Confirmational Identification of Escherichia coli, a Comparison of Genotypic and Phenotypic Assays for Glutamate Decarboxylase and beta-d-Glucuronidase.
[This corrects the article on p. 3350 in vol. 62, PMID: 8795225.].
Journal Article↗
Biomedical subjects
C H Johnson
Publications and source records attributed to C H Johnson.
Effect of different full-limb casts on in vitro bone strain in the distal portion of the equine forelimb.
OBJECTIVE: To determine the ability of a full-limb transfixation pin cast to protect the distal portion of the equine forelimb from weight-bearing forces by measuring bone strain in vitro on cadaver limbs loaded in a mechanical testing machine. SAMPLE POPULATION: 6 forelimbs from 6 horses. PROCEDURE: Each limb was instrumented with 3 unidirectional metal foil electrical resistant strain gauges. Gauges were placed on the dorsal aspect of the distal portion of the radius and the mid-dorsal portion of the cortex of the third metacarpal bone and the first phalanx. Each limb was tested 3 times, once supported with a transfixation pin cast, once supported by a standard full-limb cast, and finally, uncast. The limbs were tested in a mechanical testing machine under axial loads ranging from 100 to 1,000 lb, and bone strains were recorded at each load. RESULTS: Compared with values for the uncast limb, the transfixation pin cast and the standard full-limb cast significantly (P < 0.001) reduced bone strain on the distal portion of the radius, third metacarpal bone, and first phalanx. Compared with the standard full-limb cast, the transfixation pin cast significantly (P < 0.001) reduced bone strain on the first phalanx. CONCLUSION AND CLINICAL RELEVANCE: Compared with the standard full limb cast, the full-limb transfixation pin cast is more protective of the first phalanx.
Animals↗
Effect of pin hole size and number on in vitro bone strength in the equine radius loaded in torsion.
OBJECTIVE: To determine the effect of pin hole size and number on the breaking strength of the adult equine radius when loaded in torsion to failure. SAMPLE POPULATION: 54 pairs of equine radii from adult horses. PROCEDURE: For test one, 12 pairs of radii were used to determine the effect of pin hole size on torsional breaking strength. A 6.35-mm hole was drilled in 1 radius, and a 9.5-mm hole was drilled in the contralateral radius. For test two, 36 pairs of radii were randomly assigned to 1 of 3 treatment groups (n = 12) to determine the effect of pin hole number on the torsional breaking strength of the equine radius. One radius of each pair served as a control, and one, three, or six 6.35-mm transcortical holes were drilled in the contralateral radius. For test three, 6 pairs of radii had torsional forces applied directly to the transfixation pins, as opposed to the bone itself. One radius of a pair served as a control, and three 6.35-mm smooth Steinman pins were placed in the contralateral radius. All radii were loaded in torsion to failure, and the breaking strengths were recorded. RESULTS: Compared with the 6.35-mm hole, the 9.5-mm hole significantly decreased torsional strength of the radius. There was no significant difference in mean torsional strength between the control radii and the radii with 1, 3, or 6 transcortical holes or when the transfixation pins were loaded. CONCLUSION: Use of up to three 6.35-mm transfixation pins can be used in a full-limb transfixation pin cast to optimize stiffness without a significant decrease (12%) in bone strength.
Animals↗
Mitochondrial protein synthesis is not required for efficient excision of intron aI5 beta from COX1 pre-mRNA in Saccharomyces cerevisiae.
Splicing of the group I intron aI5 beta from the yeast mitochondrial COX1 transcript requires at least four proteins, encoded by the nuclear genes PET54, MRS1/PET157, SUV3 and MSS18. These proteins either act directly to facilitate intron aI5 beta excision, or indirectly in some manner. One possible indirect mode of action of these nuclear gene products is in stimulation of expression of a mitochondrial protein, such as a maturase, that is necessary for intron aI5 beta excision. To test this possibility, splicing of intron aI5 beta was examined in a rho-strain, which is incapable of mitochondrial protein synthesis. A quantitative RT-PCR assay was set up to compare levels of spliced COX1 mRNA present in three strains: a wild-type rho + strain; the rho-strain 7-49b-11, which retains the entire COX1 transcription unit; and a strain bearing a null mutation in the nuclear PET54 gene. The results showed that excision of aI5 beta occurs relatively efficiently in the rho-strain, and therefore does not require any mitochondrial-encoded proteins.
DNA Restriction Enzymes↗
Delayed entry into prenatal care: effect of physical violence.
OBJECTIVE: To assess whether women who experienced physical violence by their partner during the 12 months before delivery were more likely to delay entry into prenatal care than were women who had not experienced physical violence. METHODS: We analyzed data from the Pregnancy Risk Assessment Monitoring System. The sample included 27,836 women who delivered live infants during 1993-1994 in nine states and were surveyed 2-6 months after delivery. We calculated risk ratios and 95% confidence intervals (CIs) to measure the association between physical violence within the 12 months before delivery and entry into prenatal care. RESULTS: The prevalence of delayed entry into prenatal care (entering after the first trimester) was 18.1% and that of reported physical violence was 4.7%. Overall, women who experienced physical violence were 1.8 times more likely (95% CI 1.5, 2.1) to have delayed entry into prenatal care than women who had not experienced such violence. When stratifying by selected maternal characteristics, this association was found only for groups of women who were 25 years of age or older or were of higher socioeconomic status. CONCLUSION: Older women and women of higher socioeconomic status who reported physical violence were more likely to delay entry into prenatal care than younger or less affluent women.
Adult↗
Massive abdominal-wall hernia reconstruction with expanded external/internal oblique and transversalis musculofascia.
We describe a technique for expansion and primary closure of massive and large recalcitrant abdominal-wall hernias in the middle and lower abdomen utilizing expanders placed in the lateral abdominal wall between the external oblique and the deeper complex of the internal oblique and transversalis fasciae. Since this technique describes expansion of the lateral abdominal wall, insertion incisions are made in the lateral abdominal wall away from the primary zone of injury surrounding the abdominal hernia and without interrupting the blood supply or innervation to the abdominal-wall muscle, fascia, or skin. This technique, described in four patients with massive abdominal-wall hernias, has been used successfully for primary closure with vascularized autogenous abdominal-wall fascia, obviating the need for interposition of prosthetic material or extraabdominal flaps.
Abdominal Muscles↗
Inactivation of Helicobacter pylori by chlorination.
Three strains of Helicobacter pylori were studied to determine their resistance to chlorination. The organisms were readily inactivated by free chlorine and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water.
Chlorine↗
Chest wall reconstruction.
Chest wall resection and reconstruction continue to provide a formidable challenge. Prolonged hospitalization of 2 to 3 weeks in often necessary, and patients at our institution have undergone an average of two operations to achieve final closure. However, in multiple reviews of the senior author's personal experience, 85% of patients alive 30 days after operation had a healed, asymptomatic chest wall. Most late deaths occur as a result of the underlying disease process, usually malignancy.
Female↗
Tissue expansion in the upper extremities.
In this article, the authors demonstrate current concepts of soft-tissue reconstruction using tissue expansion principles. History, pathophysiology, and biomechanics of tissue expansion are reviewed. Anatomic areas of expander use in the upper extremity have been delineated, as well as new concepts of nerve and arterial elongation using intraoperative expansion techniques. The authors outline their current technique of upper extremity tissue expansion in the preoperative, intraoperative, and postoperative settings, allowing the reader to appreciate the technique of tissue expansion and its role in soft-tissue reconstruction of the upper extremity.
Adult↗
Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.
To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.
Animals↗
A sigma factor that modifies the circadian expression of a subset of genes in cyanobacteria.
We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70-like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.
Amino Acid Sequence↗
Transcription of CABII is regulated by the biological clock in Chlamydomonas reinhardtii.
The small gene family encoding the chlorophyll a/b-binding proteins of photosystem II (CABII or lhcb) is known to exhibit circadian rhythms of mRNA abundance in Chlamydomonas reinhardtii. In this study we investigated the role of transcription in the phenomenon. We used as reporters Chlamydomonas genes that encode nitrate reductase (NITI) and arylsulfatase (ARS2) transcriptionally fused to sequences upstream of one of the CABII genes (called CABII-1). We found that both reporters exhibited the same circadian rhythm of mRNA abundance in phase, period, and amplitude as does the endogenous CABII-1 gene. We also evaluated the efficacy of arylsulfatase enzymatic activity as a reporter and found that its half-life is too long to make it a useful reporter of rhythmic transcription during a circadian or diurnal cycle. The amount of mRNA synthesis from the CABII-1 gene was examined by in vivo labeling experiments and a circadian rhythm in transcription rate was demonstrated. In vivo labeling also revealed a circadian rhythm of mRNA synthesis for the CABII gene family as a whole. The results from the transcriptional reporter assays together with the in vivo labeling experiments strongly support the conclusion that the biological clock regulates the transcriptional activity of the CABII-I gene, and moreover that regulation at the transcriptional level is the predominant mode by which the clock regulates this gene.
Animals↗
Circadian clocks in prokaryotes.
Prokaryotes have long been thought incapable of expressing circadian (daily) rhythms. Recently, however, such biological 'clocks' have been discovered in several species of cyanobacteria. These endogenous timekeepers control gene expression on a global level in cyanobacteria. Even in cyanobacterial cultures that are growing with average doubling times more rapid than one per 24 h, the circadian clock controls gene expression and cell division. We have isolated mutants of the cyanobacterial circadian pacemaker and are currently characterizing the loci responsible for their altered period phenotypes.
Cell Division↗
Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp. strain PCC 7942.
Extensive circadian (daily) control over gene expression in the cyanobacterium Synechococcus sp. strain PCC 7942 is programmed into at least two differentially phased groups. The transcriptional activity of the smaller group of genes is maximal at about dawn and minimal at about dusk. We identified one of the genes belonging to this latter group as purF, which encodes the key regulatory enzyme in the de novo purine synthetic pathway, glutamine PRPP amidotransferase (also known as amidophosphoribosyltransferase). Its expression pattern as a function of circadian time was confirmed by both luminescence from a purF::luxAB reporter strain and the abundance of purF mRNA. By fusing sequences upstream of the purF coding region to promoterless luxAB genes, we identified a limited upstream region, which potentially regulates purF circadian expression patterns in vivo. We also identified the purL gene immediately upstream of purF. The purL gene encodes FGAM synthetase, the fourth enzyme in the purine nucleotide biosynthesis pathway. Although these genes are expressed as part of a larger operon in other bacteria, reporter gene fusions revealed that purF and purL are transcribed independently in Synechococcus and that they are expressed at different phases of the circadian cycle. This differential expression pattern may be related to the oxygen sensitivity of amidophosphoribosyltransferase.
Amino Acid Sequence↗
Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.
Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.
Base Sequence↗
Detection of Escherichia coli O157:H7 in water from coliform enrichment cultures.
Environmental water samples were seeded with Escherichia coli O157:H7 and the bacterium was recovered using a traditional coliform enrichment procedure followed by selective plating on sorbitol MacConkey agar and biochemical and serological characterization. Assays for beta-glucuronidase and glutamate decarboxylase were found to be useful procedures for screening suspected isolates. The organism was not recovered in a survey of various water samples.
Bacteriological Techniques↗
Circadian oscillations of cytosolic and chloroplastic free calcium in plants.
Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.
Aequorin↗
Circadian orchestration of gene expression in cyanobacteria.
We wanted to identify genes that are controlled by the circadian clock in the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942. To use luciferase as a reporter to monitor gene expression, bacterial luciferase genes (luxAB) were inserted randomly into the Synechococcus genome by conjugation with Escherichia coli and subsequent homologous recombination. The resulting transformed clones were then screened for bioluminescence using a new developed cooled-CCD camera system. We screened approximately 30,000 transformed Synechococcus colonies and recovered approximately 800 clones whose bioluminescence was bright enough to be easily monitored by the screening apparatus. Unexpectedly, the bioluminescence expression patterns of almost all of these 800 colonies clearly manifested circadian rhythmicity. These rhythms exhibited a range of waveforms and amplitudes, and they also showed a variety of phase relationships. We also found bioluminescence rhythms expressed by cyanobacterial colonies in which the luciferase gene set was coupled to the promoters of several known genes. Together, these results indicate that control of gene expression by circadian clocks may be more widespread than expected thus far. Moreover, our results show that screening organisms in which promoterless luciferase genes have been inserted randomly throughout the genome by homologous recombination provides an extremely sensitive method to explore differential gene expression.
Circadian Rhythm↗