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Biomedical subjects

C G Miller

Publications and source records attributed to C G Miller.

At least 19 recordsLinked to original sources
PubMed1992

Modified extravesical ureteral reimplantation in pediatric renal transplantation: 5 years of experience.

Numerous surgical procedures have been described to reimplant the ureter into the bladder during renal transplantation. Since November 1985 we have used a modified extravesical technique in 19 children. At the time of transplantation patient age ranged from 2 to 17 years (average age 10 years). Of these patients only 2 received a cadaveric kidney. Postoperative followup ranged from 4 to 54 months (average 32 months). No immediate or delayed urological complications were noted, and all but 1 graft has continued to function. This procedure is not only expeditious and safe but it also eliminates a long cystostomy suture line and requires a short ureteral length. Urinary leakage and ureteral obstruction, 2 of the most common urological complications, have not been observed in our patients. Although further experience and longer followup are required, this technique has become our procedure of choice for ureteral reimplantation in children undergoing renal transplantation.

Adolescent↗
PubMed1992

Local production of interleukin-8 is associated with nosocomial pneumonia.

One hundred five (70%) of 151 patients hospitalized in the intensive care unit and undergoing mechanical ventilation had bronchial secretions that tested positive for interleukin-8 within 36 hours of admission. Arterial blood, mixed venous blood, and urine collected simultaneously all tested negative, except for 11 patients admitted with intra-abdominal septic foci. The presence of interleukin-8 in the pulmonary air space early in the course of hospitalization was significantly associated with patients with multiple injuries, the need for greater ventilatory support, the occurrence of pulmonary dysfunction, and a 66% incidence of nosocomial bacterial pneumonia. We conclude that the early local production of interleukin-8 in the lungs is an early marker of pulmonary injury and may be involved in the pathogenesis of nosocomial bacterial pneumonia.

Adult↗
PubMed1992

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.

Amino Acid Sequence↗
PubMed1992

Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene.

Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations. The Salmonella typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22. Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles of prlC complemented all phenotypes associated with an S. typhimurium opdA mutation. A plasmid carrying an amber mutation in prlC [prlC31(AM)] was unable to complement except in an amber suppressor background. Tn1000 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA mapped to the region of the plasmid shown by deletion analysis and subcloning to contain prlC. The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein. The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S. typhimurium opdA, showing that these genes are homologs. The nucleotide sequence of the prlC1 suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the prlC suppressor phenotype results from changes in the endopeptidase OpdA.

Alleles↗
PubMed1992

Cloning and nucleotide sequence of the Salmonella typhimurium dcp gene encoding dipeptidyl carboxypeptidase.

Plasmids carrying the Salmonella typhimurium dcp gene were isolated from a pBR328 library of Salmonella chromosomal DNA by screening for complementation of a peptide utilization defect conferred by a dcp mutation. Strains carrying these plasmids overproduced dipeptidyl carboxypeptidase approximately 50-fold. The nucleotide sequence of a 2.8-kb region of one of these plasmids contained an open reading frame coding for a protein of 77,269 Da, in agreement with the 80-kDa size for dipeptidyl carboxypeptidase (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration). The N-terminal amino acid sequence of dipeptidyl carboxypeptidase purified from an overproducer strain agreed with that predicted by the nucleotide sequence. Northern (RNA) blot data indicated that dcp is not cotranscribed with other genes, and primer extension analysis showed the start of transcription to be 22 bases upstream of the translational start. The amino acid sequence of dcp was not similar to that of a mammalian dipeptidyl carboxypeptidase, angiotensin I-converting enzyme, but showed striking similarities to the amino acid sequence of another S. typhimurium peptidase encoded by the opdA (formerly optA) gene.

Amino Acid Sequence↗
PubMed1992

Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium.

The opdA gene (formerly called optA) of Salmonella typhimurium encodes a metallopeptidase, oligopeptidase A (OpdA), first recognized by its ability to cleave and allow utilization of N-acetyl-L-Ala4 (E. R. Vimr, L. Green, and C. G. Miller, J. Bacteriol. 153:1259-1265, 1983). Derivatives of pBR328 carrying the opdA gene were isolated and shown to express oligopeptidase activity at levels approximately 100-fold higher than that of the wild type. These plasmids complemented all of the phenotypes associated with opdA mutations (failure to use N-acetyl-L-Ala4, defective phage P22 development, and diminished endopeptidase activity). The opdA region of one of these plasmids (pCM127) was defined by insertions of Tn1000 (gamma delta), and these insertions were used as priming sites to determine the nucleotide sequence of a 2,843-bp segment of the insert DNA. This region contained an open reading frame coding for a 680-amino-acid protein, the N terminus of which agreed with that determined for purified OpdA. This open reading frame contained both a sequence motif typical of Zn2+ metalloproteases and a putative sigma 32 promoter. However, no induction was detected upon temperature shift by using a beta-galactosidase operon fusion. The predicted OpdA sequence showed similarity to dipeptidyl carboxypeptidase, the product of the S. typhimurium gene dcp, and to rat metallopeptidase EC 3.4.24.15., which is involved in peptide hormone processing.

Amino Acid Sequence↗
PubMed1991

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.

Adenosine Triphosphatases↗
PubMed1991

Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression.

Salmonella typhimurium contains three distinct transport systems (CorA, MgtA, and MgtB) that move Mg2+ across the cytoplasmic membrane. Mutant strains containing only one of these three systems have been constructed and used to study each system in isolation. Characterization of these systems has been hampered, however, by the need to use 28Mg2+, a relatively unavailable, extremely expensive, and short lived radioisotope. This paper reports that 63Ni2+ is transported into the cell by all three of the S typhimurium Mg2+ transport systems. In a strain deficient in all three systems, uptake of 63Ni2+ was undetectable under the conditions used. Comparison of 63Ni2+ uptake kinetics and inhibition of 63Ni2+ transport by other divalent cations suggest that Ni2+ can be used as an analog of Mg2+ in the study of these three transport systems. Using 63Ni2+ to measure uptake, the effect of Mg2+ levels in the growth medium on transport by each system was tested. Transport by the CorA system was unaffected by changes in the amount of Mg2+ in the growth medium. In contrast, uptake via MgtA and MgtB was significantly increased in cells grown in 10 microM extracellular Mg2+ compared to cells grown in 10 mM Mg2+. The increases in uptake were the result of increases in Vmax without change in Km. This result suggests that, in low Mg2+ medium, cells contained higher levels of the transporters. Production of beta-galactosidase from mgtA::lacZ and mgtB::lacZ but not corA::lacZ fusions was also increased when cells were grown in low extracellular concentrations of Mg2+ indicating that the regulation occurs at the level of transcription. Expression of beta-galactosidase was also inhibited by the addition of other divalent cations including Ca2+ and Mn2+. Regulation of transcription from the mgtA and mgtB promoters was similar over the range of extracellular Mg2+ concentrations from 10 microM to 10 mM. At 1 microM, however, transcription from the mgtB promoter, as measured by beta-galactosidase levels in a mgtB::lacZ transcriptional fusion strain, was increased over 800-fold, and Ca2+ could no longer inhibit transcription effectively. In contrast, growth at 1 microM extracellular Mg2+ increased transcription from the mgtA promoter only about 30-fold and Ca2+ could still inhibit this increase. These results suggest that at least two distinct mechanisms are responsible for regulation of the mgtA and mgtB transcription in response to extracellular cation concentration.

Biological Transport↗
PubMed1991

A double-blind study of the speed of onset of analgesia following intramuscular administration of ketorolac tromethamine in comparison to intramuscular morphine and placebo.

A double-blind, randomised, parallel group, placebo-controlled study was performed in 85 patients to compare the speed of onset of analgesia following the intramuscular administration of a single dose of 30 mg of ketorolac tromethamine, 10 mg of morphine or placebo. A new, sensitive, method was used to measure the latency of analgesia. The onset of analgesia was defined by the time taken for the pain intensity score to reach a specified percentage of the baseline value. Twenty-five percent of patients achieving a 50% reduction in baseline pain intensity score appears to be the most appropriate parameter to assess the speed of onset of analgesia of ketorolac and morphine in the postoperative setting. Paired comparison demonstrated that ketorolac had a significantly faster onset of analgesia (p = 0.03) when compared to placebo, whilst comparison of morphine to placebo analgesic latency (p = 0.06) just failed to reach significance. There was no significant difference between the analgesic onset time of ketorolac and morphine (p = 0.73). Intramuscular ketorolac and intramuscular morphine have comparable analgesic onset times in the postoperative pain context. However, the sensitive method of measuring onset of analgesia described, highlights the slow onset of analgesia when analgesics of known efficacy are given by the intramuscular route in the postoperative period. More attention should be given to the speed of onset of analgesia in future assessments of analgesics.

Adult↗
PubMed1991

Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system.

The CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5 min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95 min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild-type and corA mutations. Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co(2+)-resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild-type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficient to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild-type allele of at least one of the corB, corC or corD loci.

Bacterial Proteins↗
PubMed1991

Cloning and nucleotide sequence of the anaerobically regulated pepT gene of Salmonella typhimurium.

The anaerobically regulated pepT gene of Salmonella typhimurium has been cloned in pBR328. Strains carrying the pepT plasmid, pJG17, overproduce peptidase T by approximately 70-fold. The nucleotide sequence of a 2.5-kb region including pepT has been determined. The sequence codes for a protein of 44,855 Da, consistent with a molecular weight of approximately 46,000 for peptidase T (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration). The N-terminal amino acid sequence of peptidase T purified from a pJG17-containing strain matches that predicted by the nucleotide sequence. A plasmid carrying an anaerobically regulated pepT::lacZ transcriptional fusion contains only 165 bp 5' to the start of translation. This region contains a sequence highly homologous to that identified in Escherichia coli as the site of action of the FNR protein, a positive regulator of anaerobic gene expression. A region of the deduced amino acid sequence of peptidase T is similar to segments of Pseudomonas carboxypeptidase G2, the E. coli peptidase encoded by the iap gene, and E. coli peptidase D.

Amino Acid Sequence↗
PubMed1991

Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium.

oxrB8, a mutation that diminishes the anaerobic induction of pepT and other anaerobically regulated, oxrA (fnr)-dependent Salmonella typhimurium genes, is an allele of rpoA, the gene for the alpha subunit of RNA polymerase. Four additional rpoA mutations that affect anaerobic pepT expression have been isolated after localized mutagenesis of the rpoA region. All but one of these rpoA mutations appear to have relatively specific effects on genes that require the OxrA (FNR) protein, a positive transcriptional regulator of a family of anaerobically expressed genes. All of these mutations lead to amino acid substitutions in the C-terminal region of the alpha subunit. These results taken with a number of previous observations suggest a role for the alpha subunit in the interaction between RNA polymerase and positive transcriptional regulatory proteins. They also suggest that the C-terminal region of alpha is important for these interactions.

Amino Acid Sequence↗
PubMed1990

Prediction of hip fracture in elderly women: a prospective study.

OBJECTIVE: To assess the relative importance of osteoporosis of the os calcis, cognisance, and mobility in the risk of subsequent fracture of the hip in elderly women. DESIGN: Prospective study of elderly women in residential care over two years. SETTING: 21 Private or 38 local authority residential homes for the elderly and 4 geriatric hospitals in Doncaster and Hull. SUBJECTS: 1414 Ambulant women aged over 69, in private or local authority residential care or geriatric care. Those who had had bilateral hip surgery were excluded. MAIN OUTCOME MEASURES: Broad band ultrasonic attenuation (BUA) index, Clifton assessment procedures for the elderly test (for cognisance), and mobility on a six point scale, and fracture of the hip in the subsequent two year period. RESULTS: 73 Women fractured their hip during the two years. Their mean age was not significantly different from that of the women who did not have a fracture (85.3 (SD 5.6) v 83.9 (6.3); p = 0.07), but their mean BUA index (40.3 (19.3) v 50.9 (22.2) db/MH2), and score for cognisance (median 19 (interquartile range 10.5-27.0) v 24 (17-30)) were significantly lower (both p less than 0.001). These variables had independent associations with fracture of the hip. Women with fractures had a significantly lower score for the psychomotor component of the cognisance test (4.5 (1-8) v 7 (2-10); p less than 0.0025 and were significantly more mobile (1(1-3) v 3 (1-6); p less than 0.02). Subdividing women according to high, medium, and low scores for BUA index and cognisance testing disclosed a high risk group (118 women) with low BUA index and cognisance score, whose incidence of fracture was 12.8%; in the group at lowest risk (136 women) with high BUA index and cognisance score, the incidence of fracture was only 1.5% (relative risk 8.4 (95% confidence interval -2.0 to 35.5]. Further analysis showed that those most at risk were, additionally, most mobile but that less mobile women with good cognisance had a low incidence of fractures, regardless of the BUA index, (1.2%, high index, v 0.9%, low index). CONCLUSIONS: Elderly women most at risk of sustaining hip fractures were those with low BUA index, low cognisance test score, and high mobility. Improving bone strength and cognisance in elderly women may reduce their incidence of hip fracture.

Aged↗
PubMed1990

A general method of optimizing automated DNA synthesis to decrease chemical consumption to less than half.

An automated DNA synthesis protocol (FC3) has been optimized to decrease the cost of chemical consumption by two- to threefold. Anion-exchange chromatography at pH 12.5 in a Mono Q (Pharmacia) column was used to analyze the oligonucleotides we synthesized. From these analyses, we formulated the FC3 program and a procedure by which a DNA synthesizer designer or operator can use 18-base-long homopolymers of A, G, C, and T to optimize the reaction times, reagent concentrations, solvent wash conditions, and the many steps in the synthesis cycle.

Automation↗
PubMed1990

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene.

The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely--35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.

Alleles↗
PubMed1989

Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium.

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.

Amino Acid Sequence↗