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At least 1,405 records · Page 78Linked to original sources
PubMed1994

A role for intronic sequences on expression of thyroid hormone receptor alpha gene.

We have cloned and characterized the organization of the rat thyroid hormone receptor alpha (THR) gene. Multiple transcription start sites were mapped by RNA primer extension analyses. The promoter of the rat THR alpha gene does not contain a TATA or CAAT box. Deletion analyses of the 5' region of THR alpha gene and transfection assays, using NIH3T3 and NG108-15 cells, revealed that the sequences from -137 to +205 (+205 resides in the first intron) are necessary for efficient expression of this gene. This region contains two positively acting elements, the sequence -137 to -60 upstream from the major start of transcription and three copies of an AGG sequence located in the first intron. In contrast, two octamer-binding motifs in the first intron function as the negative regulatory elements. Gel mobility shift assays showed that the purine-rich sequence and the octamer-binding motifs bind to a protein(s) present in NIH3T3 and NG108-15 cells, the recipients in transient transfection assays. Genomic sequence comparison of THR alpha and beta revealed the presence of the purine-rich track in both genes, while the octamer-binding motifs were found only in the alpha gene. These results might explain the differential regulation of THR alpha and beta gene expression previously noted.

3T3 Cells↗
PubMed2001

Expression of humanized Fab fragments that recognize the IgE-binding domain of human Fc(epsilon)RIalpha in COS and CHO cells.

Interfering with the binding of IgE to high-affinity IgE receptor alpha chain (Fc(epsilon)RIalpha) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc(epsilon)RIalpha inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc(epsilon)RIalpha Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc(epsilon)RIalpha binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc(epsilon)RIalpha will provide useful information for the design of therapeutic reagents for allergy and asthma.

Animals↗
PubMed1996

Cell type-specific mechanisms regulate hepatitis B virus transgene expression in liver and other organs.

Intracellular expression of hepatitis B virus (HBV) was analysed in transgenic HBV mouse lines designated G7 and G26, the former lacking hepatitis B surface antigen (HBsAg) promoters. HBsAg mRNA expression was greater in the G26 line than in the G7 line, although in situ hybridization showed a qualitatively similar expression pattern in specific cell types. HBsAg mRNA was most abundant in hepatocytes, followed in magnitude by proximal renal tubular epithelial cells, pancreatic acinar cells, and epithelial cells of the gastric, small intestinal, and bronchiolar mucosae. In biliary epithelial cells, brain, spleen, large intestine, testis, heart, and skeletal muscle, HBsAg mRNA was undetectable. In cell transfection assays, the HBV enhancer/preS1 promoter efficiently expressed a luciferase reporter with appropriate upregulation by HNF-3 alpha and C/EBP alpha transcription factors in hepatocyte-derived cells but not in non-parenchymal epithelial liver cells or fibroblasts. These results suggest that cell-type specificity of HBV expression is regulated by interactions between viral elements and cellular transactivators. Variable expression of G7 and G26 HBV transgenes in epithelial cells combined with differences in transgene expression in similar sets of cells suggests at least two levels of regulation: one directing cell specificity of HBV expression and the other governing quantitative expression of HBV mRNA.

Animals↗
PubMed1990

Hemodynamic evaluation of common cardiac arrhythmias.

Hemodynamic effects of arrhythmias are considered to be relatively unimportant unless the heart rate is very slow or very rapid. The present study describes a simple method which enables determination of beat-to-beat changes in stroke volume, cardiac output, double product and cardiac efficiency expressed as the interrelationship between the last two parameters. It is obtained by calculating stroke volume from directly measured values of arterial pressure, using a modification of a formula described for irregular rhythms. The calculated parameters are expressed as the percentage of the values of the sinus beat or state occurring with normal cardiac rhythm. The results confirmed that atrial ectopic beats have a less deleterious effect on myocardial function than those from ventricular or nodal origin. The change in stroke volume or cardiac output may be accompanied by either a decreased or increased double product and/or cardiac efficiency. This depends on the type of arrhythmia, the number of ectopic beats per minute and the condition of the patient's heart. The method provides measurement of this parameter in a given patient at a given state as well as at other different frequencies of the same arrhythmia. It demonstrates then which frequency induces hemodynamic changes of sufficient degree to justify antiarrhythmic therapy. The method may be useful in optimising care of patients in units for critical care, or even in outpatient departments.

Arrhythmias, Cardiac↗
PubMed1992

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗
PubMed1987

Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli.

Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.

Amino Acids↗
PubMed2003

[Expression and purification of the N-domain of human canstatin and its bioactivity].

Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3). The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3% of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6%. CAM assay showed that N-domain effectively inhibited the angiogenesis of chicken embryo microcapillary vessel.

Angiogenesis Inhibitors↗
PubMed2001

Defective Gi protein coupling in two formyl peptide receptor mutants associated with localized juvenile periodontitis.

The formyl peptide receptor (FPR) is a prototypical chemoattractant receptor expressed in neutrophils. It is well known that the FPR couples to G(i) proteins to activate phospholipase C, chemotaxis, and cytotoxic cell functions, but the in vivo role of the FPR in man has remained elusive. Recently, F110S and C126W mutations of the FPR have been associated with localized juvenile periodontitis. We studied FPR-F110S and FPR-C126W in comparison with wild-type FPR (FPR-WT) by coexpressing epitope-tagged versions of these receptors with the G protein Galpha(i2)beta(1)gamma(2) in Sf9 insect cells. FPRs were efficiently expressed in Sf9 membranes as assessed by immunoblotting using the beta(2)-adrenoreceptor as a standard. FPR-C126W differed from FPR-WT and FPR-F110S in migration on SDS-polyacrylamide gels and tunicamycin-sensitive glycosylation. FPR-WT efficiently reconstituted high-affinity agonist binding and agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i2)beta(1)gamma(2). In contrast, FPR-F110S only weakly reconstituted agonist-stimulated GTPgammaS binding, and FPR-C126W was completely inefficient. Collectively, our data show almost complete and complete loss of G(i) protein coupling in FPR-F110S and FPR-C126W, respectively. The severe functional defects in FPR-F110S and FPR-C126W contrast with the discrete clinical symptoms associated with these mutations, indicating that loss of FPR function in host defense is, for the most part, readily compensated.

Aggressive Periodontitis↗
PubMed1999

Phenotypic consequence of the gene abnormality in the platelet glycoprotein IX gene observed in a patient with Bernard-Soulier syndrome through mammalian cell expression system.

We previously reported a patient with Bernard-Soulier syndrome (BSS) who had a homozygous missense mutation in the GPIX gene Phe55 (TTT) to Ser (TCT) replacement in the leucine-rich motif (LRM) of the GPIX polypeptide, as a probable cause of BSS phenotype. To study the effect of this mutation on the surface expression of the GPIb/IX complex and GPIX, we introduced this mutation into the cDNA of GPIX by site-directed mutagenesis and performed in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex. Mutant GPIX could not increase the surface expression of GPIb-alpha, but also surface expression of GPIX itself. Immunostaining of the CHO-K1 cells transfected with two plasmids for mutant GPIX and GPIb-beta showed that mutant GPIX was weakly but certainly detected in the cytoplasm of the transfected cells. These findings indicate that this substitution is responsible for BSS phenotype and that the LRM of GPIX is a critically important element for an efficient expression of the GPIb/IX complex. They also suggest that the failure of GPIb/IX complex expression can be caused by a mechanism other than synthetic defect of the mutant protein.

Animals↗
PubMed2001

cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450.

The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.

Amino Acid Sequence↗
PubMed2003

Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line.

AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 +/- 0.025 nmol.min(-1).mg(-1) S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

Base Sequence↗
PubMed2002

Expression and purification of recombinant bone morphogenetic protein-2 in E.coli.

OBJECTIVE: To explore the method for producing human bone morphogenetic protein-2 (hBMP-2) by gene engineering techniques. METHODS: E.coli BL21 was transformed with recombinant plasmid pYR (pBV220-hBMP-2) under different conditions, and SDS-PAGE analysis was conducted to observe the effects of the activation status and induction time of the bacterium on the target protein expression. The inclusion bodies obtained from E.coli were purified by anion exchange chromatography DEAE and molecular sieve S-300, and the recombinant protein was renatured by dialyse. RESULTS: SDS-PAGE analysis showed a conspicuous band after induction signifying a new foreign protein with relative molecular mass of approximately 13 000. After activation of the bacteria when D600 was about 0.45, most efficient expression of rhBMP-2 was achieved which reached the peak 4 h after induction with heat. Implantation of the purified recombinant hBMP-2 resulted in proliferation of mesenchymal cells and new cartilage and bone formation, as shown by histological analysis 4 weeks after implantation. CONCLUSION: hBMP-2 produced by gene engineering techniques possesses the biological capacity of ectopic bone formation.

Animals↗
PubMed2006

Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli.

Antibody molecules bind to antigen with six complementary determining region (CDR) loops, three of which are located on each variable heavy (V(H)) and light (V(L)) chains. Discovery and optimization of antibodies that bind antigen using in vitro techniques require diversification of one or more of these CDRs. Since antibodies are dimeric, simultaneous diversification of heavy and light chains on separate genetic elements would allow "chain shuffling" to occur simply and efficiently. Efficient expression of antibody V(H) and V(L) requires that the two separate replicons be compatible with one another, but also have similar properties, such as copy number in E. coli. Standard plasmids that are compatible with one another in E. coli exist at widely variable copy numbers. Recently we described the isolation of ColE1 mutants that have similar copy numbers but different incompatibility characteristics. Thus, new compatibility groups in the ColE1 family were established. Herein we describe the E. coli expression of V(H) and V(L) genes to form a functional Fab. The ability to express antibody heavy and light chains from separate but compatible high copy plasmids should allow new opportunities in antibody engineering, such as rapid chain shuffling and generation of more complex antibody libraries.

Biological Evolution↗
PubMed2004

Construction of eukaryotic expression plasmid of human PRX3 and its expression in HEK-293FT cells.

To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.

Cell Line, Transformed↗
PubMed2006

Use of ribosomal promoters from Burkholderia cenocepacia and Burkholderia cepacia for improved expression of transporter protein in Escherichia coli.

Expression of heterologous protein in Escherichia coli usually based on the IPTG-inducible expression systems. The use of these systems for membrane protein production, however, usually caused cytotoxic problem that affected the yield and functional characterization of the protein. Optimization of these systems for transporter protein production is time-consuming and is usually ineffective. Here, we described the use of the ribosomal promoters P(s12) from Burkholderia cenocepacia LMG16656 and from Burkholderia cepacia MBA4 for efficient expression of functional transporter protein in E. coli. These promoters were used to drive the expression of a transmembrane protein, Deh4p, which help transport monohaloacetates into B. cepacia MBA4 for metabolism. Production of Deh4p in E. coli using an IPTG-inducible promoter resulted in no expression in uninduced condition and cell lysis in the presence of IPTG. Moreover, it has been reported that IPTG increased the endogenous production of other permeases such as LacZ and MelB. Cells expressing Deh4p from a P(s12) promoter grew normally in rich medium and which did not increase the expression of other permease. Uptake of (14)C-monochloroacetic acid has confirmed the production of the transporter protein in these cells. The results showed that the constitutive ribosomal protein promoters from the Burkholderia sp. could be used for effective expression of transporter protein in E. coli without causing any detrimental and unnecessary effect.

Bacterial Proteins↗
PubMed2006

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels.

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.

Amino Acid Sequence↗
PubMed1991

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.

Amino Acid Sequence↗
PubMed2003

Murine cytomegalovirus abortively infects human dendritic cells, leading to expression and presentation of virally vectored genes.

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.

Blotting, Western↗