The visual analog scale in the immediate postoperative period: intrasubject variability and correlation with a numeric scale.

UNLABELLED: The visual analog scale (VAS) has been used to assess the efficacy of pain management regimens in patients with acute postoperative pain, but its usefulness has not been confirmed in postoperative pain studies. We studied 60 subjects in the immediate postoperative period. The specific data collected were: VAS scores versus an 11-point numeric pain scale; repeatability in VAS scores over a short time interval; and change in VAS scores from one assessment period to the next versus a verbal report of change in pain. The correlation coefficients for VAS scores with the 11-point pain scale were 0.94, 0.91, and 0.95. The repeatability coefficients were 17.6, 23.0, and 13.5 mm. Of the 56 patients who completed all three assessments, only 16 (29%) had repeatability within 5 mm on all three. Some of the changes in VAS scores between assessments were in the direction opposite the verbally reported changes in pain (31%); however, most (92%) were within 20 mm. There was no correlation between the level of sedation, previous pain experience, anxiety, or anticipated pain with consistency in VAS scores. We conclude that any single VAS score in the immediate postoperative period should be considered to have an imprecision of +/- 20 mm. IMPLICATIONS: The visual analog scale was developed for assessing chronic pain but is often used in studies of postoperative pain. This study finds that the visual analog scale correlates well with a verbal 11-point scale but that any individual determination has an imprecision of +/- 20 mm.

Effects of Osmoprotectants upon NaCl Stress in Rice.

Plants accumulate a number of osmoprotective substances in response to NaCl stress, one of them being proline (Pro). While characterizing some of the changes in solute accumulation in NaCl-stressed rice (Oryza sativa L.), we identified several other potential osmoprotectants. One such substance, trehalose, begins to accumulate in small amounts in roots after 3 d. We performed a series of experiments to compare the effects of Pro and trehalose on ion accumulation to determine whether the two chemicals protect the same physiological processes. We found that Pro either has no effect or, in some cases, exasperates the effect of NaCl on growth inhibition, chlorophyll loss, and induction of a highly sensitive marker for plant stress, the osmotically regulated salT gene. By contrast, low to moderate concentrations of trehalose reduce Na+ accumulation, salT expression, and growth inhibition. Somewhat higher concentrations (10 mM) prevent NaCl-induced loss of chlorophyll in blades, preserve root integrity, and enhance growth. The results of this study indicate that during osmotic stress trehalose or carbohydrates might be more important for rice than Pro.

Heat-inducible rice hsp82 and hsp70 are not always co-regulated.

We have characterized several heat-shock-induced genes in rice (Oryza sativa L.) and compared their expression under a variety of conditions. Three of these genes, which are analogs of the hsp82/90 family, lie within a cloned 18-kilobase (kb) region of the genome. The middle member of this cluster, designated hsp82B, has been fully sequenced. The gene uses a promoter containing six putative heat-shock elements as well as several unusual sequence motifs including a stretch of 11 thymidines alternating with 11 adenosines. The mRNA for this gene reaches its highest relative level of expression within 120 min after plants are shifted to 42 degrees C; no other conditions induce this gene. By contrast, we found that during heat stress the expression of hsp70 correlates well with increases in internal ion concentrations, and can also be induced by excess salt or ethanol at normal growth temperatures. These results appear to indicate that whereas hsp70 is induced by all stresses that lead to protein denaturation-including heat stress-HSP82 mRNA accumulates only upon heat stress.

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots.

PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a cDNA library prepared from mRNAs of rice shoots utilizing the product of PCR as probe. Two cDNA clones, prxRPA and prxRPN, were isolated. They encode distinct isozymes of peroxidase. Sequence analysis indicated that the clones encode mature proteins of approximately 32 kDa, both of which possess a putative signal peptide. Comparison of the amino acid sequences of the two rice peroxidases showed that they are about 70% similar to each other but are only 40% to 50% similar to other plant peroxidases. RNA blot hybridization revealed that mRNAs that corresponded to prxRPA and prxRPN cDNAs accumulate at high levels in roots but only at low levels in stems and leaves. In various tissues of rice plants, levels of both mRNAs were stimulated by wounding and by ethephon. These results indicate that at least two isozymes of peroxidase are expressed not only in shoots but also in roots of rice plants, and that the expression of these genes is influenced by ethylene which is the simplest plant hormone.

Molecular characterization and regulation of the phosphoglycerate kinase gene from Trichoderma viride.

We have isolated the phosphoglycerate kinase gene (pgk) of Trichoderma viride and characterized its expression. Comparison of genomic and cDNA clones allowed the correct deduction of the intron boundaries and the 3'-end cleavage site of this gene. Primer extension analysis showed that transcription initiated at three start points between -296 and -298 bp upstream of the translational start codon. The promoter sequence contained a number of cis-acting sequences commonly found in eukaryotic promoters. The pgk transcript analysis of T. viride grown on defined carbon sources showed that neither rate nor growth phase greatly affects pgk expression. By contrast, when Trichoderma spp. were grown in the presence of cell walls of a phytopathogenic fungus as carbon source, pgk messenger levels dropped markedly. This suggests that pgk mRNA accumulation is specifically repressed in the simulated mycoparasitic state.

Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria.

Differential effects of elicitors on the viability of rice suspension cells.

We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid-treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases.

Transient Gene Expression in Intact and Organized Rice Tissues.

Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a [beta]-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light-harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.

Dissociation of peptidyl-tRNA from ribosomes is perturbed by streptomycin and by strA mutations.

Peptidyl-tRNA may dissociate preferentially from ribosomes during protein synthesis when it is inappropriate to, does not correctly complement, the messenger RNA. To test this idea, growing cultures of Escherichia coli were treated with streptomycin to increase the frequency of errors during protein synthesis. Since the treated cells had a temperature-sensitive peptidyl-tRNA hydrolase and could not destroy dissociated peptidyl-tRNA, it was possible to measure the rate of its accumulation after raising the temperature to non-permissive conditions. Both low and high doses of streptomycin enhanced the rate of dissociation and accumulation of peptidyl-tRNA. The rank order of rates of dissociation/accumulation of various isoaccepting tRNA families was not significantly altered by the drug treatment. We concluded that streptomycin stimulated a normal pathway for dissociation of peptidyl-tRNA. Two streptomycin- resistant strains of E. coli had higher rates of dissociation of peptidyl-tRNA than did their sensitive parent strain. When treated with high doses of the drug, the resistant strains showed slightly reduced rates of dissociation of peptidyl-tRNA. These results were interpreted in terms of a two state, two site model for protein synthesis: streptomycin enhances the binding of aminoacyl-tRNA to a tight state of the ribosome A site; the strA mutation enhances translocation to a loose state of the ribosome P site.

Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes.

Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.