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Biomedical subjects

N E Tumer

Publications and source records attributed to N E Tumer.

At least 19 recordsLinked to original sources
PubMed2001

A C-terminal deletion mutant of pokeweed antiviral protein inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition without depurinating the sarcin/ricin loop of rRNA.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. Here, a series of PAP mutants were used to examine the relationship between depurination of the S/R loop and inhibition of +1 programmed ribosomal frameshifting (PRF) and to define PAP sequences critical for inhibition of +1 PRF and Ty1 retrotransposition in the yeast Saccharomyces cerevisiae. Using three different classes of mutants we present evidence that strong binding of a C-terminal PAP mutant (PAPc) to ribosomes is sufficient to inhibit +1 PRF and Ty1 retrotransposition in the absence of S/R loop depurination. PAPc did not affect the totivirus ScV-L-A and HIV-1-directed -1 PRF efficiencies or the ability of cells to maintain the M(1)-dependent killer phenotype, demonstrating the specificity of the effect of PAPc on +1 PRF.

Cell Line↗
PubMed2001

Expression of a bacterial ice nucleation gene in a yeast Saccharomyces cerevisiae and its possible application in food freezing processes.

A 3.6 kb ice nucleation gene (ina) isolated from Erwinia herbicola was placed under control of the galactose-inducible promoter (GAL1) and introduced into Saccharomyces cerevisiae. Yeast transformants showed increased ice nucleation activity over untransformed controls. The freezing temperature of a small volume of water droplets containing yeast cells was increased from approximately -13 degrees C in the untransformed controls to -6 degrees C in ina-expressing (Ina(+)) transformants. Lower temperature growth of Ina(+) yeast at temperatures below 25 degrees C was required for the expression of ice nucleation activity. Shift of temperature to 5-20 degrees C could induce the ice nucleation activity of Ina(+) yeast when grown at 25 degrees C, and maximum ice nucleation activity was achieved after induction at 5 degrees C for approximately 12 h. The effects of Ina(+) yeast on freezing and texturization of several food materials was also demonstrated.

Bacterial Outer Membrane Proteins↗
PubMed2000

A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template.

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

Animals↗
PubMed2000

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.

Base Sequence↗
PubMed1999

Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA.

Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA.

Antiviral Agents↗
PubMed1999

Pokeweed antiviral protein accesses ribosomes by binding to L3.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein, catalytically removes an adenine residue from the conserved alpha-sarcin loop of the large rRNA, thereby preventing the binding of eEF-2.GTP complex during protein elongation. Because the alpha-sarcin loop has been placed near the peptidyltransferase center in Escherichia coli ribosomes, we investigated the effects of alterations at the peptidyltransferase center on the activity of PAP. We demonstrate here that a chromosomal mutant of yeast, harboring the mak8-1 allele of peptidyltransferase-linked ribosomal protein L3 (RPL3), is resistant to the cytostatic effects of PAP. Unlike wild-type yeast, ribosomes from mak8-1 cells are not depurinated when PAP expression is induced in vivo, indicating that wild-type L3 is required for ribosome depurination. Co-immunoprecipitation studies show that PAP binds directly to L3 or Mak8-1p in vitro but does not physically interact with ribosome-associated Mak8-1p. L3 is required for PAP to bind to ribosomes and depurinate the 25 S rRNA, suggesting that it is located in close proximity to the alpha-sarcin loop. These results demonstrate for the first time that a ribosomal protein provides a receptor site for an ribosome-inactivating protein and allows depurination of the target adenine.

Antiviral Agents↗
PubMed1998

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo.

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.

Bacterial Proteins↗
PubMed1998

Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II.

Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1-2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.

Antiviral Agents↗
PubMed1998

The pokeweed antiviral protein specifically inhibits Ty1-directed +1 ribosomal frameshifting and retrotransposition in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the alpha-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed -1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M1-dependent killer virus phenotype, suggesting that -1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.

Frameshifting, Ribosomal↗
PubMed1997

C-terminal deletion mutant of pokeweed antiviral protein inhibits viral infection but does not depurinate host ribosomes.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. In addition to its ribosome-inactivating ability, PAP has potent antiviral activity against many plant and animal viruses, including HIV. We recently described the isolation and characterization of nontoxic PAP mutants, NT123-2, which has a point mutation (E176V) in the active site that abolishes enzymatic activity, and NT124-3, which has a nonsense mutation that results in deletion of the C-terminal 25 aa (W237Stop). In vitro translation of rabbit reticulocyte lysate ribosomes was inhibited by the C-terminal deletion mutant, but not by the active site mutant. We expressed both mutants in transgenic tobacco and showed that, unlike PAP or variant PAP, neither mutant is toxic to transgenic plants. In vivo depurination of rRNA was detected in transgenic tobacco expressing variant PAP, but not in transgenic plants expressing either the active site mutant or the C-terminal deletion mutant PAP. When extracts from transgenic plants containing the mutant PAPs were exogenously applied to tobacco leaves in the presence of potato virus X (PVX), the C-terminal deletion mutant had antiviral activity, while the active site mutant had no antiviral activity. Furthermore, transgenic plants expressing low levels of the C-terminal deletion mutant showed resistance to PVX infection, while transgenic plants expressing very high levels of the active site mutant PAP were not resistant to PVX. Our results demonstrate that an intact active site of PAP is necessary for antiviral activity, toxicity, and in vivo depurination of tobacco ribosomes. However, an intact active site is not sufficient for all these activities. An intact C terminus is also required for toxicity and depurination of tobacco ribosomes in vivo, but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity.

Animals↗
PubMed1995

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

Animals↗
PubMed1995

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

Cloning, Molecular↗
PubMed1994

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus.

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.

Alfalfa mosaic virus↗
PubMed1993

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

Base Sequence↗
PubMed1991

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.

Journal Article↗
PubMed1990

Characterization of infectious transcripts from a potato virus X cDNA clone.

A full-length cDNA clone of potato virus X (PVX) has been constructed and fused to the bacteriophage T7 promoter in an in vitro transcription vector. Transcripts derived from this template (pMON 8660) were infectious when inoculated onto the local lesion host, Chenopodium amaranticolor. The infectivity of these transcripts was approximately 0.2% that of authentic PVX RNA. Lesions sampled from plants inoculated with these transcripts contained virus particles and virus aggregates typically observed in lesions from plants inoculated with authentic PVX RNA, as evidenced by electron microscopy. In addition, progeny virus isolated from these lesions was as infectious as progeny virus from an authentic PVX RNA infection when inoculated onto new local lesions plants. Infectious transcripts derived from PVX cDNA clones will facilitate analysis of the molecular aspects of PVX infection.

Antigens, Viral↗