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Results for “Functional characterization”

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PubMed2005

Biochemical and functional characterization of ORF138, a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae.

In cytoplasmic male sterility (CMS), original mitochondrial genes contribute to sex determinism by provoking pollen abortion. The function of the encoded proteins remains unclear. We studied the ORF138 protein, responsible for the 'Ogura' CMS, which is both used in hybrid seed production and present in natural populations. We analyzed the biochemical and structural properties of this protein in male-sterile plants and in E. coli. We showed that this protein spontaneously forms dimers in vitro. Truncated variants of the protein, containing either the hydrophobic or the hydrophilic moiety, also spontaneously dimerize. By fractionating mitochondria, we showed that ORF138 was strongly associated with the inner mitochondrial membrane of male-sterile plants. Our results also strongly suggest that ORF138 forms oligomers in male-sterile plant mitochondria. In E. coli, ORF138 was associated with the plasma membrane, as shown by membrane fractionation, and formed oligomers. The production of this protein strongly inhibited bacterial growth, but not by inhibiting respiration. The observed toxic effects required both the hydrophilic and hydrophobic moieties of the protein.

Base Sequence↗

PubMed2005

Identification and functional characterization of a novel interleukin 17 receptor: a possible mitogenic activation through ras/mitogen-activated protein kinase signaling pathway.

Interleukin-17 receptor (IL-17R) is increasingly emerged as a distinct receptor family functioning in diverse cellular processes including inflammation and cancer. In this study, we uncovered a novel member of IL-17R from mouse tissue that was named mouse IL-17RE (mIL-17R). Mouse IL-17RE cDNA is composed of at least 14 exons and presents at least 6 spliced isoforms (mIL-17RE1-6) with a molecular weight ranging from 34.2 to 70.1 kD. Mouse IL-17RE is expressed in limited tissues such as lung, kidney, stomach, intestine and testis, etc., and is mainly localized in the cytoplasm and on cell membrane. IL-17RE can also be detected in numerous tumor cell lines. Importantly, a mitogenic effect was detected in BaF3 cells stably transfected with the chimeric receptor fused by the ectodomain of erythropoietin receptor (EPOR) with the transmembrane and endomain of IL-17RE in a serum-dependent but EPO-independent manner. Moreover, ERK1/2 phosphorylation was significantly up-regulated as the dose of mIL-17RE increased. Specific RNAi targeting at mIL-17RE dramatically inhibited the activation of ERK1/2, indicating that mIL-17RE could functionally activate RAS/MAPK signaling pathway. Using dominant negative MEK (Dn-MEK) or RAS (Dn-RAS) as a signaling blocker, we were able to show that mIL-17RE probably activated RAS/MAPK signaling at or upstream of RAS. Overall, our results strongly indicate that mIL-17RE may belong to a novel growth-receptor like molecule that has the capability to support cellular mitogenesis through RAS/MAPK pathway.

Alternative Splicing↗

PubMed1998

Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: characterization of long tetracycline-inducible transcripts reading through the attachment site.

A detailed transcriptional analysis of the conjugative transposon Tn916 was carried out, which revealed that transcription of the transfer functions requires excision of the element and dramatically increases in the presence of tetracycline. The key components of this regulatory system are two contiguous transposon-borne genes, orf7 and orf8, located downstream from and having the same polarity of transcription as the tetracycline resistance determinant tetM. The gene orf7 encodes a 140-amino-acid (aa) protein exhibiting limited homology with sigmaF of Bacillus subtilis, whereas orf8 encodes a 76-aa peptide that does not share any sequence homology with any cognate proteins. In the presence of tetracycline, an attenuation mechanism enables the transcription of orf7 and orf8 from the tetM promoter. The resulting increased synthesis of ORF7 and ORF8 activates the promoter Porf7 located upstream from orf7, which then directs the expression of the transfer functions in the transposon circular intermediate through long transcripts encompassing the attachment site. The apparently non-regulated promoter Pxis located upstream of the excisionase encoding gene xis could also participate in the expression of the tra genes. We also demonstrate that Tn916 carries another regulated promoter, Porf9, which directs transcription of a single gene, orf9, located downstream from and transcribed counterclockwise to tetM. This gene encodes a 117-aa putative transcriptional repressor, but the exact role of this protein in the mobility of Tn916, as well as the regulation of its expression, remains to be elucidated. Our results constitute the molecular basis for the observation that tetracycline increased the transfer frequency of this type of element.

Bacillus subtilis↗

PubMed2007

Identification and functional characterization of genetic variants of human organic cation transporters in a Korean population.

Genetic variants of three human organic cation transporter genes (hOCTs) were extensively explored in a Korean population. The functional changes of hOCT2 variants were evaluated in vitro, and those genetic polymorphisms of hOCTs were compared among different ethnic populations. From direct DNA sequencing, 7 of 13 coding variants were nonsynonymous single-nucleotide polymorphisms (SNPs), including four variants from hOCT1 (F160L, P283L, P341L, and M408V) and three from hOCT2 (T199I, T201M, and A270S), whereas 6 were synonymous SNPs. The linkage disequilibrium analysis presented for three independent LD blocks for each hOCT gene showed no significant linkage among all three hOCT genes. The transporter activities of MDCK cells that overexpress the hOCT2-T199I, -T201M, and -A270S variants showed significantly decreased uptake of [(3)H]methyl-4-phenylpyridinium acetate (MPP(+)) or [(14)C]tetraethylammonium compared with those cells that overexpress wild-type hOCT2, and the estimated kinetic parameters of these variants for [(3)H]MPP(+) uptake in oocytes showed a 2- to 5-fold increase in K(m) values and a 10- to 20-fold decrease in V(max) values. The allele frequencies of the five functional variants hOCT1-P283L, -P341L, and hOCT2-T199I, -T201M, and -A270S were 1.3, 17, 0.7, 0.7, and 11%, respectively, in a Korean population; the frequency distributions of these variants were not significantly different from those of Chinese and Vietnamese populations. These findings suggest that genetic variants of hOCTs are not linked among three genes in a Korean population, and several of the hOCT genetic variants cause decreased transport activity in vitro compared with the wild type, although the clinical relevance of these variants remains to be evaluated.

1-Methyl-4-phenylpyridinium↗

PubMed1990

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons.

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.

Amino Acid Sequence↗

PubMed1996

Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

Amino Acid Sequence↗

PubMed2006

Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane.

Previously, in our laboratory a 40-kDa zinc transporter protein was purified and functionally reconstituted in proteoliposomes (Kumar R, Prasad R. Biochim Biophys Acta 1419: 23-32, 1999). Furthermore, we now report the identification of Slc39a10 cDNA encoding the 40-kDa zinc transporter protein by isolating a cloned DNA complementary to zinc transporter mRNA. cDNA was constructed from immunoenriched mRNA encoding the zinc transporter. cDNA was inserted into pBR322 using poly(dC)- poly(dG) tailing. Escherichia coli DH5alpha cells were transformed, and colonies were screened for zinc transporter cDNA by insertional inactivation. Plasmid DNA was purified from the ampicillin-sensitive clones, and the cDNA was sequenced from both strands. A basic local alignment research tool (BLAST) search of cDNA revealed that it belongs to the Slc39 gene family of zinc transporters and was designated as Slc39a10. Zinc transporter protein deduced on the basis of cDNA sequence was named rZip10 and consists of 385 amino acids with 9 predicted transmembrane domains. The Slc39a10 gene was abundantly expressed in both rat and human tissues. Increased extracellular zinc concentration resulted in upregulation of Slc39a10 in LLC-PK(1) cells expressing rZip10, which was downregulated at higher zinc concentrations. These cells accumulated more zinc than control cells. rZip10-mediated zinc uptake activity was time-, temperature-, and concentration-dependent and saturable which followed Michaelis-Menten kinetics with a K(m) of 19.2 microM and V(max) of 50 pmol x min(-1) x mg protein(-1). This activity was competitively inhibited by cadmium with K(i) of 91 microM. rZip10-mediated zinc uptake was inhibited by COOH group-modifying agents such as DCC. Immunofluorescence studies showed that rZip10 localizes to the plasma membrane of LLC-PK(1) cells.

Amino Acid Sequence↗

PubMed2000

Effects of VEGF on hemodynamics and cardiac function: characterization and mechanisms.

Vascular endothelial growth factor (VEGF), a mitogen specific for endothelial cells, produces beneficial angiogenesis in animal models of ischemic diseases; however, its side effects on hemodynamics and cardiac function may limit clinical use for some indications. This review focuses on the VEGF-induced hemodynamic profile, including hypotension, tachycardia, decreases in cardiac output and stroke volume, and changes in preload and afterload. The hemodynamic effects of VEGF are due to vasodilation and vascular hyperpermeability, which are mainly mediated by nitric oxide. Finally, the regimen or strategy for attenuation or prevention of these side effects is discussed.

Journal Article↗

PubMed1991

Domain structure in actin-binding proteins: expression and functional characterization of truncated severin.

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.

Actins↗

PubMed2005

Functional characterization of human proapoptotic molecules in yeast S. cerevisiae.

The presence of a complete (BH1-3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells. It is unclear, however, what distinct roles the members of the large family of BH3-only proapoptotic molecules play in apoptosis. Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict. We have, therefore, established three functional categories of BH3-only human proapoptotic proteins based on their toxicity after expression in budding yeast: directly killing (tBid), sensitizing in Bax/Bcl-2 expressing cells (Bad or Puma), and non-toxic (BNip3, BNip3L, and Noxa). The mechanism of killing by the proapoptotic molecules in yeast, however, is not due to activation of the recently described yeast metacaspase MCA1.

Apoptosis↗

PubMed2005

Genomic and functional characterization of stellate cells isolated from human cirrhotic livers.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are believed to participate in liver fibrogenesis and portal hypertension. Knowledge on human HSCs is based on studies using HSCs isolated from normal livers. We investigated the phenotypic, genomic and functional characteristics of HSCs from human cirrhotic livers. METHODS: HSC were obtained from normal and cirrhotic human livers. Cells were characterized by immunocytochemistry and gene microarray analysis. Cell proliferation, Ca(2+) changes and cell contraction were assessed by 3H-thymidine incorporation and by using an epifluorescence microscope. RESULTS: HSCs freshly isolated from human cirrhotic livers showed phenotypical features of myofibroblasts. These features were absent in HSCs freshly isolated from normal human livers and become prominent after prolonged culture. HSCs from cirrhotic human livers markedly express genes involved in fibrogensis, inflammation and apoptosis. HSCs from normal livers after prolonged culture preferntially expressed genes related to fibrogenesis and contractility. Agonists induced proliferation, Ca(2+) increase and cell contraction in HSCs isolated from human cirrhotic livers. Response to agonists was more marked in culture-activated HSCs and was not observed in HSCs freshly isolated from normal livers. CONCLUSIONS: HSCs from human cirrhotic livers show fibrogenic and contractile features. However, the current model of HSCs activated in culture does not exactly reproduce the activated phenotype found in cirrhotic human livers.

Calcium↗

PubMed2003

Two Italian kindreds with familial hypocalciuric hypercalcaemia caused by loss-of-function mutations in the calcium-sensing receptor (CaR) gene: functional characterization of a novel CaR missense mutation.

OBJECTIVES: Description of two unrelated Italian kindreds with familial hypocalciuric hypercalcaemia (FHH), an autosomal dominant disease mostly caused by heterozygous inactivating mutations of the Ca2+ sensing receptor (CaR). PATIENTS AND DESIGN: We studied 11 members of the two families. Genomic DNA was isolated from peripheral blood leucocytes in all family members and in 50 unrelated Italian controls. Total serum and ionized calcium, PTH, creatinine, phosphate, magnesium, and urinary calcium clearance to creatinine clearance ratio were measured. Direct sequencing of the entire coding region of the CaR was performed in the probands. Functional studies were performed in COS-7 cells transiently expressing the mutated CaR. RESULTS: In the proband of family A direct sequencing revealed a novel heterozygous Y218C missense mutation in exon 4. The same mutation was identified in the affected but not in the unaffected family members or in any of the 50 unrelated Italian controls. Transient expression of the Y218C CaR in COS-7 cells revealed a blunted Ca2+-evoked accumulation of inositol trisphosphates, indicating that the Y218C is a loss-of-function mutation. Cotransfection experiments showed that the mutant receptor had no impact on the function of the wild-type receptor, suggesting that a reduced expression of the normal CaR, rather than a dominant-negative effect, accounted for the functional impairment. In the proband of family B an already described heterozygous P55L missense mutation in exon 2 of the CaR gene was found. The same mutation was identified in the affected family members. CONCLUSIONS: We described two familial hypocalciuric hypercalcaemia kindreds with loss-of-function mutations of the Ca2+ receptor gene and identified a novel heterozygous mutation (Y218C) characterized by a blunted response to Ca2+ stimulation compared to the wild-type receptor and no interference with the function of the wild-type Ca2+ receptor.

Adult↗

PubMedDate not supplied

Functional characterization of the mouse lymphotoxin-beta receptor promoter.

The lymphotoxin beta-receptor (LT beta R), a member of the tumor necrosis factor (TNF) receptor family, plays a crucial role in lymphoid organogenesis by signaling through its functional ligand LT alpha(1)beta(2). While the receptor is expressed on a wide range of cell types e.g. fibroblasts and monocytes, the ligand is expressed only on activated T, B and NK cells. Remarkably, no cell type has been identified so far that expresses both the receptor and the ligand. In order to characterize the mouse LT beta R gene expression on a molecular level, we isolated about 1 kb of the 5' flanking region of the LT beta R gene. Primer extension analysis revealed one transcriptional start site located at - 60 upstream of the ATG-containing first exon. Northern blot analysis showed that the LT beta R is abundantly expressed in the mouse fibroblast cell line NIH 3T3, and to a lesser extent, in the mouse macrophage-like cell line RAW 264.7. To determine whether the 5' flanking region exerts functional promoter activity, we generated deletion mutants fused to the luciferase reporter gene. Transfection experiments using these reporter gene constructs showed that the isolated 5' flanking region is transcriptionally active in NIH 3T3 and RAW 264.7 cells, and determined a minimum length required for the transcriptional activity of the LT beta R promoter in these cells. Further sequence analysis of the isolated 5' flanking region identified a number of putative DNA-binding sites for transcription factors. Interestingly, incubation of NIH 3T3 cells with dexamethasone resulted in an elevated mRNA level of the LT beta R gene. This effect was abolished by using the specific glucocorticoid receptor inhibitor RU486, indicating an increased transcriptional activity of the LT beta R promoter after glucocorticoid stimulation.

3T3 Cells↗

PubMed1990

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.

Amino Acid Sequence↗

PubMed1996

Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells.

Bile acids are taken up into human liver by Na+-dependent and Na+-independent transport mechanisms. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of organic anions is markedly reduced. We have investigated the molecular and functional derangements of bile acid transport in the human hepatoblastoma cell line HepG2. Uptake of [3H]-taurocholic acid was saturable and entirely Na+ independent, with the kinetic characteristics of the human liver organic anion transporting polypeptide (OATP). OATP, but not the Na+-dependent bile acid transporter (Na+-taurocholate-cotransporting polypeptide [NTCP]), was detectable by reverse-transcription polymerase chain reaction (RT-PCR) analysis of HepG2 RNA. The level of OATP expression in HepG2 cells was determined by Northern blot analysis and was found to be 40% in comparison with normal liver. Transfection of an OATP-derived phosphorothioate (PTO)-antisense oligonucleotide into HepG2 cells resulted in 77% inhibition of temperature-dependent bile acid uptake. Injection of HepG2 messenger RNA (mRNA) into Xenopus laevis oocytes significantly stimulated Na+-independent taurocholate uptake, indicating the expression of a bile acid transport protein. We conclude that bile acid uptake into human hepatoblastoma HepG2 cells is mediated by the multi-specific organic anion transporting polypeptide OATP. Therapeutic strategies employing bile acid-derived cytostatic agents for the treatment of hepatocellular carcinomas may therefore depend upon the expression of the Na+-independent bile acid transporter OATP in hepatic malignancies.

Animals↗

PubMed2026

Genome-wide identification and evolutionary analysis of the ERF-VII gene family in the tea plant (Camellia sinensis) and functional characterization of CsRAP2.2 in response to cold stress.

The ERF-VII gene family, a critical branch of the AP2/ERF superfamily, is central to plant stress adaptation. However, its evolutionary history and function in tea plant (Camellia sinensis) remain unclear. Here, we performed integrated evolutionary, genomic, and functional analyses of ERF-VII genes across 14 plant lineages and 20 tea plant cultivars. The phylogenetic analysis revealed that ERF-VII proteins originated after vascular plant divergence, coinciding with the emergence of the N-terminal MCGGA/I motif linked to the oxygen-dependent N-degron pathway. Gymnosperms retained few conserved members, whereas angiosperms exhibited lineage-specific expansion-extensive in monocots via whole-genome duplication, moderate in eudicots with functional diversification. Pan-genome analysis across 20 tea plant cultivars further revealed varietal differences in ERF-VII gene distribution. Transcriptome profiling via the Tea Plant Information Archive identified CsRAP2.2 as a cold-inducible ERF-VII member with sustained expression under low-temperature stress. Functional assays demonstrated that silencing CsRAP2.2 reduced cold tolerance, while overexpression in tea leaves and heterologous expression in Arabidopsis thaliana enhanced cold tolerance by maintaining photosystem II efficiency, reducing membrane lipid peroxidation, and improving antioxidant capacity. Weighted gene co-expression network analysis positioned CsRAP2.2 as a regulatory hub integrating cold, hormone, and oxygen-sensing pathways. These results clarify the evolutionary trajectory of ERF-VII genes and establish CsRAP2.2 as a core cold-tolerance regulator in tea plant. These findings may inform future breeding of cold-resilient tea cultivars.

Camellia sinensis↗

PubMed1997

Functional characterization of a kindling-like model of ethanol withdrawal in cortical cultured neurons after chronic intermittent ethanol exposure.

Chronic ethanol exposure has been reported to alter NMDA and GABA(A) receptor function and gene expression in brain regions of animals and mammalian cultured cortical neurons. In the present study, we investigated the effects of another model of chronic, but intermittent, ethanol treatment (CIE) on GABA(A) and NMDA receptor systems in cortical neurons. CIE (50 mM ethanol, 12 h exposure/12 h withdrawal, 5 cycles) exposure produced increased [3H]MK-801 binding and diazepam insensitive binding sites as measured by [3H]Ro15-4513 binding to cortical cultured neuronal membranes, at 0 h following the last treatment cycle relative to control neurons. The NMDA mediated increase in intracellular calcium [Ca2+]i was also increased following similar CIE treatment. CIE treatment also increased the ability of pentylenetetrazol (PTZ) to inhibit GABA mediated 36Cl- influx relative to control neurons. These effects were not reversible following 1 week ethanol withdrawal, implying enhanced sensitivity of PTZ to inhibit GABA(A) receptor mediated inhibition, and an increased NMDA receptor function in CIE treated cortical neurons. These alterations are consistent with the behavioral studies in animals, and suggest that both GABA(A) and NMDA receptors play an important role in ethanol withdrawal following either chronic or CIE exposure. Furthermore, this provides a feasible in vitro model for further biochemical and molecular studies of the mechanism underlying the CIE induced kindling-like phenomenon observed in humans.

Animals↗

PubMed1996

Functional characterization of a mutant thyroid hormone receptor in Xenopus laevis.

Thyroid hormone plays a causative role during frog metamorphosis, and its effect is mediated by thyroid hormone receptors (TRs). To investigate the function of Xenopus TRs, we have recently developed a thyroid hormone dependent in vivo transcription system by introducing TRs and RXRs (9-cis-retinoic acid receptors) into Xenopus oocytes. Interestingly, using this system, we have found that the TRalphaB cloned previously is defective in transcriptional activation compared with TRalphaA. In vitro DNA binding experiments show that TRalphaB.RXR heterodimers have drastically reduced affinity for a thyroid hormone response element. Site-directed mutagenesis shows that two of the seven amino acid residues that differ between TRalphaA and TRalphaB are responsible for the defect in TRalphaB function. These two residues affect the DNA binding by both TR.RXR heterodimers and TR homodimers. In contrast, heterodimer formation with RXRs is not affected as demonstrated by coimmunoprecipitation and dominant-transcriptional inhibition experiments. By cDNA and genomic DNA sequence analysis, we have demonstrated that the residues, which affect TRalphaB function when mutated, are identical between the wild type TRalphaB and TRalphaA. Thus, our experiments have discovered the first amphibian TR mutant. The DNA binding and transcription activation functions of the mutant are discussed in relation to the recently published TR crystal structure.

Amino Acid Sequence↗