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Protein kinase C-theta (PKCtheta) is a Ca(2+)-independent member of the PKC family that is selectively expressed in skeletal muscle and T lymphocytes and plays an important role in T cell activation. However, the molecular basis for the important functions of PKCtheta in T cells and the manner in which it becomes coupled to the T cell receptor-signaling machinery are unknown. We addressed the functional relationship between PKCtheta and CD28 costimulation, which plays an essential role in T cell receptor-mediated IL-2 production. Here, we provide evidence that PKCtheta is functionally coupled to CD28 costimulation by virtue of its selective ability to activate the CD28RE/activator protein-1 (AP-1) element in the IL-2 gene promoter. First, CD28 costimulation enhanced the membrane translocation and catalytic activation of PKCtheta. Second, among several PKC isoforms, PKCtheta was the only one capable of activating NF-kappaB or CD28RE/AP-1 reporters in T cells (but not in 293T cells). Third, wild-type PKCtheta synergized with CD28/CD3 signals to activate CD28RE/AP-1. In addition, PKCtheta selectively synergized with Tat to activate a CD28RE/AP-1 reporter. Fourth, CD3/CD28-induced CD28RE/AP-1 activation and NF-kappaB nuclear translocation were blocked by a selective PKCtheta inhibitor. Last, PKCtheta-mediated activation of the same reporter was inhibited by the proteasome inhibitor MG132 (which blocks IkappaB degradation) and was found to involve IkappaB-kinase beta. These findings identify a unique PKCtheta-mediated pathway for the costimulatory action of CD28, which involves activation of the IkappaB-kinase beta/IkappaB/NF-kappaB-signaling cascade.

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Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) established that TruB, a pseudouridine (psi) synthase from Escherichia coli, was able to make psi55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs. By deletion of the truB gene, we now show that TruB is the only protein in E. coli able to make psi55 in vivo. Lack of TruB and psi55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to approximately 0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make psi55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of psi55.

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The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.

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Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 degrees C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.

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The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3'AUACU(7)5' at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU(7) sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U(7) resulted in templates that did not support polyadenylation of upstream mRNA. Interestingly, we found that the loss of polyadenylation function led to antitermination of the upstream transcript and resulted in a readthrough transcript that contained the upstream and downstream mRNA sequences. Mutations that blocked polyadenylation also blocked transcription termination and generated mostly readthrough transcript. Reverse transcription-PCR of readthrough transcripts and subsequent nucleotide sequencing of the amplified product revealed no extra adenosine residues at the junction of the readthrough transcript. These results indicate that polyadenylation is required for transcription termination of VSV mRNA. The intergenic dinucleotide GA did not appear to be necessary for transcription termination. Furthermore, we found that insertion of the polyadenylation signal sequence AUACU(7) alone was sufficient to direct polyadenylation and efficient transcription termination at the inserted site. Taken together, the data presented here support the conclusion that polyadenylation is the major determinant of transcription termination at the intercistronic gene junctions of VSV.

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Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels > or = 70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P(60/62/64)) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. Increasing the amount of P(60/62/64) expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P(TTT)) had no apparent effect on transcription (113% of the wild-type level) or replication (81% of the wild-type level), substitution with phenylalanine (P(FFF)) rendered the protein much less active in transcription (< 5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P(EEE)) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P(RRR)) or transcription (P(60/62/64)) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L.

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Peritoneal lavage performed postoperatively in the treatment of purulent peritonitis was evaluated in a prospective randomized study. Patients with free purulent peritonitis due to perforated appendicitis or colonic perforation were treated with intravenous infusion of cefuroxime and metronidazole. The patients were randomly allocated to treatment with or without continuous postoperative peritoneal lavage. The patients were kept under observation for postoperative septic intra-abdominal complications. Of the 79 patients, 41 were treated with lavage postoperatively and 38 were not. No postoperative abscess or other septic intra-abdominal complication was found in any patient. In ten, the postoperative lavage was interrupted because of technical complications or complaints of discomfort by the patient. In this study, no clinical benefit of continuous peritoneal lavage postoperatively in the treatment of purulent peritonitis was noted. Lavage done postoperatively is expensive and seems to carry a risk of complications. Thorough rinsing of the infected abdominal cavity perioperatively and adequate antibiotic treatment, including an antianaerobic agent, seem to be effective in preventing intra-abdominal septic complications.

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