Rural trauma. Systems in evolution.
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Humans↗
Biomedical subjects
P R Cunningham
Publications and source records attributed to P R Cunningham.
The ripped cava.
Lacerations of the inferior vena cava are associated with a high mortality and may be difficult to repair. The majority of injuries are due to penetrating trauma. Rapid transportation to definitive surgical care with effective resuscitation may improve mortality. Surgical management includes adequate treatment of hypovolemic shock due to blood loss. Placement of intravenous infusion sites below the level of the diaphragm may be effective. Operative control of the inferior vena cava can be accomplished by directed digital compression followed by a proximal and distal control. Injuries of the inferior vena cava above the level of the renal veins are associated with an increased mortality. Retrohepatic and subdiaphragmatic injuries are highly lethal. This article discusses appropriate surgical approaches for repair of the inferior vena cava above and below the diaphragm.
Emergency Medical Services↗
Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin.
An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition.
Base Composition↗
Intravenous ethanol for alcohol detoxification in trauma patients.
Traumatic injury frequently follows alcohol abuse. Between October 1, 1988 and January 31, 1992, 2,219 patients were admitted to the Trauma Service at the University Medical Center of Eastern Carolina-Pitt County. Of the 1,602 who were tested for serum ethanol, 685 (43%) were found to have measurable levels. Thirty-seven patients had alcohol withdrawal and were treated with intravenous ethanol; 34 were male (21 black, 13 white) and 3 female (1 black, 2 white), with an average age of 46 years. Those who had withdrawal had an average serum ethanol level, on presentation, of 239 mg/dL (N = 34). Fourteen patients were involved in motor vehicle crashes, seven were pedestrians struck by cars, and the remaining 16 had various traumatic mechanisms of injury. The most common injuries were long-bone fractures and blunt abdominal trauma. The length of ethanol therapy averaged 4 days. A majority of patients had a favorable response to treatment. Relative contraindications to i.v. ethanol therapy were CNS trauma, liver disease, and pancreatitis. i.v. ethanol is a safe and effective method of alcohol detoxification in the trauma patient.
Adult↗
Functional effects of base changes which further define the decoding center of Escherichia coli 16S ribosomal RNA: mutation of C1404, G1405, C1496, G1497, and U1498.
The existence and functional importance of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, was proven recently by mutational analysis [Cunningham, P. R., Nurse, K., Bakin, A., Weitzmann, C. J., Pflumm, M., & Ofengand, J. (1992) Biochemistry 31, 12012-12022]. Here we show that the additional nearby tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are functionally important for tRNA binding to the ribosomal A and P sites. Breakage of the base pairs in turn led to a loss of activity at both A and P sites, whereas restoration in the reverse orientation led to recovery of activity. Recovery was incomplete, indicating that base pairing alone is insufficient for full restoration of function. Mutation of U1498 to G created the potential for the tertiary base pair C1403:G1498, which could stack on the aforementioned double base pair, creating a more stable helix longer by one residue. This mutation did not affect subunit association, A- and P-site binding of tRNA to 70S, fMet-tRNA binding to 30S, or poly(Phe) synthesis but did block formation of the first peptide bond, fMet-Val. Mutation of U1498 to A or C did not show this effect. Since the G1498 mutant could make both the 70S initiation complex and the peptide bond, as shown by its ability to form fMet-puromycin, the block in fMet-Val synthesis appears to involve some aspect of A-site function.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Composition↗
Chemical evidence for domain assembly of the Escherichia coli 30S ribosome.
A fragment of 16S RNA corresponding to most of the 5'-domain (residues 1-526) was prepared by in vitro run-off transcription. When this fragment was incubated with a mixture of 30S proteins under conditions known to result in the in vitro assembly of a complete, functional 30S ribosome from a full-length transcript, a discrete 16S particle was formed. This particle contained near stoichiometric amounts of ribosomal proteins S4, S16, S17, and S20. These four proteins are the same, and only, ones that have been shown to interact with the 5' domain of 16S RNA in the intact 30S ribosome in the footprinting studies of Noller and co-workers. We conclude that the 5' fragment 1-526 is capable of folding independently of the rest of the molecule so as to generate the protein binding sites for the same four proteins with which the corresponding segment of full-length 16S RNA normally interacts. These sites not only include those for S4, S17, and S20 that are known to bind directly to the RNA, but also the site for S16, which requires the prior binding of S4 and S20.
Base Sequence↗
Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli.
The regulation of the adhE gene, which encodes the trifunctional fermentative acetaldehyde-alcohol dehydrogenase of Escherichia coli, was investigated by the construction of gene fusions and by two-dimensional protein gel electrophoresis. Both operon and protein fusions of adhE to lacZ were induced 10- to 20-fold by anaerobic conditions, and both fusions were repressed by nitrate, demonstrating that regulation is at the level of transcription. Nitrate repression of phi (adhE-lacZ) expression, as well as of alcohol dehydrogenase enzyme activity, was partly relieved by a mutation in narL. Mutations in rpoN or fnr had no effect on the expression of adhE. Two-dimensional protein gels demonstrated that increases in the amount of adhE protein correlated with increases in enzyme activity, demonstrating that induction was due to synthesis of new protein, not to activation of preexisting protein. When oxidized sugar derivatives such as gluconate or glucuronate were used as carbon sources, the anaerobic expression of phi (adhE-lacZ) was greatly reduced, whereas when sugar alcohols such as sorbitol were used, the expression was increased compared with expression when glucose was the carbon source. This observation suggested that induction of phi (adhE-lacZ) might depend on the level of reduced NADH, which should be highest with sorbitol-grown cells and lowest with glucuronate-grown cells. When phi (adhE-lacZ) was present in a strain deleted for the adhE structural gene, anaerobic expression of phi (adhE-lacZ) was approximately 10-fold higher than in an adhE+ strain. Since the presence of alcohol dehydrogenase would serve to decrease NADH levels, this finding again implies that the adhE gene is regulated by the concentration of reduced NAD. Introduction of a pgi (phosphoglucose isomerase) mutation reduced the anaerobic induction of phi(adhE-lacZ) when the cells were grown on glucose, but had little effect on fructose-grown cells. Pyruvate did not overcome the pgi effect, but glycerol 3-phosphate did, which is again consistent with the possibility that adhE expression responds to the level of reduced NAD rather than to a glycolytic intermediate.
Alcohol Dehydrogenase↗
Blacks and whites on the UNOS renal waiting list: waiting times and patient demographics compared.
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Black or African American↗
Donor bone marrow infusion suppresses alloantibody response in RATG-treated recipients: a correlate of long survival.
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Animals↗
Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.
Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.
Base Sequence↗
G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes.
16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J. (1984) Biochemistry 23, 429-437]. To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined. Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions. However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions. This was also true for the deletion of G1401. Deletion of C1400 or C1402 had more complex effects. Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402. Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400. P site binding was inhibited by either deletion. HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Civilian field surgery in the rural trauma setting: a proposal for providing optimal care.
Rural trauma presents unique problems for surgical care. While military surgeons are prepared to provide care at or near the scene of battle, civilian literature is devoid of reports for care provided by surgeons at sites of injury occurrences. Although these injuries are infrequent, they are more likely to occur in rural trauma settings. This article describes two cases of extremity injury that required amputation at the scene and presents a proposal for swift mobilization of appropriately trained surgeons to the scene with adequate instrumentation and lighting, which can significantly reduce the morbidity and mortality of these victims.
Accidents, Occupational↗
Increase in renal magnesium overflow following aortotomy-induced hemorrhage in pigs.
Hemorrhage causes an increase in plasma Mg++ levels. This article identifies some of the tissues contributing to the hemorrhage induced increase in plasma Mg++. Anesthetized, splenectomized pigs were subjected to a 5 mm aortotomy. Blood samples from the abdominal aorta, inferior vena cava, pulmonary artery, hepatic portal vein, and renal vein were sampled for Mg++ changes caused by this uncontrolled hemorrhage. Aortotomy produced a rapid drop in arterial pressure and cardiac output, both of which remained depressed for the following 120 minutes (P less than 0.05). By 120 minutes, 6 of the 8 pigs survived, but three of the six survivors showed signs of cardiovascular decompensation. A significant increase in arterial plasma Mg++ was indicated by a paired t-test (P less than 0.05). Analysis of the venous samples also revealed a significant increase during the post-aortotomy period. Renal venous and hepatic portal venous plasmas both showed increases greater than that in the arterial plasma. The renal and splanchnic tissues, then, were partly responsible for the increase in the arterial Mg++ which follows hemorrhage.
Animals↗
SP6 RNA polymerase stutters when initiating from an AAA... sequence.
The 16S ribosomal RNA gene of Escherichia coli was placed under the transcriptional control of consensus and modified T7 promoters and a modified SP6 promoter. Both T7 and SP6 polymerases faithfully transcribed the coding sequence (beginning at the +1 position) of each construct, although SP6 polymerase was five-fold more effective than T7 polymerase in initiating with the AAAUUG... sequence. An appreciable fraction of the SP6 transcript molecules contained additional adenosines in the -1, -2, -3, -4, and -5 positions. The transcripts containing additional residues constituted approximately 40-50% of the total SP6 transcription products. Neither the nature nor extent of the additional residues was affected by replacing the pppA 5'-end by pA. Since the identity of the inserted residues does not correspond to the sequence of the template, these additional nucleosides must result from 'stuttering' of the SP6 enzyme at the -1 to +3 positions during initiation of transcription.
Bacteriophages↗
The absence of modified nucleotides affects both in vitro assembly and in vitro function of the 30S ribosomal subunit of Escherichia coli.
16S RNA of Escherichia coli lacking all post-transcriptional modifications and with 5'-termini of pppGGGAGA-, pppGAA-, pppAAA-, and pAAA- were prepared by in vitro transcription of appropriately engineered plasmids with T7 or SP6 RNA polymerases. These synthetic versions of 16S RNA were compared with natural 16S RNA for their ability to reconstitute 30S ribosomal subunits in vitro using varied conditions for both the isolation of the RNA and for reconstitution. Under all conditions studied, natural 16S RNA assembled correctly, as judged by velocity centrifugation comparison with an internal standard of native 30S particles, and the recovered ribosomes were 80-100% as active as native 30S ribosomes in initiation complex formation, P site binding of AcVal-tRNA, A site binding of Phe-tRNA, and formation of the first peptide bond. In contrast, all of the synthetic constructs including pAAA-, which has the same sequence as native 16S RNA, were only partially active in reconstitution and in the functional assays. We conclude that the lack of the 10 methylated nucleotides and/or the 2 pseudouridylate residues present in natural 16S RNA must be responsible for the reduced activity of the synthetic RNAs in ribosome assembly and function.
Centrifugation, Density Gradient↗
Kidney allograft tolerance in primates without chronic immunosuppression--the role of veto cells.
Posttransplant infusion of specific donor bone marrow cells into ATG-treated recipients promotes long survival of allografts in the absence of immunosuppressive drug therapy. DBMC infusion also inhibits antidonor CTL activation in allograft recipients, a trend analogous to the veto phenomenon. The present studies investigated a hypothesis that veto activity in DBMC is involved in the induction of donor-specific unresponsiveness in rhesus monkey kidney transplant recipients given ATG and DBM. Normal monkey BMC were fractionated into subpopulations by depletion with mAbs and immunomagnetic beads. The unfractionated BMC and BMC subsets were tested for veto activity in in vitro MLR-induced CTL and for in vivo tolerance-promoting activity in ATG-treated monkey kidney transplant recipients. In MLR-induced CTL assays, BMC specifically suppressed CTL activity to PBL from the BMC donor. The suppression was mediated by a small population of BMC that expressed a CD2+, CD8+, CD16+, DR-, CD3-, CD38- phenotype. Results of in vivo studies showed a striking correlation with the in vitro results. ATG-treated recipients given either DR- DBMC or DR- CD3- DBMC infusions had significantly prolonged graft survival and a 40-50% incidence of long survivors over 150 days (P less than 0.001 vs. ATG controls). In contrast, in recipients given CD2- DBMC or DR- CD16- DBMC infusions, the tolerance-promoting effect of DBMC was absent, and there were no long-term survivors. The results also showed an association between long survival and suppressed in vivo antidonor CTL activity. Thus acute rejection and in vivo and in vitro antidonor CTL responses were suppressed by a minor subpopulation of DBMC with similar phenotypic markers. We suggest that a veto mechanism may control the induction phase of allograft tolerance in this model, providing a critical period of CTL silence to allow development of host immunoregulatory mechanisms necessary for maintaining graft tolerance.
Animals↗
Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.
In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.
Adenine Nucleotides↗
Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.
The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.
Base Sequence↗