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PubMed1998

Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites.

The involvement of AP-1 and NF-kappaB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappaB sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -5301 base pairs upstream decreased gene activation by 90% in a -8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 (at -5115) or NF-kappaB (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappaB sites. By gel retardation analyses, CM increased AP-1- and NF-kappaB binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappaB site bound different complements of NF-kappaB/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas IkappaB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappaB inding activity. Thus, AP-1 and NF-kappaB are important cis-elements for induction of hiNOS gene transcription.

Amino Acid Sequence↗
PubMed1999

Identification of a hypoxia response element in the transferrin receptor gene.

Expression of the transferrin receptor, which mediates iron uptake from transferrin, is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements in the 3'-untranslated region of the transferrin receptor mRNA. Transcriptional mechanisms are also involved in receptor expression, but these are poorly understood. In this study we have characterized the transferrin receptor promoter region and identified a functional hypoxia response element that contains a binding site for hypoxia-inducible factor-1 (HIF-1). Exposure of K562 and HeLa cells to hypoxia for 16 h resulted in a 2- to 3-fold increase in transferrin receptor mRNA expression. A motif with multipartite organization similar to the hypoxia response element of a number of hypoxia-inducible genes such as erythropoietin was identified within a 100-base pair sequence upstream of the transcriptional start site. Mutation of a site similar to the consensus HIF-binding site (HBS) in this motif attenuated the hypoxic response by 80%. Transient co-expression of the two HIF-1 subunits (HIF-1alpha and HIF-1beta) enhanced the wild type transferrin receptor promoter activity, but that which contained a mutated HBS yielded no such response. Electrophoretic mobility shift assays revealed that HIF-1 was stimulated and bound to the transferrin receptor HBS upon hypoxic challenge. Our results indicate that the transferrin receptor is a target gene for HIF-1.

Base Sequence↗
PubMed1990

A voltage-clamp analysis of gene-dosage effects of the Shaker locus on larval muscle potassium currents in Drosophila.

Mutations of the Shaker (Sh) locus of Drosophila reduce, eliminate, or otherwise alter a transient potassium current, IA, in muscle. Recent molecular studies indicate that the Sh locus produces several proteins by alternative splicing, but the relationships of the variety of Sh gene products to IA channels in the various excitable membranes still remain to be determined. In Drosophila, many enzymes have been shown to exhibit gene-dosage effects; their amounts vary in direct proportion to the number of structural genes present. We describe a physiological isolation of IA in larval muscle which allowed precise quantification of gene-dosage effects on IA in Sh heterozygotes and aneuploids. We found that doubling the number of Sh genes in aneuploids increased IA to twice that of normal, consistent with the notion that the Sh locus encodes the entire IA channel in larval muscle. We further examined heterozygous combinations of different Sh mutations for evidence of interactions among Sh gene products within the IA channel, which may yield clues to the possible subunit composition of the channel. Combinations among 5 Sh mutations plus their normal counterpart followed a simple gene-dosage effect; in each case the resulting IA was about the average of the homozygous currents, compatible with the notion of additive contributions from 2 independent populations of IA channels. Two additional Sh mutations caused pronounced departures from the simple dosage effect; the amplitude of IA in heterozygotes was significantly smaller than that expected from gene dosage, a strong dominant effect attributable to interactions among protein subunits. These contrasting observations may be accounted for by certain hetero- or homo-multimeric arrangements of Sh products in the IA channel.

Aneuploidy↗
PubMed2006

Dissemination of a Sjögren's syndrome-associated extranodal marginal-zone B cell lymphoma: circulating lymphoma cells and invariant mutation pattern of nodal Ig heavy- and light-chain variable-region gene rearrangements.

OBJECTIVE: Both the genesis and outgrowth of extranodal marginal-zone B cell lymphomas (MZLs) of the mucosa-associated lymphoid tissue (MALT) type are generally thought to represent antigen-driven processes. We undertook this study to analyze lymphoma progression and dissemination outside of the MALT-type lesions. METHODS: Histopathologic and Ig heavy- and light-chain variable-region gene (V(H/L)) analyses were performed in sequential tissue samples from a patient with primary Sjögren's syndrome (SS) with glandular (parotid) manifestations and subsequent nodal dissemination of a low-grade MZL. RESULTS: This MZL expressed a CD20+,CD27+,sIgM/kappa+,IgD-,CD5-,CD10-,Bcl-6-,CD23-,p53-,p21-,MDM2- phenotype and mutated V(H)1-69/D2-21/J(H)4alpha-V(kappa)A27/J(kappa)2 Ig rearrangements. Notably, circulating lymphoma cells from the parotid glands occurred transiently in the patient's blood, as detected by single-cell polymerase chain reaction. In addition, 2 minor B cell clones (clones 2 and 3, with V(H)3-07/D3-22/J(H)3b-V(lambda)3L/J(lambda)2/3 and V(H)3-64/D3-03/J(H)2-V(kappa)A19/J(kappa)2 rearrangements, respectively) were also detected in the parotid glands and blood, and 1 of these (clone 2) was also detected in the lymph nodes. Ig V(H/L) analyses revealed ongoing (antigen-driven) mutations of the glandular lymphoma rearrangements, but an invariant mutation pattern of their nodal counterparts. CONCLUSION: These data indicate coexpansion and transient (re)circulation of the lymphoma clone and 2 additional glandular B cell clones in a primary SS-associated extranodal MZL. Combined histologic and molecular features of the nodal lymphoma subclone reflect a process of "follicular colonization" that eventually froze the mutation machinery after accumulation of additional (antigen-driven) Ig V(H/L) mutations.

Female↗
PubMed2005

A critical role for Gly25 in the B chain of human thrombin.

We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25(330)S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25(330)S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by Taipan snake or Echis carinatus venoms. We show that the G25(330)S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25(330) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin.

Animals↗
PubMed2002

Promoter analysis of the gene encoding the beta-subunit of the rat amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial Na(+) channel (ENaC), found in the apical membrane of Na(+)-absorptive epithelia, is made up of three differentially regulated subunits: alpha, beta, and gamma. We undertook a study of the 5'-end of the gene encoding the beta-ENaC subunit in the rat. 5'-Rapid amplification of cDNA ends and RNase protection assays indicated multiple transcription start sites over a 50-bp region. Sequencing 1.3 kb of the 5'-flanking DNA revealed putative binding sites for PEA3, Sp1, activator protein (AP)-1 and Oct-1 but neither a TATA box nor consensus sites for steroid hormone receptor binding. Transient transfections of reporter constructs driven by beta-ENaC 5'-flanking DNA in the representative epithelial cell lines Madin-Darby canine kidney, MLE-15, and Caco-2 revealed a negative element present between positions -424 and -311 that affected basal transcription rates. Gel shift assays showed protein-DNA binding activity of an AP-1 consensus site in this region; however, mutation of the AP-1 site did not abrogate the repressive activity of the region in transient transfections. Deletion of two clusters of Sp1 consensus binding sites between -1 and -51 bp and between -169 and -211 bp indicated that the proximal cluster was essential to basal promoter activity in transfected cell lines. In a comparison of these data with those in published studies on alpha- and gamma-ENaC promoters, the beta- and gamma-subunit promoters appear to be more similar to each other than to the alpha-promoter.

Animals↗
PubMed2005

Severe congenital hyperinsulinism caused by a mutation in the Kir6.2 subunit of the adenosine triphosphate-sensitive potassium channel impairing trafficking and function.

CONTEXT: The ATP-sensitive potassium (K(ATP)) channel, assembled from the inwardly rectifying potassium channel Kir6.2 and the sulfonylurea receptor 1, regulates insulin secretion in beta-cells. A loss of function of K(ATP) channels causes depolarization of beta-cells and congenital hyperinsulinism (CHI), a disease presenting with severe hypoglycemia in the newborn period. OBJECTIVE: Our objective was identification of a novel mutation in Kir6.2 in a patient with CHI and molecular and cell-biological analysis of the impact of this mutation. DESIGN AND SETTING: We combined immunohistochemistry, advanced life fluorescence imaging, and electrophysiology in HEK293T cells transiently transfected with mutant Kir6.2. PATIENT AND INTERVENTION: The patient presented with macrosomia at birth and severe hyperinsulinemic hypoglycemia. Despite medical treatment, the newborn continued to suffer from severe hypoglycemic episodes, and at 4 months of age subtotal pancreatectomy was performed. MAIN OUTCOME MEASURE: We assessed patch-clamp recordings and confocal microscopy in HEK293T cells. RESULTS: We have identified a homozygous missense mutation, H259R, in the Kir6.2 subunit of a patient with severe CHI. Coexpression of Kir6.2(H259R) with sulfonylurea receptor 1 in HEK293T cells completely abolished K(ATP) currents in electrophysiological recordings. Double immunofluorescence staining revealed that mutant Kir6.2 was partly retained in the endoplasmic reticulum (ER) causing decreased surface expression as observed with total internal reflection fluorescence. Mutation of an ER-retention signal partially rescued the trafficking defect without restoring whole-cell currents. CONCLUSION: The H259R mutation of the Kir6.2 subunit results in a channel that is partially retained in the ER and nonfunctional upon arrival at the plasma membrane.

ATP-Binding Cassette Transporters↗
PubMed2004

Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.

Clustering of nicotinic muscle acetylcholine receptors (AChRs) requires association with intracellular rapsyn, a protein with an intrinsic ability to self-cluster. Previous studies on sofa potato (sop), an AChR null line of zebrafish, have suggested that AChRs may play an active role in subsynaptic localization of rapsyn clusters. To test this proposal directly, we identified and cloned the gene responsible for the sop phenotype and then attempted to rescue subsynaptic localization of the receptor-rapsyn complex in mutant fish. sop contains a leucine to proline mutation at position 28, near the N terminus of the zebrafish AChR delta subunit. Transient expression of mutant delta subunit in sop fish was unable to restore surface expression of muscle AChRs. In contrast, expression of wild-type delta subunit restored the ability of muscle to assemble surface receptors along with the ability of fish to swim. Most importantly, the ability of rapsyn clusters to localize effectively to subsynaptic sites also was rescued in large part. Our results point to direct involvement of the AChR molecule in restricting receptor-rapsyn clusters to the synapse.

Animals↗
PubMed2004

Evolution of precore/core promoter mutations in hepatitis B carriers with hepatitis B e antigen seroreversion.

The evolution of precore stop codon mutation (A1896) and dinucleotide mutation (T1762/A1764) in the basic core promoter (BCP) of hepatitis B virus (HBV) genome during transient seroconversion and seroreversion of hepatitis B e antigen (HBeAg) remains unclarified. Five HBeAg-positive HBV carriers who experienced transient seroconversion followed by seroreversion of HBeAg (Group I, 3.3%) and 3 HBeAg-negative HBV carriers with documented reversion of HBeAg (Group II, 2.5%) in a prospective cohort of 272 patients with chronic hepatitis B were thus identified. The sequential changes at the precore nucleotide 1896 and BCP dinucleotide 1762/1764 were determined by polymerase chain reaction and direct sequencing. At enrollement, precore A1896 and BCP T1762/A1764 were noted in 4 (50%) and 1 (13%) of the eight patients. During a median follow-up period of 58 months (range: 31-76 months), 12 episodes of transient HBeAg seroconversion followed by seroreversion were encountered in Group I patients and 3 episodes of HBeAg seroreversion in Group II patients. Accompanying acute exacerbations were found in two-thirds of patients with either HBeAg seroconversion or seroreversion. Overall, precore nucleotide A1896 remained identical in 73% and 83% of the seroconversion and seroreversion events, respectively. BCP dinucleotide T1762/A1764 remained unchanged in 94% and 92% of the seroconversion and seroreversion events, respectively. At the end of follow-up, only one had both precore and BCP mutations. In conclusion, these data suggested that HBeAg seroreversion might be due to the lack of sustained precore and BCP mutations after HBeAg seroconversion. Although uncommon, HBeAg seroreversion can be associated with hepatitis exacerbation.

Adolescent↗
PubMed2006

Molecular characterization of two founder mutations causing long QT syndrome and identification of compound heterozygous patients.

BACKGROUND: Mutations of at least six different genes have been found to cause long QT syndrome (LQTS), an inherited arrhythmic disorder characterized by a prolonged QT interval on the electrocardiogram (ECG), ventricular arrhythmias and risk of sudden death. AIM: The aims were to define the yet undetermined phenotypic characteristics of two founder mutations and to study clinical features in compound heterozygotes identified during the course of the study. METHODS: To maximize identification of the compound heterozygotes, we used an extended group of LQTS patients comprising 700 documented or suspected cases. Functional studies were carried out upon transient expression in COS-7 or HEK293 cells. RESULTS: The KCNQ1 IVS7-2A>G (KCNQ1-FinB) mutation associated with a mean QTc interval of 464 ms and a complete loss-of-channel function. The HERG R176W (HERG-FinB) mutation caused a reduction in current density as well as slight acceleration of the deactivation kinetics in vitro, and its carriers had a mean QTc of 448 ms. The HERG R176W mutation was also present in 3 (0.9%) out of 317 blood donors. A total of six compound heterozygotes were identified who had the HERG R176W mutation in combination with a previously reported LQTS mutation (KCNQ1 G589D or IVS7-2A>G). When present simultaneously with an apparent LQTS-causing mutation, the HERG R176W mutation may exert an additional in vivo phenotypic effect. CONCLUSIONS: The HERG R176W mutation represents a population-prevalent mutation predisposing to LQTS. Compound heterozygosity for mutant LQTS genes may modify the clinical picture in LQTS.

Adult↗
PubMed1997

HSDJ, a human homolog of DnaJ, is farnesylated and is involved in protein import into mitochondria.

The role of HSDJ, a human homolog of bacterial DnaJ and yeast YDJ1p/MAS5, in mitochondrial protein import was examined. Recombinant HSDJ was purified and an antibody was prepared. HSDJ mRNA was heat-induced in cultured cells. In pulse-labeling and chase experiments using COS-7 cells, the endogenous HSDJ homolog was prenylated. Transiently expressed HSDJ was also prenylated, whereas its mutant C394S in which cysteine of the "CaaX box" was mutated to serine, was not. HSDJ, but not C394S, synthesized in rabbit reticulocyte lysate was farnesylated. The HSDJ antibody inhibited import of ornithine transcarbamylase precursor (pOTC) into isolated mitochondria when added prior to pOTC synthesis, but not when added prior to import assay. In transient expression of pOTC in COS-7 cells, pOTC was synthesized and processed to the mature form with an apparent half-life of 2-3 min. Coexpression of HSDJ or C394S resulted in slight retardation of the pOTC processing. These results indicate that HSDJ is involved in an early step(s) of protein import into mitochondria.

Animals↗
PubMed2005

Induction of NR4A orphan nuclear receptor expression in macrophages in response to inflammatory stimuli.

Oxidized lipids and inflammatory cytokines are believed to play a causal role in atherosclerosis through the regulation of gene expression in macrophages and other cells. Previous work has implicated the nuclear receptors peroxisome proliferator-activated receptor and liver X receptor in the control of lipid-dependent gene expression and inflammation. Here we demonstrate that expression of a third group of nuclear receptors, the NR4A ligand-independent orphan receptors, is highly inducible in macrophages by diverse inflammatory stimuli. Treatment of macrophages with lipopolysaccharide (LPS), cytokines, or oxidized lipids triggers the transcriptional induction of Nur77 (NR4A1), Nurr1 (NR4A2), and NOR1 (NR4A3) expression. Several lines of evidence point to the NF-kappaB signaling pathway as a principal mediator of inducible NR4A expression in macrophages. Analysis of the murine and human Nur77 promoters revealed two highly conserved NF-kappaB response elements. Mutation of these elements inhibited LPS-dependent expression of the Nur77 promoter in transient transfection assays. Furthermore, induction of Nur77 expression by LPS was severely compromised in fibroblasts lacking the three NF-kappaB subunits, Nfkb1, c-Rel, and RelA. Consistent with its ability to be induced by oxidized lipids, Nur77 was expressed in macrophages within human atherosclerotic lesions. These results identified NR4A nuclear receptors as potential transcriptional mediators of inflammatory signals in activated macrophages.

Animals↗
PubMed1990

A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells.

We have identified a previously undetected cis-acting element in the mouse beta-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the beta-globin direct-repeat element (beta DRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The beta DRE motif is highly conserved in all adult mammalian beta-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the beta DRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the beta DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in beta DRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the beta DRE have implications for the mechanism of action of the element in regulating beta-globin transcription and for mutational studies of other repetitive or redundant transcription elements.

Animals↗
PubMed2006

An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.

A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.

Amino Acid Motifs↗
PubMed2000

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

Animals↗
PubMed2001

Novel mutation in the PML/RARalpha chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia.

OBJECTIVE: All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS: We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS: A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS: These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.

Amino Acid Sequence↗
PubMed2000

Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.

Animals↗
PubMed1999

Constitutive activation of the delta opioid receptor by mutations in transmembrane domains III and VII.

We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129 (domain III) [corrected], and Tyr308 (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPgammaS (where GTPgammaS is guanosine 5'-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [35S]GTPgammaS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents alpha-aminobutyric acid) efficiently reduced basal [35S]GTPgammaS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPgammaS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp128 (III) and Tyr308 (VII) interact with each other and that Tyr129 (III) undergoes H bonding with His278 (VI). Thus, Asp128, Tyr129, and Tyr308 may be involved in a network of interhelical bonds, which contributes to maintain the delta receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.

Amino Acid Substitution↗