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PubMed2001

Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver.

BACKGROUND & AIMS: Hepatic uptake of cholephilic organic compounds is mediated by members of the organic anion-transporting polypeptide (OATP) family. We aimed to characterize the novel OATP-B with respect to tissue distribution and hepatocellular localization and to compare its substrate specificity with those of OATP-A, OATP-C, and OATP8. METHODS: Tissue distribution and hepatocellular localization of OATP-B were analyzed by Northern blotting and immunofluorescence, respectively. Transport of 16 substrates was measured for each individual human OATP in complementary RNA-injected Xenopus laevis oocytes. RESULTS: Expression of OATP-B was most abundant in human liver, where it is localized at the basolateral membrane of hepatocytes. OATP-B, OATP-C, and OATP8 mediated high-affinity uptake of bromosulphophthalein (K(m), approximately 0.7, 0.3, and 0.4 micromol/L, respectively). OATP-B also transported estrone-3-sulfate but not bile salts. Although OATP-A, OATP-C, and OATP8 exhibit broad overlapping substrate specificities, OATP8 was unique in transporting digoxin and exhibited especially high transport activities for the anionic cyclic peptides [D-penicillamine(2,5)]enkephalin (DPDPE; opioid-receptor agonist) and BQ-123 (endothelin-receptor antagonist). CONCLUSIONS: OATP-B is the third bromosulphophthalein uptake system localized at the basolateral membrane of human hepatocytes. OATP-B, OATP-C, and OATP8 account for the major part of sodium-independent bile salt, organic anion, and drug clearance of human liver.

Animals↗
PubMed2002

Transport of a neurotoxicant by molecular mimicry: the methylmercury-L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2.

Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an L-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1-4F2 heavy chain (4F2hc) and LAT2-4F2hc. Oocytes expressing LAT1-4F2hc or LAT2-4F2hc demonstrated enhanced uptake of [(14)C]MeHg when administered as the L-cysteine or D,L-homocysteine complexes, but not when administered as the D-cysteine, N -acetyl-L-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities ( K (m)) of MeHg-L-cysteine uptake by LAT1 and LAT2 (98+/-8 and 64+/-8 microM respectively) were comparable with those for methionine (99+/-9 and 161+/-11 microM), whereas the V (max) values were higher for MeHg-L-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [(3)H]methionine and [(14)C]MeHg-L-cysteine were trans -stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg-L-cysteine is both a cis - and trans -substrate. In addition, [(3)H]methionine efflux was trans -stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.

Amino Acid Transport System y+↗
PubMed1989

The block to transcription elongation at the SV40 attenuation site is decreased in vitro by oligomers complementary to segments of the attenuator RNA.

We have previously reported that a mechanism resembling attenuation in prokaryotes regulates simian virus 40 (SV40) late gene expression. We have suggested that modulation of the attenuator RNA secondary structure is an integral element regulating the elongation block at the attenuation site [Hay et al., Cell 29 (1982) 183-193]. In the present study, oligodeoxyribonucleotides (oligos), 13-19 nucleotides long, were used to probe the involvement of the attenuator RNA secondary structure in the control of elongation block at the SV40 attenuation site. These oligos are complementary to segments of the attenuator RNA suggested to play a role in the regulation of attenuation. The oligos were added to an in vitro transcription reaction containing SV40 transcription complexes, and their effect on transcription through the attenuation site was measured. As predicted, the three oligos caused specific decreases in the elongation block at the SV40 attenuation site. These results provide direct evidence for the involvement of RNA secondary structure in the attenuation mechanism in SV40.

Animals↗
PubMed2002

Identification and expression of deoxyribonuclease (DNase) I alternative transcripts in the rat.

Deoxyribonuclease (DNase) I has been implicated in the induction of DNA fragmentation and cell death, however little is known about its regulation in vivo. In the present study, we describe that DNase I messenger RNA (mRNA) is alternatively spliced in rat kidney, and the activity of the DNase I correlates with the alternative splicing during the course of renal ischemia/reperfusion. Northern blot analysis with mRNA from control rat kidneys and kidneys subjected to ischemia/reperfusion in vivo yielded two bands of approximately 1.3 and 1.5 kb, suggesting the possibility of alternative splicing. However, prolonged reperfusion up to 16 h resulted in the predominant expression of 1.3 kb transcript. The disappearance of the 1.5 kDa band was associated with the increased DNase I activity in the kidney during ischemia/reperfusion. To study the alternative splicing of the DNase I mRNA, rat kidney cortex DNA complementary to RNA library was screened using rat DNase I probe. Twenty-one positive clones were obtained and were compared with the reported DNase I mRNA transcript cloned from rat parotid gland. All clones showed 100% homology with the reported DNase I coding sequence and part of 5'-untranslated region (5'-UTR), named exon 1a by us. Twelve out of 21 isolated clones had longer 5'-UTR than previously described, and DNase I pre-mRNA was alternatively spliced in this region. Six out of these 12 clones contained extra up to 153 bp in extreme 5' end, whereas, in six other clones, an internal 132 bp segment (exon 1b) of this additional sequence was absent, and only the extreme 5'UTR sequence (exon 1c) was found in these clones. The nucleotide analysis showed that alternating exon 1b has the possibility of a secondary structure with high internal homology and potential for at least one major stable stem-loop. Both newly identified segments, exons 1b and 1c, were also identified in genomic DNA. The long splice variant, containing exon 1b, is expressed only in the kidney among different tissues tested. Exon 1b inhibited translational activity of DNase I mRNA in vitro. Our data suggest that alternative splicing in 5'-UTR in the kidney may provide a prompt DNA-independent regulation of DNase I activity when DNA is damaged during ischemic injury.

Alternative Splicing↗
PubMed1985

Expression of cellular oncogenes in teratoma-derived cell lines.

The expression of ten proto-oncogenes was studied in cell lines derived from transplantable mouse teratomas. The cell lines represent different forms of early embryonic cell specialization. The analysis included two embryonal carcinoma (EC) lines (PCC3 and F9), and four differentiated cell lines derived from teratocarcinoma, namely trophoblastoma (3-TDM), parietal endoderm (PYS-2), visceral endoderm (PSA5-E) and skeletal myoblasts (Cl10). The expression of c-oncogenes was studied by analysing poly(A)+RNA for complementary sequences by dot blot and Northern blot hybridization. The results were related to the rate of cell multiplication and the state of differentiation by examining [3H]thymidine incorporation, growth curves and tissue-specific differentiation markers. Expression of c-myc and c-Ki-ras was found in all cell lines. In dot blot assays, poly(A)+RNA from all cell lines also hybridized with v-abl and v-sis probes. A marked decrease in c-myc expression was found in teratoma-derived myoblasts differentiating into myotubes. A similar reduction was found when 'nullipotent' F9 cells were induced by retinoic acid (RA) to form primitive endoderm. However, reduction of the growth rates of the parietal and visceral endodermal cell lines were not accompanied by decreased expression of c-myc or c-Ki-ras. Hybridization signals obtained with a v-sis probe was low in all teratoma-derived cell lines tested, except for the myogenic cell line Cl10. Both in exponentially growing and differentiated cultures of this line, two size classes of transcripts hybridized strongly to the v-sis probe. However, these transcripts, 7 and 3 kb, most likely represent endogenous retroviral transcripts and not c-sis transcripts. Expression of c-myb, c-mos, c-fes, c-src and c-erb A and c-erb B could not be detected in any of the cell lines studied.

Animals↗
PubMed1968

Double-stranded Ribonucleic Acid from Cytoplasmic Polyhedrosis Virus of the Silkworm.

Ribonucleic acid (RNA) was extracted by phenol treatment from cytoplasmic polyhedrosis virus isolated from the midgut of infected silkworms. This RNA appears as threads when precipitated in alcohol. Two components having different sedimentation constants were observed. The molecular weight of the RNA preparation obtained by sedimentation coefficient (weight-averaged) and intrinsic viscosity was about 2 x 10(6) to 3 x 10(6). It was one-half to one-third the size of the calculated molecular weight for an entire RNA molecule in a virion. Electron micrographs of this RNA preparation showed two peaks in the distribution of contour length, at 0.4 and 1.3 mum, which would correspond to molecular weights of 10(6) and 3 x 10(6), respectively. The extracted RNA seemed to split into segments at a preferential breaking point. This RNA was soluble in concentrated salt solution, differing from single stranded high-molecular-weight RNA. The base composition of this RNA was complementary in the ratios of adenosine to uridine and guanosine to cytosine. It contained 43% guanosine plus cytosine. Based on its filamentous appearance by electron microscopy, typical pattern of optical rotatory dispersion and circular dichroism, sharp transition of the optical properties on heating, great hyperchromicity on degradation, nonreactivity with formaldehyde, and resistance to ribonucleases, it is concluded that this RNA is double-stranded and has regular base pairings of guanosine-cytosine and adenosine-uridine.

Journal Article↗
PubMed1999

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8. 4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8. 0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8. 0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8' and 8. Its synthesis was inhibited by cycloheximide but not by cytosine beta-D-arabinofuranoside, which suggests that the 2. 0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

Animals↗
PubMedDate not supplied

Nucleosides and nucleotides. 170. Synthesis and properties of oligodeoxynucleotides containing 5-[N-[2-[N,N-bis(2-aminoethyl)- amino]ethyl]carbamoyl]-2'-deoxyuridine and 5-[N-[3-[N,N-bis(3-aminopropyl) amino]propyl]carbamoyl]-2'-deoxyuridine.

The synthesis of oligodeoxynucleotides (ODNs) containing 5-[N-[2-[N,N-bis(2-aminoethyl)amino]ethyl]-carbamoyl]-2'-deoxyuridine (BAE) and 5-[N-[3-[N,N-bis(3-aminopropyl)amino]propyl]carbamoyl]-2'- deoxyuridine (BAP) is described. The thermal stabilities of duplexes containing these ODNs and either the complementary DNA or RNA strand and of triplexes consisting of these ODNs and the target duplex were studied by thermal denaturation. ODNs containing BAE or BAP stabilize duplex formation with either the complementary DNA or RNA strands but destabilize triplex formation with the target duplex. Furthermore, the resistance of these ODNs to nuclease hydrolysis was studied by using snake venom phosphodiesterase (a 3'-exonuclease) and nuclease S1 (an endonuclease). It was found that ODNs containing either BAE or BAP were more resistant to nucleolytic hydrolysis by either of the nucleases than the unmodified ODN.

Base Sequence↗
PubMed1997

RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.

Amanitins↗
PubMed1995

Synthesis of novel products in vitro by an RNA-dependent RNA polymerase.

RNA-dependent RNA polymerase from turnip crinkle virus-infected turnip transcribes both strands of a virus-associated satellite RNA, sat-RNA C (356 bases), in vitro. While both plus- and minus-strand sat-RNA C can direct the synthesis of full-length complementary-strand products, transcription of minus-strand RNA also generates two non-template-sized products, L-RNA and S-RNA (C. Song and A. E. Simon, Proc. Natl. Acad. Sci. USA 91:8792-8796, 1994). Here we report that synthesis of L-RNA and S-RNA results from terminal elongation of the 3' end of the template. L-RNA has a panhandle structure and is composed of minus-strand template covalently linked to newly synthesized RNA complementary to its 5' 190 bases. S-RNA is composed of template covalently linked to its full-length complementary strand. All minus-strand templates tested yielded S-RNA. However, synthesis of L-RNA was affected by deletion of the 3' end of the minus-strand template or several internal regions and base alterations near the 5' end or in an internal sequence immediately upstream from the template-product junction that could potentially form a heteroduplex with the 3' end. Furthermore, mutations that disrupted or restored a stem-loop involved in RNA recombination in vivo affected the level of L-RNA produced in vitro, suggesting that the mechanisms for intramolecular formation of panhandle RNAs and intermolecular RNA recombination involve similar features.

Base Sequence↗
PubMed1971

Mumps virus replication in chick embryo lung cells: properties of ribonucleic acids in virions and infected cells.

Ribonucleic acid (RNA) species in mumps virions and in infected cells were compared. The predominant RNA species in virions labeled with (3)H-uridine sedimented at 50S; RNA species sedimenting at 28, 18, and about 10S were also present. The virion-associated RNA species sedimenting slower than 50S contained some nucleotide sequences similar to 50S virion RNA. Although mumps virus replication was severely inhibited by high concentrations of actinomycin D, some virus was made, and virus-specific RNA species accumulated in infected cells. Mumps virus resembled other paramyxoviruses in inducing, in infected cells, synthesis not only of 50S RNA but also of slower sedimenting RNA species with a peak distribution at about 18S, complementary in base sequences to 50S virion RNA. In addition, base sequences of the parental type were relatively abundant in the RNA species sedimenting slower than 50S; these may represent precursors of the slowly sedimenting RNA species associated with virions. Ribonuclease-resistant RNA was detected in infected cells; this may represent replicative or transcriptive intermediates. Inhibition of protein synthesis with cycloheximide severely depressed accumulation of labeled 50S RNA in infected cells but did not interfere with accumulation of RNA species sedimenting slower than 50S. Actinomycin D treatment had a similar effect. Annealing of genomes and virus-induced complementary RNA species of Newcastle disease virus, Sendai virus, and mumps virus did not reveal any base sequence homologies.

Animals↗
PubMed1971

Chloroplast RNA populations in dark-grown, light-grown, and greening Euglena gracilis.

RNA preparations from dark-grown, light-grown, and greening Euglena gracilis have been compared by polyacrylamide gel electrophoresis and by hybridization to Euglena chloroplast DNA. Chloroplast ribosomal RNA is not detected in dark-grown cells; its abundance increases in greening cells over a 72 hr period until the concentration characteristic of light-grown cells is reached. Other RNA species complementary to chloroplast DNA are present in comparable abundance in light-grown, dark-grown, and greening cells.

Autoradiography↗
PubMed2004

Novel single nucleotide polymorphisms of organic cation transporter 1 (SLC22A1) affecting transport functions.

Organic cation transporter OCT1 (SLC22A1) plays an essential role in absorption, distribution, and excretion of various xenobiotics including therapeutically important drugs. In the present study, we analyzed the functional properties of the single nucleotide polymorphisms (SNPs) in SLC22A1 gene found in Japanese control individuals. Four mutations resulting in the amino acid changes (F160L, P283L, R287G, and P341L) were functionally characterized in Xenopus oocyte expression system. Two new SNPs, identified in Japanese population, P283L and R287G exhibited no uptake of both [14C]TEA and [3H]MPP+, although their protein expressions were detected in the plasma membrane of the oocytes injected with their cRNAs. Uptake of [14C]TEA by P341L was reduced to 65.1% compared to wild type, whereas F160L showed no significant change in its transport activity. This study suggests that the newly found OCT1 variants will contribute to inter-individual variations leading to the differences in cationic drug disposition and perhaps certain disease processes.

Animals↗
PubMed2004

Aromatic amino acid transporter AAT-9 of Caenorhabditis elegans localizes to neurons and muscle cells.

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

Amino Acid Transport Systems↗
PubMed1981

Mouse complement components C3 and C4. Characterization of their messenger RNA and molecular cloning of complementary DNA for C3.

Mouse liver mRNA species which direct the cell-free synthesis of pro-C3 and pro-C4 polypeptides with an apparent molecular weight of 175,000 and 190,000, respectively, were shown to sediment faster than 28 S. By electrophoresis in a denaturing gel the mRNA for C3 was determined to contain approximately 7,500 ribonucleotides. cDNA was synthesized from size-fractionated mouse liver mRNA and cloned in the plasmid pBR 322. Among the cDNA clones recovered three were identified as being complementary to portions of the mRNA for C3.

Animals↗
PubMed1986

Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage.

A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma.

Cloning, Molecular↗
PubMed2005

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1).

OBJECTIVES: The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. METHODS: As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. RESULTS: Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at > or = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. CONCLUSIONS: These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Adenine↗
PubMed2005

Establishment and utilization of a tetracycline-controlled inducible RNA interfering system to repress gene expression in chronic myelogenous leukemia cells.

RNA interference (RNAi), a posttranscriptional gene silencing process mediated by small double-stranded RNA specifically complementary to the targeted transcript, has been used extensively in the development of novel therapeutic approaches against various human diseases including chronic myelogenous leukemia (CML). Here, we report the successful construction of a tetracycline-controlled siRNA in CML cell line K562. A K562 cell line stably expressing the reverse tetracycline-controlled transactivator (rtTA) was constructed. A tetracycline responsive element (TRE) was integrated into the RNA polymerase III promoter region of pBS/U6 that was used to drive specific siRNA to target the novel cytokine receptor-like factor 3 (CRLF3) gene. The results show that rtTA was able to recognize the TRE to prevent siRNA-mediated exogenous and endogenous CRLF3 gene repressions. Moreover, CRLF3-siRNA mediated gene repression could be induced in a dose-dependent manner in the presence of doxycycline. Thus, the inducible siRNAi system in K562 cells might be useful for the study of RNAi-mediated therapeutic approaches against CML.

Base Sequence↗

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