The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn.

BACKGROUND: The long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. METHODS: Rats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. RESULTS: Spinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3' untranslated region. CPEB1 knockdown decreased the expression of MCU protein. CONCLUSIONS: These findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.

A comparison of the Pentax Airway Scope™ with the Airtraq™ in an infant manikin.

We compared the Pentax Airway Scope(TM) with the Airtraq(TM) optical laryngoscope in an infant manikin. Twenty-three anaesthetists randomly performed tracheal intubation: at rest, (a) with the Airway Scope and (b) with the Airtraq; and during chest compressions, (c) with the Airway Scope and (d) with the Airtraq. The success rate, modified Cormack and Lehane classification for glottic view, time taken to view the glottis, and time to place the tracheal tube were recorded. There was no difference in intubation success rate or quality of glottic view between the two devices. The median (IQR [range]) time taken to obtain a view of the glottis was 4.5 (3.7-6.4 [1.8-14.0]) s using the Airway Scope compared with 7.1 (5.5-9.6 [3.3-12.0]) s using the Airtraq (p = 0.001), and to successful placement of the tracheal tube was 8.3 (6.8-9.4 [3.7-20.7]) s using the Airway Scope compared with 11.2 (10.4-13.8 [4.9-23.7]) s using the Airtraq (p = 0.001). During chest compressions, the median (IQR [range]) time taken to view the glottis was 5.1 (4.0-7.2 [2.0-12.4]) s using the Airway Scope compared with 7.5 (5.0-13.2 [4.2-26.4]) s using the Airtraq (p = 0.006), and to successful placement of the tracheal tube was 9.5 (6.6-13.7 [4.5-16.2]) s using the Airway Scope compared with 11.7 (9.1-18.1 [6.2-37.4]) s using the Airtraq (p = 0.022). We conclude that both devices provided good quality views of the glottis and successful tracheal intubation in an infant manikin both at rest and during external chest compressions. Use of the Airway Scope resulted in a shorter time to view the glottis and perform successful tracheal intubation compared with the Airtraq.

The Pentax-AWS((R)) rigid indirect video laryngoscope: clinical assessment of performance in 320 cases.

The Pentax-AWS airway scope system is a rigid indirect video laryngoscope with integrated tube guidance. Laryngoscopy and intubation are visualised using a built in LCD monitor which displays the view obtained by a CCD camera mounted in the tip of the laryngoscope. We describe its clinical performance in 320 patients. The Pentax-AWS significantly improved the laryngeal view compared to the Macintosh laryngoscope. Forty-six patients (14%) who were classified as Cormack Lehane glottic view grade 3 or 4 using the Macintosh laryngoscope were classified as grade 1 (45 cases) or 2a (1 case) using the Pentax-AWS airway scope. Laryngeal views measured by percentage of glottic opening score were improved significantly using the Pentax-AWS. Intubation using the Pentax-AWS was successful in all cases, 96% at the first and 4% at the second attempt. The mean (SD) time required to place the tracheal tube was 20 (10) s. The Cormack Lehane grade obtained with the Macintosh blade did not affect the total time to correctly position the tube using the Pentax-AWS. Intubation difficulty scale (score = 0 in 305 patients, score = 1 in 14 and score = 2 in one patient) indicates that tracheal intubation was performed easily in most cases. The Pentax-AWS not only improves the laryngeal view, but its tube guide also facilitates rapid, easy and reliable tracheal intubation under vision. It can be useful in routine anesthesia care and may be advantageous in the situation of unanticipated difficult intubation.

Intravenous dexmedetomidine for cesarean delivery and its concentration in colostrum.

BACKGROUND: Dexmedetomidine is a sedative agent with high α2-adrenoreceptor selectivity. We investigated intravenous dexmedetomidine administration during scheduled cesarean delivery under neuraxial anesthesia; and its concentration in the colostrum. METHODS: Twenty-seven participants having elective cesarean delivery under combined spinal-epidural anesthesia were enrolled. After delivery and cord clamping, 6µg/kg/h of intravenous dexmedetomidine was administered for 10minutes, followed by a dose of 0.7µg/kg/h until peritoneal closure. Sedation, vital signs and side effects were recorded. Blood and colostrum samples were collected from each participant at 6, 12, and 24h after dexmedetomidine administration. Samples were analysed using liquid chromatography tandem-mass spectroscopy. RESULTS: Colostrum samples were collected from 10 patients. The median [95% CI] plasma dexmedetomidine concentration was 333 [303-534] pg/ml at 0h and 19.7 [13.5-25.8] pg/ml at 6h. The colostrum concentration was 12.3 [8.1-20.1] pg/ml at 6h. The dexmedetomidine completely disappeared from both within 24h. The calculated milk-to-plasma ratio at 6h was 0.76 [0.57-0.86]. The relative infant dose was 0.034% [0.020-0.062%]. At dexmedetomidine discontinuation, the Richmond Agitation-Sedation Scale score was -2 (range,-4 to -1). During surgery, no patients complained of nausea, peritoneal irritation or afterbirth pain. CONCLUSIONS: The dexmedetomidine milk-to-plasma ratio did not exceed 1 in any participant, and the relative infant dose was very low. Maternal sedation using dexmedetomidine is unlikely to be harmful for the infant.

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.

Parametric genome rearrangement.

Algorithms inspired by comparative genomics calculate an edit distance between two linear orders based on elementary edit operations such as inversion, transposition and reciprocal translocation. All operations are generally assigned the same weight, simply by default, because no systematic empirical studies exist verifying whether algorithmic outputs involve realistic proportion of each. Nor do we have data on how weights should vary with the length of the inverted or transposed segment of the chromosome. In this paper, we present a rapid algorithm that allows each operation to take on a range of weights, producing an relatively tight upper bound on the distance between single-chromosome genomes, by means of a greedy search with look-ahead. The efficiency of this algorithm allows us to test random genomes for each parameter setting, to detect gene order similarity and to infer the parameter values most appropriate to the phylogenetic domain under study. We apply this method to genome segments in which the same gene order is conserved in Escherichia coli and Bacillus subtilis, as well as to the gene order in human versus Drosophila mitochondrial genomes. In both cases, we conclude that it is most appropriate to assign somewhat more than twice the weight to transpositions and inverted transpositions than to inversions. We also explore segment-length weighting for fungal mitochondrial gene orders.

[Anesthetic management of two patients with insulinoma using propofol--in association with rapid radioimmunoassay for insulin].

We experienced anesthetic management of two patients with insulinoma in whom frequent hypoglycemic episodes with blood glucose levels of 39-42 mg.dl-1 had been observed. Each patient received epidural analgesia with a catheter inserted at the T 9/10 intervertebral space. Anesthesia was induced with propofol 80-100 mg and fentanyl 200 micrograms. Tracheal intubation was facilitated with vecuronium 6 mg. Anesthesia was maintained with continuous infusion of propofol and epidural anesthesia. Rapid measurements of immunoreactive insulin (IRI) were useful for localization of insulinoma during surgery. Perioperative plasma glucose levels could be maintained within normal ranges by continuous infusion of glucose. Rebound hyperglycemic episodes were not observed, and IRI was reduced after removal of the insulinoma. General anesthesia using propofol and epidural block is a useful choice for the anesthetic management of patients undergoing an operation for removal of an insulinoma.

Effect of age on pulmonary gas exchange during laparoscopy in the Trendelenburg lithotomy position.

BACKGROUND: Physiological changes in respiratory mechanics caused by aging may lead to a deterioration in pulmonary gas exchange, an increase in the alveolar-arterial oxygen gradient [(A-a)D(O2)] and a difference between the arterial carbon dioxide (CO(2)) tension (P(a)(CO(2))) and expired end-tidal CO(2) tension (P(ET)(CO(2))) [P((a-ET))(CO(2))] during laparoscopy in the Trendelenburg lithotomy position (TLP). METHODS: The subjects were 51 gynecologic patients. Pressure-controlled ventilation was used to maintain P(ET)(CO(2)), measured by the side stream method, within the range 4-4.67 kPa. During laparoscopy with CO(2) insufflation in TLP, the tidal volume was increased to keep P(ET)(CO(2)) within +/- 20% of the pre-insufflation value. The subjects were divided into three groups by age: young group (< 45 years); middle-aged group (45-64 years); and elderly group ( > or = 65 years). RESULTS: Before pneumoperitoneum (PPN), significant differences were found between the young and elderly groups in the arterial oxygen tension (P(a)(O(2))), (A-a)D(O(2)), P(a)(CO(2)) and P((a-ET))(CO(2)). In all groups, the peak inspiratory pressure and P(a)(CO(2)) increased progressively during PPN in TLP. P((a-ET))(CO(2)) increased gradually after starting CO(2) insufflation in TLP only in the elderly group. CONCLUSIONS: An increase in P((a-ET))(CO(2)) was seen during PPN in TLP in the elderly group. With CO(2) insufflation in TLP, the setting of mechanical ventilation based on the value of P(ET)(CO(2)) (measured by the side stream method) should be determined with caution in elderly patients.

Synonymous codon preferences in bacteriophage T4: a distinctive use of transfer RNAs from T4 and from its host Escherichia coli.

Codon usage data of bacteriophage T4 genes were compiled and synonymous codon preferences were investigated in comparison with tRNA availabilities in an infected cell. Since the genome of T4 is highly AT rich and its codon usage pattern is significantly different from that of its host Escherichia coli, certain codons of T4 genes need to be translated by appropriate host transfer RNAs present in minor amounts. To avoid this predicament, T4 phage seems to direct the synthesis of its own tRNA molecules and these phage tRNAs are suggested to supplement the host tRNA population with isoacceptors that are normally present in minor amounts. A positive correlation was found in that the frequency of E. coli optimal codons in T4 genes increases as the number of protein monomers per phage particle increases. A negative correlation was also found between the number of protein monomers per phage and the frequency of "T4 optimal codons", which are defined as those codons that are efficiently recognized by T4 tRNAs. From these observations it was proposed that tRNAs from the host are predominantly used for translation of highly expressed T4 genes while tRNAs from T4 tend to be used for translation of weakly expressed T4 genes. This distinctive tRNA-usage in T4 may be an optimization of translational efficiency, and an adjustment of T4-encoded tRNAs to the synonymous codon preferences, which are largely influenced by the high genomic AT-content, would have occurred during evolution.

Identification and chromosomal distribution of DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis.

DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis were identified, using the GenBank sequence database. Chromosomal locations of the conserved segments were compared between the two bacteria, and the following three features were observed. (1) Although the two genomes are nearly identical in size, chromosomal arrangements of the conserved segments are considerably different from each other. (2) In many cases, chromosomal locations of a conserved segment in the two species have deviated from each other by a multiple of 60 degrees. (3) There are many instances in which a contiguous segment in one genome is split into two or more segments located at distinct positions in the other genome, and these split segments were found to tend to lie on the E. coli or B. subtilis genome separated by distances of multiples of 60 degrees. On the basis of these observations, genome organizations of the two bacteria were discussed in terms of genome doublings as well as random chromosomal rearrangements.

Gene arrangements and phylogeny in the class Proteobacteria.

A simple method is presented for reconstructing phylogenetic trees on the basis of gene transposition. It is shown that differences in gene arrangements among genomes could allow us to determine whether a gene transposition event has occurred before or after species divergence from parsimonious considerations. The method is applied to evolutionary relationships among the bacterial class Proteobacteria, for which complete genomic sequences most densely accumulate and comprehensive gene order comparisons are possible. We were able to infer the emergence order of proteobacterial subclasses as epsilon-->beta-->gamma. This order is consistent with sequence-based inferences, which conversely confirms the usefulness of the approach presented here.

Identification of regulatory building blocks in Escherichia coli genome.

The nucleotide sequences of extragenic regions in the Escherichia coli genome are statistically analyzed. Sequence elements with high occurrence frequencies are identified; these elements are: (1) extragenic palindromic sequences, which are markedly distinguishable from the already identified repetitive extragenic palindromic sequences; (2) promoter sequences of purine biosynthetic genes; and (3) rho-independent terminator sequences. The repetitious occurrence and extensive sequence similarities suggest that these elements share common evolutionary origins. Copies of one sequence element would have become distributed to various positions on the genome during evolution and have been fixed at locations that provide a selective advantage. The extragenic regions of the E. coli genome seem to consist of various regulatory 'building blocks', similar to a protein which consists of modules or domains.

Periodic distribution of homologous genes or gene segments on the Escherichia coli K12 genome.

A computer search for homologous relationships among Escherichia coli proteins has been carried out with the use of databases. Homologous genes or gene segments thus identified at the amino acid sequence level have a tendency to lie on the genome separated by distances of multiples of 7 min. This relatively regular pattern of gene distribution on the E. coli genome is interpreted as reflecting the early history of multiple genome doublings. Evolutionary advantages of duplications of the total genome are discussed in relation to the acquisition of new multistep pathways such as the Krebs cycle and to the generation of a variety of proteins in existence today.

A possible mode of protein evolution. Role of the anti-sense strand in the generation of new proteins.

The possibility that a new functional protein may be generated during evolution by using the anti-sense strands of pre-existing genes is proposed. Sequence similarity between the anti-sense strand of one gene and the sense strand of another gene may provide evidence for this generation. The computer search for the "sense/anti-sense" strand similarities between nucleotide sequences for receptors and sequences available in a database was carried out. This type of similarity was found for various receptor genes, such as the low density lipoprotein receptor, lymphocyte receptor for IgE, asialoglycoprotein receptor (hepatic lectin), T-cell receptor, epidermal growth factor receptor, insulin receptor and estrogen receptor genes. Sequence similarities between the sense strands of receptor genes were also found. These results may suggest that the repertoire of proteins was increased by the utilization of the anti-sense or sense strands of pre-existing genes.

Conformational change and cooperative ligand binding in hemoglobin.

Although many studies have been carried out on the cooperativityly of ligand binding to hemoglobin, its molecular mechanism is not yet completely confirmed. Various models have been proposed on one hand, but on the other hand the direct judgement of the validity of each model is confronted with experimental difficulties. With this situation in mind, we re-examine carefully various kinds of experimental data, with the intention of finding any hints for future studies on the molecular mechanism of the cooperativity of ligand binding not only to hemoglobin but also to other allosteric proteins composed of subunits. As a result of these re-examinations most of the experimental data can be consistently explained from the viewpoint that the strain due to ligation is transmitted to the interfaces of the subunits and the degree of the strain stored in the interfaces between the subunits controls the cooperativity of of ligand binding. The subjects selected to the present study are molecular structural differences between the deoxy- and oxy-hemoglobin, temperature dependence of oxygen equilibrium constants, and recombination curves of carbon monoxide in flash photolysis experiments. In the first section, the difference of the interfaces of the subunits between deoxy- and oxy-hemoglobin is investigated on the basis of the atomic coordinates determined by Perutz et al. and we examine energetically which of the segment's pairs contributes mainly to the difference in the molecular structure between the two forms. On the basis of this investigation, it is shown in the next section that the oxygen equilibrium constants of human adult hemoglobin under normal physiological conditions are well explained by the following sequence of the conformational changes. The local changes in the interfaces of the subunits are reflected directly on the small differences among the first three Adair constants and the succeeding rearrangement to the four subunits causes the outstandingly larger value of the fourth Adair constant. In more alkaline pH, this rearrangement begins to occur at the earlier stages of ligation. In the succeeding section, it is pointed out that the property of this rearrangement can be studied in more detail through the recombination curves followed by flash photolysis under special conditions.

Computer-assisted analysis of chromosomal locations and transcriptional directions of Escherichia coli genes.

We present a computer-assisted method for locating and orienting nucleotide sequence segments on a large restriction map by comparing restriction fragment lengths. This method is based on the observation that long restriction fragments are rare and, therefore, the longest restriction fragments serve as effective discriminators. The method was applied to Escherichia coli genes, and chromosomal locations and transcriptional directions for more than 500 genes were determined.

Theoretical analysis of single-round transcription experiments on trp leader region.

A kinetic model is proposed to reproduce the time courses of the concentration change in paused leader RNA, terminated leader RNA, and readthrough RNA in the single-round transcription experiments on trp leader region of Escherichia coli and its mutants, L132, L75, and L75L135 (Winkler, M. E., and C. Yanofsky, 1981, Biochemistry, 20:3738-3744; Fisher, R., and C. Yanofsky, 1983, J. Biol. Chem., 258:9208-9212). This model fits the experimental results well and also captures the essential aspects of the processes of transcriptional pausing and termination. In the wild type template, under optimal conditions, it is found that the transcription rate at the pause and attenuation sites is of the same order of magnitude, 10,000-fold lower than the transcription rate at the other sites, and the high termination level at the attenuation site is attributable to a higher dissociation rate. This analysis also provides a clue as to how the template base change, various concentrations of ribonucleoside triphosphates, and the presence or absence of L-factor affect the transcription and dissociation rates to yield different termination levels at the pause or attenuation site. It also discusses the molecular mechanism of the transcriptional pausing and termination.

Regulation in the synthesis of Escherichia coli RNA polymerase proposed from sequence analysis.

A computer analysis of the operons containing Escherichia coli RNA polymerase subunits was carried out to obtain information about the regulation in the synthesis of these subunits, identifying intercistronic promoter and terminator sequences and searching for ribosome-binding sites and possible secondary structures of the corresponding mRNAs. This investigation showed an extensive secondary structure of beta-mRNA, which provides a molecular basis for the mechanism of the phenomenologically known post-transcriptional regulation in the synthesis of beta subunit. Since a similar secondary structure was also recognized in the mRNA corresponding to the upstream part of alpha subunit gene, it is proposed that the synthesis of alpha subunit is autogenously regulated by RNA polymerase itself, probably at the translational level, in the same way as in the synthesis of beta subunit. This regulation not only guarantees the suppression of overproduction of RNA polymerase subunits but also throws light on the problem of how the syntheses of RNA polymerase and ribosome respond similarly to the shift of nutrients and temperature, but differently to the starvation for amino acids.