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PubMed1999

ATP modulates anti-IgE-induced release of histamine from human lung mast cells.

Adenosine 5'-triphosphate (ATP) is released from the cytoplasm under physiologic and pathophysiologic conditions and enters the extracellular space, where it acts on a group of recently cloned cell-surface receptors termed P2-purinoceptors (subtypes P2X and P2Y). We examined the effects of extracellular ATP, uridine triphosphate (UTP), the stable ATP analogues alpha,betamethylene-ATP (alpha,betamATP), beta,gammamethylene-ATP (beta,gammamATP), and 2-methylthio-ATP (2mSATP), and adenosine (10(-6)-10(-3) M) on histamine release from human lung mast cells (HLMC) induced by anti-IgE and the calcium ionophore A23187. None of the nucleotides or adenosine directly induced histamine release. Adenosine exhibited a bimodal effect, enhancing histamine release at 10(-6) to 10(-4) M (P > 0.05, NS) and inhibiting it at 10(-3) M (P < 0.05). ATP (10(-4) M) enhanced anti-IgE-induced histamine release (10.9 +/- 2.7% to 19. 2 +/- 2.9%, n = 20, P < 0.01), but not ionophore A23187-induced histamine release (n = 10). The adenine nucleotides consistently enhanced anti-IgE-induced histamine release; the rank order for this action was: ATP > 2mSATP > alpha,betamATP > beta,gammamATP, suggesting mediation by a P2Y-purinoceptor subtype. The selective P2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid failed to influence the effect of ATP, further supporting P2Y-purinoceptor mediation of anti-IgE-induced histamine release. UTP, an agonist at P2Y-purinoceptors, also significantly enhanced anti-IgE-induced histamine release. Application of the reverse transcription-polymerase chain reaction indicated that HLMC constitutively express the messenger RNAs encoding the P2Y1- and P2Y2-purinoceptor subtypes, and not that encoding the P2X7-purinoceptor (i.e., P2Z), a subtype implicated in ATP-induced histamine release in rodent peritoneal mast cells. The data produced in the study suggest that ATP plays an important modulatory role in histamine release from HLMC, and that it may therefore be mechanistically involved in human allergic/asthmatic reactions.

Adenine Nucleotides↗
PubMed1997

Expression and characterization of a putative high affinity human soluble leptin receptor.

Leptin, a circulating 16-kDa protein secreted by adipocytes, decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of 125I-leptin (Ki approximately 200 pM); no detectable binding was present in the medium. In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific 125I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins. Data from cross-linking studies identified two specific bands in the 125I-leptin/soluble leptin receptor complex with molecular masses of approximately 130-150 kDa and 300 kDa. The 130-150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the 125I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized 125I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.

Animals↗
PubMed2026

Biomolecular Condensates Integrate Transcriptional and Epigenetic Responses to Hypoxia.

Hypoxia is a defining feature of physiological stress and the core of solid tumors, where aberrant vascularization limits oxygen delivery; cells respond through mechanisms that extend beyond the canonical stabilization of hypoxia-inducible factors (HIFs). Recent studies suggest that hypoxia can promote the formation of specific biomolecular conden-sates, membraneless compartments generated through liquid-liquid phase separation in which regulatory proteins and RNAs become locally enriched at genomic regions, while chromatin mainly serves as an organizational scaffold. Transcription factors, the coacti-vators p300/CBP, Mediator, and BRD4, chromatin-modifying enzymes, and architectural RNAs such as NEAT1 and MALAT1 partition into these compartments, and their con-densation can help reorganize local chromatin structure and enhancer-promoter interac-tions. Because molecular oxygen is a shared co-substrate for the Jumonji-C histone demethylases and the ten-eleven translocation (TET) DNA dioxygenases, hypoxia reshapes histone methylation and DNA methylation in parallel, and readers that bridge these marks, including UHRF1, may participate in condensate-associated chromatin regulation. Hypoxia-driven condensation of ZHX2 rewires enhancer-promoter contacts and higher-order genome architecture, influencing cell identity, stemness, and metastatic potential, and Polycomb condensates represent another candidate epigenetic compartment that may be influenced by hypoxic signaling. These processes may be particularly important in cancer, where chronic hypoxia provides a sustained stimulus for condensate formation and epigenetic remodeling. Together, these findings support a model in which phase separation and epigenetic reprogramming are not separate layers but one integrated response to low oxygen, offering opportunities to target maladaptive condensates in disease.

Epigenesis, Genetic↗
PubMed1991

Defective induction of Jun and Fos-related proteins in phorbol ester-resistant EL4 mouse thymoma cells.

Treatment of sensitive EL4 mouse thymoma cells with phorbol esters causes growth inhibition, adherence to substrate and production of several lymphokines including Interleukin 2. Resistant cells lack all of these responses. Since production of Interleukin 2 mRNA is dependent on protein synthesis, and the Interleukin 2 gene has a phorbol ester responsive element, we examined both cell lines for expression of the various Jun and Fos species which bind to this element. Phorbol ester induced c-fos, jun-B, and jun-D RNAs within 20 min in both cell lines. Fos-B was similarly induced in sensitive cells but induction was delayed and greatly enhanced in resistant cells. C-jun RNA induction was detected only in sensitive cells. Western analysis confirmed the induction of c-Jun and a Fos-related protein in sensitive cells only. Southern analysis indicated that both cell lines contain c-jun and fra-1 genes. These results suggest that defective induction of c-Jun and/or Fos-related proteins may contribute to the absence of phorbol ester-induced lymphokine production in resistant EL4 cells.

Animals↗
PubMed2001

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.

Alternative Splicing↗
PubMed1992

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Amino Acid Sequence↗
PubMed1999

Enhanced duck hepatitis B virus gene expression following aflatoxin B1 exposure.

Epidemiological studies have suggested synergistic interactions between chronic hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure in the etiology of hepatocellular carcinoma (HCC), although the molecular mechanisms of their interactions are still not understood. The aim of this study was to use the Pekin duck model to investigate the impact of AFB1 exposure on duck hepatitis B virus (DHBV) replication during the early stages of virus-carcinogen interactions. Six-week-old chronic DHBV-carrier or uninfected ducks were exposed to AFB1 for 5 weeks or treated with dimethylsulfoxide (DMSO) as a control. Animals were observed for 6 to 13 weeks after AFB1 treatment to study the influence of AFB1 exposure on DHBV replication and liver pathologies. Histological analysis showed more marked changes in the livers of AFB1-treated ducks, and these were enhanced by DHBV infection. A significant increase in serum and liver DHBV DNA level was observed in AFB1-treated ducks as compared with DMSO-treated controls. In addition, viral RNAs, in particular the pregenomic RNA that is the template of viral replication, and intrahepatic DHBV DNA replicative intermediates, were significantly increased by AFB1 treatment. Moreover, an overexpression and accumulation of DHBV large envelope (L) protein was observed in the hepatocytes of AFB1-exposed animals. The in vitro study has further confirmed an increase in intracellular viral DNA and in virus release in AFB1-treated primary duck hepatocytes. Taken together, our results indicate that AFB1 exposure leads to an increase in virus gene expression associated with intrahepatic accumulation of DHBV L protein and enhanced liver pathology.

Aflatoxin B1↗
PubMed2026

The emerging impact of CRISPR and gene editing on global crop improvement.

The advent of CRISPR-based genome editing has revolutionized crop improvement, offering unprecedented precision and efficiency in modifying key agronomic traits. This review comprehensively examines the mechanisms, applications, and future potential of CRISPR technology in enhancing global crop production. CRISPR-Cas systems, originally identified as adaptive immune mechanisms in bacteria and archaea, have been repurposed for targeted genome editing in plants. The CRISPR-Cas9 system, in particular, has emerged as a powerful tool for introducing site-specific double-strand breaks, enabling precise genetic modifications. The three-stage process of adaptation, expression, and interference underlies the CRISPR mechanism, with guide RNAs directing Cas endonucleases to specific genomic loci. Advances in CRISPR technology have expanded its applications beyond gene knockouts, encompassing base editing, prime editing, and epigenome editing. These innovations have facilitated the development of crops with enhanced yield, stress tolerance, disease resistance, nutritional content, and post-harvest quality. However, challenges related to off-target effects, regulatory hurdles, ethical concerns, and public acceptance must be addressed to fully harness the potential of CRISPR in agriculture. Integration of CRISPR with other cutting-edge technologies, such as synthetic biology, artificial intelligence, and high-throughput phenotyping, holds immense promise for accelerating crop improvement efforts. As research continues to refine CRISPR tools and expand their applicability across diverse plant species, this transformative technology is poised to play a pivotal role in shaping a sustainable, resilient, and productive global food system for future generations.

Gene Editing↗
PubMed1998

The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly.

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.

Base Sequence↗
PubMed1991

Nucleotides in precursor tRNAs that are required intact for catalysis by RNase P RNAs.

Precursor tRNAAsp molecules, containing a 26-base 5' leader, were treated with diethylpyrocarbonate, 50% hydrazine or anhydrous hydrazine/3M NaCl and then subjected to processing by RNase P RNAs from Escherichia coli or Bacillus subtilis. Fully processed tRNAs and material not successfully cleaved by the catalytic RNAs were analyzed for their content of chemically altered nucleotides. Several bases were identified as being required intact for optimal activity as substrate as judged by exclusion of chemically modified residues from processed molecules, and simultaneous enhancement in material that was not recognized as substrate. Such nucleotides cluster near the site of cleavage at the mature 5' end and in the T stem and loop. Purines at residues 1 and 2 adjacent to the site of cleavage, position 57 in the T loop, and site 64 in the T stem exhibited the most pronounced effects. These results suggest a model of recognition of substrate by RNase P RNAs in which the ribozyme interacts with the corner of the precursor tRNA's three dimensional structure, where the T- and D-loops are juxtaposed, and extends along the top of the molecule back towards the site of catalysis.

Autoradiography↗
PubMed2002

Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein.

BACKGROUND/AIMS: Epidemiological studies have shown that coinfection or superinfection with hepatitis B virus (HBV) and C virus (HCV) frequently leads to the suppression of hepatitis B virus replication. The mechanism of this phenomenon is still unclear. Shih et al. [J Virol 1993;67:5823] reported a direct suppression of HBV replication by the core protein of HCV. The target structure of HCV core protein in this system remained unclear. METHODS: As HCV core protein has been shown to influence expression from transcriptional elements, we studied whether HCV core protein altered the activity of the two HBV enhancers 1 and 2. Luciferase vectors for HBV enhancers 1 or 2 were cotransfected with expression constructs for HCV core protein in murine and human hepatocyte lines. RESULTS: Full-length HCV core protein suppressed the HBV enhancer 1 up to 11-fold, the enhancer 2 3-4-fold. Suppression of HBV enhancer 1 by HCV core from genotype 1b was stronger than by HCV core of genotypes 3a or 1a. Carboxyterminally truncated core proteins had lower or no suppression activity. CONCLUSIONS: These data suggest that HCV core protein may directly repress transcription of the HBV RNAs. This trans-repression may contribute to suppression of HBV replication in patients coinfected with both viruses.

Animals↗
PubMed2003

The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.

The type 1 insulin-like growth factor receptor (IGF1R) is often overexpressed by tumors and mediates growth and apoptosis protection. We previously showed that antisense reagents complementary to the IGF1R translation start site enhance radio- and chemosensitivity and impair Atm function. However these agents induce relatively modest IGF1R down-regulation and affect insulin receptor levels. To identify alternative sites for molecular targeting, we utilized scanning oligonucleotide arrays to probe the secondary structure of IGF1R mRNA. This strategy enabled selection of antisense oligonucleotides that generated high heteroduplex yield with IGF1R but not insulin receptor transcripts. Antisense oligonucleotides that hybridized strongly to IGF1R mRNA caused IGF1R down-regulation within intact tumor cells, whereas weakly hybridizing oligonucleotides were inactive. Furthermore, the ability of small interfering RNAs (siRNAs) to block IGF1R expression correlated with the accessibility of the target sequence within the transcript. Thus, siRNAs corresponding to weakly hybridizing oligonucleotides caused minor IGF1R down-regulation, whereas siRNAs homologous to accessible targets induced profound sequence-specific IGF1R gene silencing, blocked IGF signaling, and enhanced tumor cell radiosensitivity. This indicates that secondary structure in the target transcript has a major effect on siRNA efficacy. These findings have implications for siRNA design and suggest that IGF1R-targeting agents incorporating this mode of action have potential as anticancer therapy.

Gene Silencing↗
PubMed2004

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA.

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.

Bacterial Proteins↗
PubMed2005

Each genomic RNA in HIV-1 heterozygous virus generate new virions.

Retrovirus are unique because they present two complete copies of the genomic RNA in each virion. It is believed that only one proviral DNA is formed from the two genomic RNAs. To check this hypothesis, we constructed two deleterious HIV-1 variants in gag gene which upon transfection in Cos-1 cells were able, by complementation, to form heterozygous viruses, used to infect MT4 cells in a plaque assay. Analysis of the proviral DNA of the eight plaques obtained indicated that five were recombinants between the two deleterious mutants. Three other plaques showed three bands corresponding to the reverse transcription of both strands of one heterozygous virion and to the recombination of the two genomes. These results demonstrate that the two genomic RNAs in HIV-1 heterozygous virions could be used in the generation of new viruses. This mechanism permits the recovery of deleterious mutants and enhances the evolutive potential of HIV-1.

Amino Acid Sequence↗
PubMed1978

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro.

Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the alpha-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5'-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5'-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro.

Globins↗
PubMed2005

Discovery of a gene family critical to wyosine base formation in a subset of phenylalanine-specific transfer RNAs.

A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3'-side of the tRNA(Phe) anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N(1)-methylguanosine (m(1)G(37)) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m(1)G(37) is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNA(Phe)-m(1)G(37) to tRNA(Phe)-yW.

Chromatography, High Pressure Liquid↗
PubMed2006

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes.

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis.

Adenoviridae↗
PubMed1980

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

Amanitins↗