Syndrome↗

PubMed2005

Functional characterization of prostate cancer by integrated magnetic resonance imaging and oxygenation changes during carbogen breathing.

OBJECTIVE: The objective of this study was to evaluate the changes in oxygenation of prostate cancer induced by carbogen breathing using blood oxygen level-dependent (BOLD) magnetic resonance image (MRI) with an endorectal coil (eMRI). MATERIALS AND METHODS: In 32 patients with biopsy-proven prostate cancer, endorectal MRI was performed at 1.5 Tesla using the BOLD method. Images were acquired during 4 x 4-minute episodes alternating between room air and carbogen (95% O2/5% CO2) breathing. In each episode, 40 images were acquired (T2*-weighted EPI sequence, 12-14 slices, 3-mm thickness). All patients underwent radical prostatectomy; BOLD-MRI findings were correlated with the histopathologic results. RESULTS: BOLD-MRI could be evaluated in 29 patients, and revealed heterogeneous signal changes of normal prostate and cancer tissue similar to the heterogeneity of prostate tissue in anatomic/pathologic preparation. A significant signal intensity increase (P = 0.004) was found in normal central gland and peripheral zone during carbogen breathing. Signal enhancement in carcinoma was significantly lower (P = 0.004) compared with the contralateral normal side. CONCLUSION: Intrinsic blood-tissue contrast-functional MRI during carbogen breathing may help detect and characterize prostate carcinoma from normal tissue, particularly in small 1-sided carcinomas. This may be useful for identifying candidates for radiotherapy and monitoring noninvasive therapeutic approaches.

Aged↗

PubMed2003

Functional characterization of the mammalian mRNA decapping enzyme hDcp2.

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.

Amino Acid Motifs↗

PubMed2005

Functional characterization of brain mitochondrial nitric oxide synthase during hypertension and aging.

Nitric oxide (NO*) plays an important role in various physiological processes. The aim of the present study was to investigate if brain mitochondrial nitric oxide synthase (mtNOS) is active and functional during hypertension. L-citrulline production, an indicator of nitric oxide synthesis, was concentration-dependent on L-arginine in all strains and all ages tested, and was inhibited by 7-Nitroindazole (7-NI). Brain mitochondria of 1 month-old (prehypertensive) spontaneously hypertensive rats (SHR) exhibited a significantly (p < 0.05) low basal L-citrulline content as compared to age-matched Wistar (W) and Wistar-Kyoto (WKY) rats. L-citrulline synthesis in SHR rats showed a significant (p < 0.01) low response to L-arginine in 3 and 7 months-old rats. Respiratory rates in states 3 and 4 increased with low L-arginine concentration in all strains and all ages. The results suggest that in rat brain mitochondria, L-citrulline synthesis is constant once age-related hypertension is installed and NO* does not regulate oxidative phosphorylation.

Aging↗

PubMed1986

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle.

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.

Animals↗

PubMed2005

Functional characterization of the promoter of the human glucose transporter 10 gene.

The human SLC2A10 gene encodes the high-affinity glucose transporter 10 (GLUT10) and is widely expressed in adult tissues, including organs which play major roles in glucose homeostasis. Its function and genomic location in a region linked to Type 2 diabetes susceptibility are consistent with a potential role in Type 2 diabetes. Analysis of the CpG-rich promoter revealed the presence of two major transcription start points with differential use in tissues and cell lines. Mapping of transcriptionally active regions in the 5' flanking sequence identified a region, located between nucleotides -70 and -14 (relative to the major transcription start point) as the SLC2A10 basal promoter. This sequence harbors consensus binding sites for Sp, AP2alpha, and other transcription factors. A juxtaposed Sp/AP2alpha motif located between -25 and -11 is critical for core promoter function. In cells expressing Sp and AP2 factors, the two motifs are required for maximal activation of the basal promoter. In cells lacking AP2alpha, transcription is dependent on the integrity of the Sp site. Using electrophoresis mobility shift assays, we demonstrate that Sp1 and Sp3 bind to the GC-box in site 5 forming specific complexes. In addition, a silencer region is present upstream of -696 which down-regulates SLC2A10 promoter activity independently of its distance to the transcript start site.

Base Sequence↗

PubMed1999

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.

Arabidopsis↗

PubMed2005

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

ATP-Binding Cassette Transporters↗

PubMed2002

Structural and functional characterization of the human DNA repair helicase XPD by comparative molecular modeling and site-directed mutagenesis of the bacterial repair protein UvrB.

A molecular model for the human nucleotide excision repair protein, XPD, was developed based on the structural and functional relationship of the protein with a bacterial nucleotide excision repair (NER) protein, UvrB. Whereas XPD does not share significant sequence identity with UvrB, the proteins share seven highly conserved helicase motifs that define a common protein structural template. They also have similar functional roles in their ATPase activity and the ability to unwind DNA and verify damaged strands in the process of NER. The validity of using the crystal structure of UvrB as a template for the development of an XPD model was tested by mimicking human disease-causing mutations (XPD: R112H, D234N, R601L) in UvrB (E110R, D338N, R506A) and by mutating two highly conserved residues (XPD, His-237 and Asp-609; UvrB, H341A and D510A). The XPD structural model can be employed in understanding the molecular mechanism of XPD human disease causing mutations. The value of this XPD model demonstrates the generalized approach for the prediction of the structure of a mammalian protein based on the crystal structure of a structurally and functionally related bacterial protein sharing extremely low sequence identity (<15%).

Amino Acid Sequence↗

PubMed1991

Construction and functional characterization of mutants of the bovine leukaemia virus trans-activator protein p34tax.

The p34tax protein [p38tax, p34, p38(XBL), XBL-I] of bovine leukaemia virus (BLV) activates transcription from the BLV long terminal repeat (LTR) promoter. To analyse the functional properties of this protein, inframe insertions and internal deletions were systematically introduced in a plasmid-encoded copy of the p34tax gene. The abilities of wild-type and mutant genes to activate gene expression from the LTR promoter linked to the chloramphenicol acetyltransferase gene and to inhibit trans-activation by the wild-type protein were studied. The trans-activating activity of 14 of the 18 mutants tested was completely abolished, but four mutants each containing a lesion in the internal portion of the polypeptide retained activity. Taken together, these results suggest the presence of an internal region of the polypeptide where structural integrity is less strictly required for the functional activity of this protein. Among the mutants incompetent in the transactivation assay, only two with mutations in the N-terminal region of the polypeptide inhibited transactivation by the wild-type protein in a dose-dependent manner. These results facilitate understanding of the physiological function of the tax protein family.

Amino Acid Sequence↗

PubMed2006

Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis.

A stem-loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R(1)NNNAAR(2)NNNNNNR(3), is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an in vitro translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (A(5)G) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.

Animals↗

PubMed2000

In vivo splicing and functional characterization of Mycobacterium leprae RecA.

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.

Mycobacterium leprae↗

PubMed2006

Four novel missense mutations in the CYP21A2 gene detected in Russian patients suffering from the classical form of congenital adrenal hyperplasia: identification, functional characterization, and structural analysis.

CONTEXT: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The most frequent cause is the deficiency of steroid 21-hydroxylase (CYP21) due to mutations in the CYP21A2 gene. OBJECTIVE: We analyzed the functional and structural consequences of the four CYP21A2 missense mutations (C169R, G178R, W302R, and R426C) to prove their clinical relevance and study their impact on CYP21 function. RESULTS: Analyzing the mutations in vitro revealed an almost absent or negligible CYP21 activity for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. Protein translation and intracellular localization were not affected by the mutants, as could be demonstrated by Western blotting and immunofluorescence studies. Analysis of these mutants in a three-dimensional model structure of the CYP21 protein explained the observed in vitro effects because all the mutations severely interfere either directly or indirectly with important structures of the 21-hydroxylase protein. CONCLUSION: The in vitro expression analysis of residual enzyme function is a complementary method to genotyping and an important tool for improving the understanding of the clinical phenotype of 21-hydroxylase deficiency. This forms the foundation for accurate clinical and genetic counseling and for prenatal diagnosis and treatment. Moreover, this report demonstrates that the combination of in vitro enzyme analysis and molecular modeling can yield novel insights into CYP450 structure-functional relationships.

Adolescent↗

PubMed1996

Phenotypic and functional characterization of peripheral blood and bone marrow natural killer cells prior to autologous transplantation.

NK cells can exert potent anti-leukemia activity after either autologous or allogeneic BMT. However, in autologous blood or marrow transplant patients, NK cell number and/or function could be reduced, and also may vary according to the sampling site. In order to evaluate the hypothesis that blood or marrow grafts from autologous transplant patients exhibit impaired NK cell activity that could contribute to disease recurrence, we evaluated the immunologic characteristics of NK cells in the bone marrow (BM) and peripheral blood (PB) from 27 patients undergoing autologous BMT, and also from 20 normal donors. We measured baseline and interleukin-2 (IL-2)-activated NK cell cytotoxicity, as well as expression of IL-2 receptors (IL-2R) (alpha-chain (p55) and beta-chain (p75)), and adhesion molecules. The cytotoxic activity of PB NK cells was significantly lower in autologous transplant patients than in normal donors (P < 0.0005) and this difference was not mitigated following IL-2 activation. In contrast, BM from autologous patients showed normal NK cell cytotoxicity, but contained higher numbers of NK cells (P < 0.025), with more intense CD56 expression (P < 0.05). Expression of p75 was lower on BM than on PB NK cells in both patients and normal donors. In addition, induction of p55 by IL-2 was abrogated in autologous PB NK cells. Therefore, depending on the site of harvest and the nature of donor cells (pre-BMT vs normal), our results show significant differences in NK cell number, function, and IL-2 receptor expression. This may affect relapse rates following autologous transplants performed with either PB or BM grafts.

Adolescent↗

PubMed2006

Functional characterization of serotonin receptor subtypes in human duodenal secretion.

Serotonin (5-HT) stimulates ion secretion in the gastrointestinal tract and the sensitivity for 5-HT might be altered in dyspeptic patients infected with Helicobacter pylori. The purpose of the present study was to characterize the 5-HT-induced electrogenic ion transport in the duodenum of dyspeptic patients with or without Helicobacter pylori infection, and to determine the 5-HT receptor subtypes functionally involved. Biopsies from the second part of duodenum were obtained from 43 dyspeptic patients during routine endoscopy. Biopsies were mounted in modified Ussing chambers with air suction for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured before and after application of graded cumulative doses of 5-HT and a single dose of bumetanide (an inhibitor of chloride/bicarbonate transport), or one of the selective 5-HT receptor antagonists: ketanserin, ondansetron, or SB-204070 (1-butyl-4 piperidinmethyl-8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-carboxylate HCl). Histological examination was performed on duodenal biopsies. Helicobacter urease testing and histological examination determined Helicobacter pylori infection. 5-HT induced a dose-dependent and bumetanide-sensitive short-circuit current, which was independent of the presence of Helicobacter pylori infection. All the three 5-HT receptor antagonists failed to significantly effect basal and 5-HT-induced short-circuit current. Our results indicate that in human duodenum 1) 5-HT is a potent stimulator of bumetanide-sensitive secretion, 2) the serotonergic receptor subtype, which acts as the main mediator of 5-HT-induced secretion is different from the 5-HT(2), 5-HT(3), and the 5-HT(4) subtype and, 3) the sensitivity to 5-HT is not altered by Helicobacter pylori infection.

Adult↗