Role of spinal cyclooxygenase-2 and prostaglandin E2 in fentanyl-induced hyperalgesia in rats.

BACKGROUND: Accumulated evidence suggests that spinal cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) may be implicated in the development of opioid-induced hyperalgesia. METHODS: Rats received subcutaneous fentanyl injections at different doses (20-80 µg kg-1), four separate times at 15-min intervals. Some rats only received fentanyl (60 µg kg-1 × 4 doses) with or without surgical incision. Fentanyl-induced hyperalgesia was evaluated via a tail-pressure or paw-withdrawal test. The concentrations of spinal COX-2, EP-1 receptor (EP-1R) mRNA, and PGE2 were measured. The effects of the COX-2 inhibitor, parecoxib (intraperitoneal 10 mg kg-1), or the EP-1R antagonist, SC51089 (intraperitoneal 100 µg kg-1), on hyperalgesia and spinal PGE2 were examined. RESULTS: Acute repeated injections of fentanyl dose-dependently induced mechanical hyperalgesia, which reached a peak at the 1st day and persisted for 1-4 days postinjection. This hyperalgesia could be partly or totally prevented by the pretreatment of either parecoxib or SC51089. Consistently, the levels of spinal COX-2 mRNA and PGE2 were also dose-dependently increased, reaching a peak at the first day and persisting for 2 days postinjection. Pretreatment with parecoxib could block the increase in spinal PGE2 and had no effects on spinal COX-2 and EP-1R mRNA. Fentanyl injection enhanced incision-induced mechanical and thermal hyperalgesia. CONCLUSIONS: Acute repeated fentanyl administration dose-dependently produced mechanical hyperalgesia and augmented surgery induced postoperative hyperalgesia. This behavioural change was paralleled with an increase in spinal COX-2 mRNA and PGE2 after fentanyl administration. Inhibition of COX-2 or blockade of EP-1R can partly or totally prevent hyperalgesia.

Trypanosoma brucei mitochondrial ribonucleoprotein complexes which contain 12S and 9S ribosomal RNAs.

Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria. The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed. Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane. Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent. We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle. We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts. The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size. This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17.5S, containing the 9S rRNA, and 20S containing the 12S rRNA. A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2:3 ratio (20S:17.5S).

Guide RNA molecules not engaged in RNA editing form ribonucleoprotein complexes free of mRNA.

Mitochondrial pre-mRNAs in kinetoplastid organisms undergo uridine additions and deletions after transcription, a phenomenon termed kRNA editing. The reaction involves small, mitochondrial DNA transcripts, so called guide RNAs which provide the editing information via base pairing to the pre-mRNAs and furthermore may act as the U-nucleotide donors. Guide RNAs are not maintained as free molecules within the mitochondrial organelle, instead form several high molecular weight ribonucleoprotein complexes. Here we report the identification of two new gRNA containing RNP complexes, 8S and 15S in size, that only assemble with upstream gRNA molecules which require editing of their cognate pre-mRNA before they can base pair. The two complexes do not contain pre-mRNA molecules and the 8S RNP can be assembled in vitro. It contains two polypeptides under these conditions with apparent molecular weights of 90 and 21 kDa that can be cross-linked to the gRNA molecule. Our observation suggests the existence of structurally simple gRNA/protein complexes that might function as building blocks for the assembly of a high molecular weight editing machinery.

Large angle spin-echo imaging.

A study was undertaken to assess the use of excitation flip angles greater than 90 degrees for T1 weighted spin-echo (SE) imaging with a single 180 degrees refocusing pulse and short TR values. Theoretical predictions of signal intensity for SE images with excitation pulse angles of 90-180 degrees were calculated based on the Bloch equations and then measured experimentally from MR images of MnCl2 phantoms of various concentrations. Liver signal-to-noise ratios (SNR) and liver-spleen contrast-to-noise ratios (CNR) were measured from breathhold MR images of the upper abdomen in 16 patients using 90 and 110 degrees excitation flip angles. The theoretical predictions showed significant improvements in SNR with excitation flip angles > 90 degrees, which were more pronounced at small TR values. The phantom studies showed reasonably good agreement with the theoretical predictions in correlating the excitation pulse angle with signal intensity. In the human imaging studies, the 110 degrees excitation pulse angle resulted in a 7.4% (p < .01) increase in liver SNR and an 8.2% (p = .2) increase in liver-spleen CNR compared to the 90 degrees pulse angle at TR = 275 ms. Increased signal intensity resulting from the use of large flip angle excitation pulses with a single echo SE pulse sequence was predicted and confirmed experimentally in phantoms and humans.

MR imaging evaluation of knee collateral ligaments and related injuries: comparison of T1-weighted, T2-weighted, and fat-saturated T2-weighted sequences--correlation with clinical findings.

The objectives of this study were to compare the ability of T1-weighted (T1W), proton density/T2-weighted (PD/T2W), and fat saturation (FS) PD/T2W magnetic resonance (MR) sequences for depiction of the knee collateral ligaments and related injuries, and to compare MR findings with clinical findings. Ten subjects with normal knee ligaments and 64 patients with suspected collateral ligament injuries underwent coronal T1W, PD/T2W, and FS PD/T2W imaging. Abnormalities ranged from edema surrounding the collateral ligaments (grade I) to complete disruption of ligamentous fibers (grade III). FS PD/T2W images improved definition of the medial collateral ligament (MCL) and lateral collateral ligament (LCL) compared with other sequences in 78% and 81% of patients, respectively. While the apparent grade of collateral ligament injury was similar with all pulse sequences in most patients, depiction of such injury was usually most conspicuous on FS PD/T2W images (MCL, 92% of patients; LCL, 38% of patients). In no patients were clinically diagnosed collateral ligament injuries undetected or understaged with MR imaging. MR findings indicated higher-grade MCL and LCL injuries than did clinical examination in 24 and 15 patients, respectively.

Mitochondrial transcripts are processed but are not edited normally in Trypanosoma equiperdum (ATCC 30019) which has kDNA sequence deletion and duplication.

Analyses of the Trypanosoma equiperdum (ATCC 30019) maxicircle reveals deletions, duplications and rearrangement compared to T. brucei. The genes for 9S rRNA and 12 proteins are absent. The 12S rRNA and cytochrome oxidase subunit I (COI) genes lack their 3' ends and are adjacent indicating deletion of intervening genes. The remaining two NADH dehydrogenase subunit genes (ND4 and ND5), the ribosomal protein RPS12 gene and the CR5 gene are duplicated and rearranged. ND4, RPS12 and the CR4 transcripts are abundant in steady state RNA while 12S rRNA and COI transcripts are not detected. Full length ND5 transcripts are rare, if present, but chimeric ND5/ND4 transcripts are abundant. The CR4 and RPS12 transcripts are the size of unedited RNAs suggesting that they are processed. However, they are not edited normally, presumably due to the absence of minicircle gRNA genes.

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Neurofibromatosis: MR imaging findings involving the head and spine.

MR imaging can be used to identify abnormalities of the head and spine in patients with neurofibromatosis. In this pictorial essay, we illustrate the craniospinal MR imaging findings in a large series of patients with neurofibromatosis.

RNase P RNA in Candida glabrata mitochondria is transcribed with substrate tRNAs.

The biosynthesis of some mitochondrial enzymes requires contributions of both the mitochondrial and nuclear genomes. The ribonucleoprotein enzyme Ribonuclease P (RNase P) is composed of a mitochondrial encoded RNA and nuclear coded protein in many yeasts, including C. glabrata. We have determined that there are at least two sites of transcription initiation that contribute to the expression of the mitochondrial RNase P RNA. A nonanucleotide promoter sequence is located upstream of the initiator tRNA while the other site of initiation of transcription is at an undetermined upstream site. An analysis of the transcripts from the region of the RNase P gene demonstrates directly that the RNase P RNA is present in large primary transcripts and located between the precursors to the initiator tRNAf(Met) and tRNA(Pro) genes. Thus this enzyme subunit is synthesized with some of its substrate tRNAs. An activity with cleavage site specificity like a previously described endonuclease that cleaves near the 3' end of tRNAs, RNase P activity and one or more additional endonucleases or exonucleases not described previously are required to convert the primary transcript to its final functional RNAs.

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.

Sequence and expression of four mutant aspartic acid tRNA genes from the mitochondria of Saccharomyces cerevisiae.

Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs. By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene. The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp. The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp. The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end. We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts. The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

Polymorphism in the structure of the yeast mitochondrial tRNA synthesis locus.

Yeast mitochondrial DNA contains a genetic locus, called the tRNA synthesis locus, which codes for information necessary for mitochondrial tRNA biosynthesis. A 9S RNA molecule coded by this locus is thought to be the trans-acting element required for the removal of 5' extensions from tRNA precursors. The DNA coding for this RNA maps to a region of mitochondrial DNA known to contain strain specific restriction site polymorphisms. Comparison of the tRNA synthesis locus in two such strains by sequence analysis demonstrates that the restriction enzyme polymorphisms are due to the deletion/insertion of a 50 base pair GC-rich element in the 5' flanking sequence of the 9S RNA coding region. There are also several differences between the 9S RNA coding region of these two strains which do not interfere with the tRNA synthesis function.