Genetic code in evolution: switching species-specific aminoacylation with a peptide transplant.

The genetic code is established in aminoacylation reactions whereby amino acids are joined to tRNAs bearing the anticodons of the genetic code. Paradoxically, while the code is universal there are many examples of species-specific aminoacylations, where a tRNA from one taxonomic domain cannot be acylated by a synthetase from another. Here we consider an example where a human, but not a bacterial, tRNA synthetase charges its cognate eukaryotic tRNA and where the bacterial, but not the human, enzyme charges the cognate bacterial tRNA. While the bacterial enzyme has less than 10% sequence identity with the human enzyme, transplantation of a 39 amino acid peptide from the human into the bacterial enzyme enabled the latter to charge its eukaryotic tRNA counterpart in vitro and in vivo. Conversely, substitution of the corresponding peptide of the bacterial enzyme for that of the human enabled the human enzyme to charge bacterial tRNA. This peptide element discriminates a base pair difference in the respective tRNA acceptor stems. Thus, functionally important co-adaptations of a synthetase to its tRNA act as small modular units that can be moved across taxonomic domains and thereby preserve the universality of the code.

Injection techniques to anesthetize the difficult tooth.

Failure to achieve anesthesia can be a significant problem in the day-to-day practice of dentistry. The usual strategy following an anesthetic failure is to reinject. Therefore, a good understanding of conventional anesthetic techniques is important. But the practitioner also needs to have a broad armamentarium of injection strategies available for the "difficult-to-anesthetize cases". These strategies include the use of 5 percent lidocaine, intrapulpal injection, periodontal ligament injection and intraosseous injection. This paper will be a brief discussion of those techniques with an emphasis on the intraosseous injection.

Analysis of peptide-binding motifs for two disease associated HLA-DR13 alleles using an M13 phage display library.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate 'sequence motif' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.

Evidence that two present-day components needed for the genetic code appeared after nucleated cells separated from eubacteria.

The trinucleotide/amino acid relationships of the present-day genetic code are established by the amino-acylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides. Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree. In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl- and tryptophanyl-tRNA synthetases. Ij particular, the eukaryotic tyrosyl- and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts. In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid. Our results raise the possibility that present day tyrosyl- and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria. The results have implications for the development of the genetic code.

Species-specific microhelix aminoacylation by a eukaryotic pathogen tRNA synthetase dependent on a single base pair.

We report here that tyrosyl-tRNA synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA synthetase and aligns with the prokaryotic tyrosyl-tRNA synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region. The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr). Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices. However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair. We show that P. carinii tyrosyl-tRNA synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr. In contrast, the enzyme does not charge E. coli microhelixTyr. Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P. carinii tyrosyl-tRNA synthetase. Conversely, we found that E. coli tyrosyl-tRNA synthetase can charge an E. coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair. The results demonstrate that the common structural framework of homologous tRNA synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation. We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA synthetases.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Expression and function of HLA-B27 in lipid-linked form: implications for cytotoxic T lymphocyte-induced apoptosis signal transduction.

Major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL-induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA-B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl-anchored HLA-B27 (PI-B27), a 42-kDa molecule which can be cleaved off the cell surface by PI-specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI-B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL-induced apoptosis signal transduction in target cells.

Evaluation of a clinical recovery score after general anesthesia.

A clinical recovery score (CRS) assessing recovery after general anesthesia was compared with the Digit-Symbol Substitution Test (DSST), Trieger Test (TT), a patient-completed visual analogue scale for alertness (VAS), and an independent observer's evaluation of recovery. The CRS included ratings of the following parameters: activity, respiration, circulation, consciousness, ambulation, color, and nausea and vomiting. Forty patients requiring the removal of three or four third molars participated in the study. All patients received the same general anesthetic technique. Each patient was evaluated by the five methods preoperatively, on admission to the recovery room, and at 15-min intervals until discharge. The four recovery tests (CRS, DSST, TT, VAS) were evaluated using chi 2 analysis to determine if there was any overall difference among the tests using the observer's determination of home readiness as the standard for discharge. The CRS was significantly more in agreement with the observer's determination than were the paper and pencil tests. The recovery tests were also evaluated with regard to instances of early dismissal or prolonged retention of the patient, again using the observer's determination as the "gold standard." The CRS was the only recovery test devoid of early dismissals. We conclude that the CRS provides a valid, simple measure of recovery that can be readily used in offices providing outpatient anesthesia and in studies measuring clinical recovery from anesthesia or sedation.

Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon.

A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons with the corresponding sequences from numerous other species are presented. The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A-dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines. Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B. subtilis cells grown in the presence of excess uracil. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence. The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined. However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid.

Electronic dental anesthesia in a patient with suspected allergy to local anesthetics: report of case.

A 56-year-old patient with alleged allergy to local anesthetics required restorative dental treatment. Electronic dental anesthesia was used successfully, in lieu of injectable local anesthetics, to manage intraoperative pain associated with the restoration of vital mandibular teeth.

A comparison of biochemical effects of nitrogen dioxide, ozone, and their combination in mouse lung.

Swiss Webster mice were exposed to either 4.8 ppm (9024 microgram/m3) nitrogen dioxide (NO2), 0.45 ppm (882 microgram/m3) ozone (O3), or their combination intermittently (8 hr daily) for 7 days, and the effects were studied in the lung by a series of physical and biochemical parameters, including lung weight, DNA and protein contents, oxygen consumption, sulfhydryl metabolism, and activities of NADPH generating enzymes. The results show that exposure to NO2 caused relatively smaller changes than O3, and that the effect of each gas alone under the conditions of exposure was not significant for most of the parameters tested. However, when the two gases were combined, the exposure caused changes that were greater and significant. Statistical analysis of the data shows that the effects of combined exposure were more than additive, i.e., they might be synergistic. The observations suggest that intermittent exposure to NO2 or O3 alone at the concentration used may not cause significant alterations in lung metabolism, but when the two gases are combined the alterations may become significant.

Comparison of pulmonary biochemical effects of low-level ozone exposure on mice and rats.

The biochemical effects of a 5-d continuous exposure to 0.45 ppm (882 microgram/m3) O3, were studied in the lungs of 2-mo-old male, specific-pathogen-free mice (Swiss Webster) and three strains of rats (Long-Evans, Wistar, and Sprague-Dawley). The results, expressed per lung, indicated a general increase in lung weight, DNA and protein contents, oxygen consumption, sulfhydryl metabolism, and the activities of several NADP+-reducing enzymes for all exposed animals relative to their controls. When the increases in the two species (mice versus three strains of rats) were compared, the mice showed significantly higher increases than the rats in several parameters. The responses among the three strains of rats were variable, but the differences were not significant. These observations suggest that Swiss Webster mice may offer a more sensitive animal model than rats for studying the pulmonary effects of a given low-level O3 exposure.