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At least 253 records · Page 14Linked to original sources
PubMed2006

Protein phosphatase 2A regulates bim expression via the Akt/FKHRL1 signaling pathway in amyloid-beta peptide-induced cerebrovascular endothelial cell death.

Amyloid-beta peptide (Abeta)-induced death in cerebral endothelial cells (CECs) is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in Abeta-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in Abeta-treated CECs. Furthermore, Abeta induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by Abeta before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited Abeta-induced FKHRL1 activation and bim induction. In addition, Abeta increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed Abeta-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that Abeta enhanced the binding of the PP2A regulatory subunit PP2ACalphabeta to Akt. These results implicate PP2A as an early regulator of Abeta-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA.

Amyloid beta-Peptides↗
PubMed1987

Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal.

Animals↗
PubMed1988

tRNAPhe and tRNAPro are the near-ultraviolet molecular targets triggering the growth delay effect.

The illumination of Escherichia coli cells with UVA light, 320 nm less than or equal to lambda less than or equal to 380 nm, triggers a transient growth and division delay. The built-in 4-thiouridine chromophore which absorbs light at 340 nm leads to the quantitative 8-13 crosslinking of a number of tRNA species corresponding to 50% of the bulk tRNA molecules. Determination of the tRNA acylation level by the various aminoacids shows that only the tRNA species acylated by Phe and Pro are strikingly affected in vivo. Both acylation levels decrease to less than 10% of their initial value during the illumination period, remain stable all along the growth lag and increase concomitantly with cell mass when growth resumes. Hence tRNA(Phe) and tRNA(Pro) are the UVA light molecular targets triggering growth delay and related effects of biological significance such as cell volume reduction, photoprotection and protection against UV mutagenesis (antiphotomutagenesis).

Acylation↗
PubMed2003

An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing.

Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield DeltaINS-ProRS. While DeltaINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction.

Alanine↗
PubMed1996

Azospirillum lipoferum tRNAs: fractionation and identification of tRNA species and the nucleotide sequence of tRNA(Asn) (QUU).

Transfer RNAs of Azospirillum lipoferum were separated by two-dimensional gel electrophoresis and identified by aminoacylation. Thirty-six tRNA spots were resolved by this technique and twenty-six tRNA species have been identified. There are five tRNAs for Leu, four for Val, three for Pro, two each for Arg, Ile, Lys and Tyr, and one each for Ala, Asp, His, Phe, Ser and Thr. The tRNA (Asn) (QUU) was purified and its nucleotide sequence was determined. The A. lipoferum tRNA (Asn) (QUU) is 92% similar to B. subtilis tRNA (Asn) gene and two hypermodified nucleosides, queuosine (Q) and N-(9-beta-D Ribofuranosylpurine-6-YL)-carbamoyl)-threonine (t6A) are present in this tRNA.

Azospirillum↗
PubMed1992

An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting.

Base Sequence↗
PubMedDate not supplied

Cytokine release pathway in mononuclear cells stimulated in vitro by dialysis membranes.

BACKGROUND: Among the possible variables associated with cytokine activation in hemodialysis, filter membrane has been reported to be a major factor and monocytes adherent to the membrane have been suggested as a possible source for cytokine synthesis. METHODS: In order to exclude variables other than the direct cell-to-membrane interaction, suspensions of peripheral blood monocytes isolated from donors' blood were incubated for 180 min at 37 degrees C in Petri dishes coated with cuprophan (Cu) or polyacrylonitrile (AN69S) or polysulfone (PS). Total RNA was purified, reverse transcribed in cDNA and amplified by polymerase chain reaction (PCR) primed with specific oligomers for determining IL-1beta and TNF-alpha gene expression. For Western blot analysis, cell homogenates and supernatants were electrophoresed and transferred to a polyvinylidene difluoride membrane and the membrane was then incubated with polyclonal antibodies specific for the detection of IL-1beta and TNF-alpha. RESULTS: Semi-quantitation of targets for PCR (using the technique of limiting dilutions and referring to actin and glyceraldehyde-3-phosphate dehydrogenase as noninducible molecule) revealed a comparable increase at 180 min (p < 0.05) in IL-1beta and TNF-alpha mRNA in monocytes incubated with polyacrylonitrile, PS and Cu membranes. In 2 of 10 experiments pro-IL-1beta was detected in monocytes interacting with Cu, PS and AN69S membrane. However, the extent of extracellular release of mature protein was greater for Cu. CONCLUSION: Some dissociation between transcriptional and translational events was detected in experiments using donors' blood in vitro. More specifically, synthetic membranes (both polyacrylonitrile and PS) were found to be as active as Cu in inducing IL-1beta and TNF-alpha mRNA expression, but less effective in promoting the extracellular release of proinflammatory cytokine products.

Gene Expression↗
PubMed1988

Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II.

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

Amanitins↗
PubMed1987

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Amino Acid Sequence↗
PubMed1994

Absence of MHC class II expression distinguishes fetal from adult B lymphopoiesis in mice.

Early B-lineage progenitor cells (Pro-B) isolated from murine fetal liver and adult bone marrow can differentiate to the immature B cell stage in a stromal cell-dependent culture system and to mature B cells upon transfer into immunodeficient SCID mice. By using immunofluorescence analysis, we found that progenitor cells from day 16 fetus differentiating in culture lacked MHC class II expression during the Pre-B and immature B cell stages, whereas such expression was readily apparent on the surface of corresponding adult-derived populations. RT-PCR analysis of RNA message levels for the four class II genes (A alpha, A beta, E alpha, E beta) yielded completely concordant results. B cells of fetal progeny did eventually express class II upon further maturation in vivo. Thus, the onset of class II expression is uniquely delayed during fetal B cell differentiation. This result explains an apparent paradox, i.e., that class II expression is absent from B cells in neonatal spleen but present as early as the Pre-B cell stage in adult bone marrow. Furthermore, we suggest that such distinct programs of class II expression during fetal and adult lymphopoiesis could result in differences in susceptibility to tolerance.

Animals↗
PubMed1995

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Base Sequence↗
PubMed1987

The transcriptional efficiency of clustered tRNA genes is affected by their position within the cluster.

The transcription of a mouse genomic segment containing four tRNA genes, coding for a tRNA(Ala), a tRNA(Ile), a tRNA(Pro) and a tRNA(Lys), has been studied in a HeLa cell extract, demonstrating that differences among their transcriptional efficiencies are evident using as templates either the natural cluster or an equimolecular mixture of the four isolated genes. Nevertheless, the structure of the cluster influences the transcriptional efficiency of the clustered genes. In fact, a cis-acting inhibitory sequence has been located at about 400 bp downstream of the tRNA(Pro) coding sequence. Moreover rearrangements of the reciprocal position of the various tRNA genes within the cluster results in significant changes in the transcriptional rates of the individual transcriptional units.

Animals↗
PubMed2002

Retrovirus-specific packaging of aminoacyl-tRNA synthetases with cognate primer tRNAs.

The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are, tRNA(Trp), and tRNA(Pro), respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNA(Pro) in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.

3T3 Cells↗
PubMed2004

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Animals↗
PubMed2006

Targeted in vitro and in vivo gene transfer into T lymphocytes: potential of direct inhibition of allo-immune activation.

BACKGROUND: Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-alpha and IFN-gamma mRNA expression. RESULTS: Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2) resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines. CONCLUSION: These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.

Animals↗
PubMed2005

Murine leukemia virus with a primer-binding site complementary to tRNALys,3 adapts to select new tRNAs for replication following extended in vitro culture.

The preference of MuLV for the selection of tRNA(Pro) as a replication primer was investigated by altering the primer-binding site (PBS) to be complementary to tRNA(Lys,3). MuLV-based vectors with a PBS complementary to tRNA(Lys,3) were found to be approximately 2-fold less infectious than vectors with the wild-type PBS complementary to tRNA(Pro). MuLV with a PBS complementary to tRNA(Lys,3) was replication competent and maintained the PBS during early stages of in vitro culture. Upon extended culture, PBS were isolated which were complementary to tRNA(Arg). A second MuLV was generated in which the region upstream of the PBS which is predicted to form an RNA stem loop structure was altered so that the nucleotide sequence within the loop would be complementary to the anticodon of tRNA(Lys,3). The virus with both the U5 and PBS complementary to tRNA(Lys,3) was also replication competent. Upon extended in vitro culture though, this virus reverted to utilize tRNA(Lys1,2). Analysis of the infectivity and replication of the wild-type and mutant viruses revealed that tRNA(Pro) was the preferred tRNA for high-level replication. Viruses with a PBS complementary to tRNA(Arg) or tRNA(Ly1,2) replicated at levels approximately 30% and 10% as effective as the wild-type virus, while virus with a PBS complementary to tRNA(Lys,3) had the slowest replication kinetics and least infectivity. Comparison of the virion tRNA content of the wild-type and mutant viruses revealed similar ratios with respect to levels of tRNA(Pro), tRNA(Arg) and tRNA(Lys). Modeling of the U5-PBS region revealed that the predicted RNA structure for the virus that selected tRNA(Arg) was more similar to the wild type virus that uses tRNA(Pro) than the virus which use tRNA(Lys1,2) or tRNA(Lys,3); the virus that uses tRNA(Lys,3) had the most profound disruption in the predicted RNA structure. The results of these studies demonstrate that MuLV has evolved to preferentially select tRNA(Pro) for high-level replication and are discussed with respect to common features of the primer selection process between MuLV and other retroviruses.

Adaptation, Physiological↗
PubMed1990

In vitro processing of transcripts containing novel tRNA-like sequences ('t-elements') encoded by wheat mitochondrial DNA.

We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.

Base Sequence↗