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PubMed1994

Molecular cloning, expression, and chromosomal localization of two isoforms of the AE3 anion exchanger from human heart.

Cl-/HCO3- exchange contributes to regulation of pHi and [Cl-] in cardiac muscle, with possible effects on excitability and contractility. We have isolated human heart cDNAs, which encode two isoforms of the anion exchanger AE3. These clones share long portions of common sequence but have different 5' ends encoding distinct amino-terminal amino acid sequences. The longer AE3 polypeptide of 1232 amino acids, bAE3, displays nearly 96% amino acid sequence identity to the rat and mouse AE3 "brain isoforms." The shorter cAE3 polypeptide of 1034 amino acids in length corresponds to the rat AE3 "cardiac isoform." The unique N-terminal 73 amino acids of the cAE3 sequence are less well conserved between rat and human. Northern blot analysis with isoform-specific probes revealed the presence of both cAE3 and bAE3 mRNAs in human heart tissue. Both AE3 protein isoforms were overexpressed in Chinese hamster ovary cells and detected by immunoblot with antipeptide antibodies. Immunoblot studies of human cardiac membranes detected only cAE3 polypeptides, which were apparently not susceptible to enzymatic deglycosylation. Injection into Xenopus oocytes of cRNAs encoding either cAE3 or bAE3 produced increased 36Cl- uptake into the oocytes, confirming the ability of both AE3 isoforms to transport Cl-. The human AE3 gene was localized to chromosome 2. AE3 may provide a new pharmacologic target for antiarrhythmic and cardioprotective drugs.

Amino Acid Sequence↗
PubMed2006

Microarray analysis of trophoblast cells.

A complex repertoire of trophoblast gene products governs the multifaceted functions performed by the placenta during the relatively short period of pregnancy. Cloning and sequencing the human as well as other mammalian genomes allow investigators to gain better insight into the function of trophoblast genes. Our ability to identify transcripts by their nucleotide sequences and determine their expression patterns enables us to glean information on gene function. Although the molecular principles underlying microarray are not new to biology, the high throughput, low reaction volumes, fluorescent labeling, accurate detection, and robust analysis software makes this approach most appealing to today's researchers, when compared with standard filter blotting techniques. This chapter focuses on DNA microarray of the human placental transcriptome as a means to identify alterations in gene expression in different physiological or pathological conditions.

DNA, Complementary↗
PubMed1999

Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures.

The present study was designed to evaluate the expression of dopamine D1 and D2 receptor mRNAs in systemic and pulmonary vasculatures. Using specific antisense riboprobes for dopamine D1 and D2 receptor cDNAs, in situ hybridization histochemistry was performed in the aorta, common carotid artery, vertebral artery, pulmonary artery, and superior vena cava of the adult male Sprague Dawley rat. In the case of the aorta, common carotid artery, and vertebral artery, dopamine D1 receptor mRNAs localized mainly in the smooth muscle cells of the tunica media. However, the signals of dopamine D2 receptor mRNAs were found in the endothelium and subendothelial layer of tunica intima, and interstitial cells of tunica adventitia. In the case of the pulmonary artery, signals of dopamine D1 receptor mRNAs were detected within the tunica intima, media, and adventitia. Expression of D2 receptor mRNAs was detected in the walls of small blood vessels within the tunica adventitia of the pulmonary artery. There were no detectable signals of dopamine D1 and D2 receptor mRNAs in the vein. The uneven distribution of dopamine D1 and D2 receptor mRNAs in the rat systemic vasculatures and pulmonary artery suggests that dopamine differentially regulates the vasodilation of the systemic and pulmonary arteries through the differential stimulation of dopamine D1 and D2 receptor.

Animals↗
PubMed1982

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

Adenoviruses, Human↗
PubMed1971

DNA in uninfected and virus-infected cells complementary to avian tumor virus RNA.

The 70S RNA component of several avian tumor viruses was hybridized with DNA extracted from avian tumor virus-infected and uninfected chicken and Japanese quail cells. Tritium-labeled 70S RNAs from Rous sarcoma virus (RSV), Rous associated virus-1 (RAV-1), RAV-60, and Schmidt-Ruppin-RSV (SR-RSV) hybridize from 3 to 10 times more with DNA from uninfected chicken cells than with DNA from Escherichia coli, calfthymus, or baby hamster kidney cells. After infection of chicken cells with RSV(RAV-1), SR-RSV, or RAV-2, the amount of 70S avian tumor virus [(3)H]RNA hybridized increases by 1.6 times. The specificity of the hybridization reaction was shown by the specific competition of 70S SR-RSV [(3)H]RNA with 70S RNA from RSV(RAV-1), and not with RNA from Sendai virus or chicken cells. There was no difference in the hybridization of 70S RNA from RSV (RAV-1), RAV-1, or RAV-60 with DNA either from chicken cells that contain RAV-60 in a nonreplicating form or from chicken cells that do not appear to contain RAV-60. These results indicate that both types of uninfected chicken cells contain DNA that is complementary to RNA from several avian tumor viruses and that the amount of complementary DNA increases in such cells after infection with an avian tumor virus. The RNAs of genetically different avian tumor viruses appear to have indistinguishable base sequences by this technique.

Alpharetrovirus↗
PubMed2001

The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp).

Hepatitis E virus (HEV) is the major cause of acute epidemic and sporadic hepatitis in the developing world. It is a positive-strand RNA virus with a genome length of about 7.2 kb. The replication mechanism of this virus is virtually unexplored. Identification of the regulatory elements involved in initiation of replication may help in designing specific inhibitors for therapy. In the positive-stranded RNA viruses the initiation of replication requires interaction of the 3' end of genome with its RNA-dependent RNA polymerase (RdRp) and possibly host-derived cofactors for synthesis of the minus-strand replicative intermediate. Secondary structure prediction of the conserved 3' end of the infectious HEV genome was carried out to identify possible stem-loop structures necessary for RNA-protein interaction and the model was confirmed by structure probing experiments. Electrophoretic mobility-shift assays showed specific binding of purified and refolded recombinant HEV RdRp protein to the 3' end of its RNA genome containing the poly(A) stretch. Mutations at the 3' end, in which the stem-loop structures were partially or completely destroyed or recreated revealed that the two stem-loop structures SL1 and SL2 at the 3' end and the poly(A) stretch are necessary for this binding. The interacting nucleotides in such an interaction were further identified by generating footprints of the complex by Pb(II)-induced hydrolysis. This specific binding of viral RdRp to the 3' end of HEV RNA directs the synthesis of complementary-strand RNA and thus such a binding domain might assume the role of a possible cis-acting element as a potential site for the initiation of replication.

3' Untranslated Regions↗
PubMed1976

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.

Base Sequence↗
PubMedDate not supplied

DNA-linked RNase H for site-selective cleavage of RNA.

ADNA-linked RNase H (Hybrid Enz-1) (Kanaya et al. (1992) J. Biol. Chem. 267, 8492-8498), in which dGTCATCTCC was attached to E. coli RNase H via a covalent linker of 21 A, was altered to improve the site-specific RNA cleavage by increasing the linker length. The sizes of the linkers on these hybrid enzymes (Hybrid Enz-2, -3, and -4) differed by 3 A, the axial rise of the DNA/RNA hybrid, to give 18-, 24-, and 27-A lengths. The conjugate with a size of A was able to cleave a synthetic 22mer RNA (5'-rAAGAUGUCUACGGAGAUGACCA-3'), containing the complementary 9mer RNA sequence (underlined), at one position, A16-U17. The kinetic parameters of Hybrid Enz-1, -2, -3, and -4 were examined using a 9mer RNA target. The results showed that longer linkers produced higher Km, kcat, and kcat/Km values, and the kcat/Km value of the conjugate with the 27-A linker reached 83% of that of the wild-type RNase H. Hybrid Enz-4 was found to be useful as an RNA restriction endonuclease.

Base Sequence↗
PubMed1994

Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis.

A small RNA encoded within the nucleus is an essential subunit of a RNA processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of replication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into question the existence of pathways for nucleo-mitochondrial transport of nucleic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techniques to assess the subcellular partitioning of MRP RNA. Cryosections of mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length from 115 to 230 nucleotides, followed by immunogold labeling. In addition, we transfected mouse C2C12 myogenic cells with constructs bearing mutated forms of the mouse MRP RNA gene and compared the relative abundance of the resulting transcripts to that of control RNAs within whole cell and mitochondrial fractions. In the former analysis we observed preferential localization of MRP RNA to nucleoli and mitochondria in comparison to the nucleoplasm and cytoplasm. In the latter series of studies we observed that wild-type MRP RNA partitions to the mitochondrial fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucleoplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA gene produced transcripts that remain competent for mitochondrial targeting. In contrast, deletion of the midportion of the coding region (nt 118 to 175) of the MRP RNA gene resulted in transcripts that fail to partition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respiring cells, and that specific structural determinants within the MRP RNA molecule permit it to be partitioned to mitochondria.

Animals↗
PubMed2003

Characterization and quantitation of differential Tsix transcripts: implications for Tsix function.

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

Animals↗
PubMed1981

Spliced and unspliced RNAs encoded by virion RNA segment 7 of influenza virus.

Cells infected with the avian influenza virus fowl plague virus, contains three species of polyadenylated RNA which are complementary to virion RNA segment 7. The largest is virtually a complete transcript of vRNA 7, and is the messenger RNA for the matrix protein, but the coding function of the two smaller species, which are approximately 320 and 285 nucleotides long (excluding poly(A)), is unknown. It is likely however that at least one of the small RNAs encodes a new virus polypeptide which has been predicted from the nucleotides sequence of vRNA 7. The major part of each RNA maps within about 300 nucleotides from the 5'-terminus of vRNA 7, but the larger species also contains additional sequences derived from the 3' terminus. Production of the two small RNAs may involve alternative patterns of splicing of the matrix protein mRNA.

Animals↗
PubMed1987

OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression through an RNase III-dependent mechanism.

OOP RNA is a major short (77 bases) transcript that is made from bacteriophage lambda DNA both in vivo and in vitro. OOP RNA is synthesized in the opposite direction to mRNA for the lambda cII gene, and the final 55 bp of the OOP region overlaps the 3' end of the cII gene. We find that a multicopy plasmid containing an OOP DNA fragment inhibits cII expression from a derepressed prophage by approximately 100-fold, using an in vivo assay in which cII protein activates galactokinase synthesis from a cII-dependent promoter on a multicopy plasmid. A large inhibitory effect is also observed when the po promoter for OOP RNA is replaced by the strong lambda pL promoter, but not when po is deleted. Plasmids that provide a large excess of "anti-OOP" RNA (RNA that is complementary to OOP RNA) make OOP RNA a less effective inhibitor of cII expression. Inhibition by the OOP DNA plasmid is not observed in an Escherichia coli strain deficient in RNase III. We propose that the 3' end of cII mRNA and OOP RNA form a double-stranded complex that is a substrate for the host enzyme RNase III, resulting in degradation of cII mRNA. Deletion studies on the OOP DNA plasmid indicate that no specific sequence between the promoter and terminator stem structure is required for the inhibitory effect. Lambda cII expression from an induced prophage is increased twofold in the presence of a large excess of anti-OOP RNA. This experiment, in which the prophage is the sole source of OOP RNA, suggests a physiological role for OOP RNA in regulating cII-gene expression.

Bacteriophage lambda↗
PubMed1966

Genetic relatedness among mycoplasmas as determined by nucleic acid homology.

Reich, Paul R. (National Institutes of Health, Bethesda, Md.), Norman L. Somerson, James A. Rose, and Sherman M. Weissman. Genetic relatedness among mycoplasmas as determined by nucleic acid homology. J. Bacteriol. 91:153-160. 1966.-A sensitive membrane filter method to detect nucleic acid homology was used to determine genetic relatedness among mycoplasma isolates. Deoxyribonucleic acid (DNA) was isolated from mycoplasmas and used as a primer for synthesis of tritium-labeled, complementary ribonucleic acid (RNA) by the enzyme RNA polymerase. DNA from each mycoplasma isolate tested was reacted separately with complementary RNA synthesized with homologous or heterologous DNA as primer. The quantity of DNA-RNA hybrids formed was assayed by the nitrocellulose membrane filter method. The amount of radioactivity bound to the membrane filter was used to measure the degree of homology between the nucleic acids. The three mycoplasma isolates from human oral cavities (DC 63, V2785, Botteicher) and the prototype strain PG21 placed in the Mycoplasma hominis type 1 group by gel diffusion and complement-fixation testing were investigated with this technique. Analysis of the data confirmed their immunological grouping with the M. hominis type 1 and their distinction from other human mycoplasmas. In contrast to the data from immunological studies, none of the four isolates tested appeared to be identical to any other. Preliminary experiments with DNA from four other mycoplasma isolates from tissue cultures inoculated with human material revealed them to be closely related, and possibly identical. The advantages of this nucleic acid homology technique for the study of relatedness among mycoplasmas are described.

DNA, Bacterial↗
PubMed1990

Molecular biology of type A endogenous retrovirus.

Intracisternal A particles (IAPs) are retrovirus-like structures consistently observable in a variety of mouse tumor cells such as myeloma and hybridoma and in early embryonic cells derived from rodents but nothing is known of their infectivity. Mouse IAPs contain a gag-like protein, a reverse transcriptase and a polyadenylated RNA molecule (IAP RNA). DNA sequences complementary to IAP RNA (IAP genome) are interspersedly present in rodent such as mice, rats, Chinese hamsters and Syrian hamsters at several hundred to a thousand copies per haploid genome. Molecularly cloned IAP genomes from two species Mus and Syrian hamster were 6 to 8 kb in length with LTRs of about 0.4 kb long. The nucleotide sequence of the Syrian hamster IAP genome, H18, predicted a typical LTR-gag-prt-pol-env-LTR structure, although many stop codons were present in the region corresponding to env. The comparison of the deduced amino acid sequences of the pol region showed IAP (type A), mouse mammary tumor virus (MMTV) (type B), and squirrel monkey retrovirus (SMRV) (type D) genomes to be closely related. By using a DNA fragment encoding the pol region of the Syrian hamster IAP genome, human endogenous retroviruses termed HERV-K, were cloned from a fetal human liver gene library. Typical HERV-K genome was 9.5 kb in length having LTRs of about 1.0 kb. The HERV-K provirus could encode gag (666 codons), prt (334 codons), pol (937 codons), and env (618 codons) genes. HERV-K was shown to be closely related to types A, B and D retroviruses. The HERV-K genomes are present at about 50 copies per haploid human genome. In several human tumor cell lines, the HERV-K genome was expressed as 8.8 kb poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression was observed following female steroids treatment. In a detailed investigation on the organization of HERV-K proviruses in human genome, we found repetitive sequences homologous to the LTR region of the HERV-K genome. They were about 630 bp in length with an A rich tail at 3' end and found to be a SINE type nonviral retroposon. These elements were present at 4,000 to 5,000 copies per haploid human genome.

Animals↗
PubMed1999

2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.

Circular Dichroism↗
PubMed2001

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines.

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Amino Acid Transport Systems↗
PubMed2003

Mitogenic actions of neuropeptide Y in vascular smooth muscle cells: synergetic interactions with the beta-adrenergic system.

Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smooth muscle cell (VSMC) growth, but which of its Y1-Y5 receptors are involved remains unclear. In quiescent rat VSMCs, NPY receptor mRNAs were undetectable (reverse transcription-polymerase chain reaction), but Y1, Y2, and Y5 expression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold [3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nM concentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low affinity peak by 50% and fully blocked NPY's response when combined. In VSMCs lacking NPY receptors and responsiveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCs with low or no NPY responsiveness, pre-exposure to beta-adrenergic receptor agonist (isoproterenol), forskolin, or dibutyryl cAMP augmented NPY's mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenol only). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is low in nonproliferating cells, amplification of NPY's mitogenic responses requires upregulation of at least the Y1 receptor by NPY itself or beta-adrenergic, cAMP-dependent activation.

Adrenergic beta-Agonists↗
PubMed1986

Coding strategy of the S genome segment of Hantaan virus.

Hantaan virus is the type species of the recently recognized Hantavirus genus of Bunyaviridae. The small (S) RNA segment of the negative-sense, tripartite genome was molecularly cloned and the nucleotide sequence was determined. The RNA sequence derived from the cDNA copy was found to contain 1696 nucleotides. A single open reading frame of sufficient size to encode the virus nucleocapsid protein was detected in the cDNA corresponding to viral complementary-sense RNA. RNA transcripts of the cDNA were synthesized with SP6 polymerase and were used to program cell-free reticulocyte lysate translation systems. Viral complementary-sense transcripts served as efficient messages in translation systems and generated Hantaan nucleocapsid protein. No translation products were detected when lysates were programmed with viral-sense transcripts. This coding assignment of the nucleocapsid protein to the viral complementary-sense RNA of the S genome segment is consistent with those of other members of this family. Unlike other Bunyaviridae, which encode both a nucleocapsid protein and a nonstructural (NSs) protein of similar sizes, a NSs protein has not been identified for Hantaan virus. Furthermore, other than the nucleocapsid protein gene sequence, the only potential open reading frame in Hantaan S RNA encoded a short, 48-amino acid polypeptide which initiated two codons beyond the termination of the nucleocapsid protein in the same reading frame. These data demonstrate that the coding strategy of the Hantaan virus S RNA is different than those reported for other viruses in this family.

Amino Acid Sequence↗