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P R Cunningham

Publications and source records attributed to P R Cunningham.

At least 37 records · Page 2Linked to original sources
PubMed1993

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↗
PubMed1993

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

Donor-specific tolerance permits burn allografting without increased sepsis.

Early excision and allografting of massive burns is beneficial. However, chronic immunosuppression, utilized to prolong allograft survival, increases the potential risk of infection. We have previously shown long-term skin allograft survival in mice with a 30% total body surface area (TBSA) burn by inducing donor-specific tolerance (DST) using only perigrafting administration of antithymocyte globulin (ATG) and donor bone marrow (DBM). Chronic immunosuppression is avoided. This study tests whether induction of DST compromises host resistance to infection. Resistance to a septic challenge created by cecal ligation and puncture (CLP) 10 days after a 30% TBSA burn was investigated in the following groups of mice: [table: see text] Positive blood cultures were documented for 97% of mortalities. Burn excision and grafting significantly (P less than or equal to 0.05) decreased mortality. No increased mortality was seen in allografted mice receiving ATG or ATG and DBM compared to isografted mice receiving no immunosuppression. These studies suggest that skin allografting with DST may permit the benefits of burn excision without the risks of infection seen with chronic immunosuppression.

Animals↗
PubMed1990

Thoracolumbar compression fractures presenting with an acute ileus.

Corticosteroids are commonly used in the treatment of connective tissue diseases such as systemic lupus erythematosus. Although they are usually efficacious, osteoporosis leading to spine compression fractures is not uncommon. In this case report, we describe an elderly patient with systemic lupus erythematosus on long-term corticosteroid therapy who presented with symptoms of acute abdomen with minimal low back symptoms. No intraabdominal process was found by abdominal studies and exploratory laparotomy. Increased lower back symptoms led to further skeletal spine studies, which initially demonstrated a compression fracture at the twelfth thoracic (T12) vertebra. Later, a T8 and a fourth lumbar (L4) compression fracture were also found. Her abdominal and lower back symptoms resolved on conservative therapy. Although the rate of these occurrences are unknown, compression spine fractures should be considered in elderly patients presenting with acute abdomen after being on long-term corticosteroid therapy.

Aged↗
PubMed1990

Transplantation and the trauma surgeon.

The number of satisfactory vascular organs suitable for organ transplantation can be increased appreciably by a close working relationship between traumatologists and transplant surgeons. Benefits will derive from more precise and rapid field management of brain-injured patients, with subsequent appropriate stabilization. If brain death is declared, prompt referral for organ donation with optimal management should result in well-functioning organs for transplantation. A defined team approach with well-defined protocols could solve most of the medical and moral dilemmas. Compassionate emotional support should be provided for families, particularly minorities, and should extend to inexperienced staff. Underlying these goals are a strong institutional commitment to staff education and an understanding of the lifesaving role that organ transplantation can play.

Adolescent↗
PubMed1989

Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli.

A 6-kb fragment of DNA, which complemented defects in the alcohol dehydrogenase (ADH)-encoding gene (adhE) of Escherichia coli, was cloned into a multicopy vector. Both ADH and coenzyme-A-linked acetaldehyde dehydrogenase (ACDH) activities were encoded by the plasmid, pHIL8. The adhE gene was identified as an open reading frame of 891 codons encoding an Mr 96,008 protein (minus the initiating methionine). Codon usage analysis indicates that adhE should be highly expressed. This gene shows no significant homology to any previously sequenced ADH-encoding gene.

Alcohol Dehydrogenase↗