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Biomedical subjects

M E Jones

Publications and source records attributed to M E Jones.

At least 19 recordsLinked to original sources
PubMed1996

A modified Luria-Delbrück fluctuation assay for estimating and comparing mutation rates.

We have investigated the accuracy with which mutation rates may be estimated using a modification of the Luria and Delbrück fluctuation experiment protocol. The modification involves growing a larger-than-usual culture, and plating out a small aliquot of it. Monte Carlo simulations of the experiments confirm that the modification leads to a decrease in the coefficient of variation of the estimated mutation rate where this is based on the median number of mutants detected in a number of cultures grown in parallel. If sets of experimental and control cultures are compared using the Mann-Whitney U-test, then fractional increases in mutation rate can be reliably detected using relatively small numbers of cultures. The modified protocols promise better estimates of mutation rates, offer a powerful test of differences in mutation rates, and are easier to implement in practice.

Computer Simulation↗
PubMed1996

Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase.

Human UMP synthase is a bifunctional protein containing two separate catalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). These studies address the question of why the last two reactions in pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme in mammalian cells, but by two separate enzymes in microorganisms. From existing data on subunit associations of the respective enzymes and calculations showing the molar concentration of enzyme to be far lower in mammalian cells than in microorganisms, we hypothesize that the covalent union in UMP synthase stabilizes the domains containing the respective catalytic centers. Evidence supporting this hypothesis comes from studies of stability of enzyme activity in vitro, at physiological concentrations, of UMP synthase, the two isolated catalytic domains prepared by site-directed mutagenesis of UMP synthase, and the yeast ODCase. The two engineered domains have activities very similar to the native UMP synthase, but unlike the bifunctional protein, the domains are quite unstable under conditions promoting the dissociated monomer.

Catalysis↗
PubMed1996

Bias caused by migration in case-control studies of prenatal risk factors for childhood and adult diseases.

Case-control studies of prenatal risk factors for disease in later life often ascertain cases from within a defined area, trace the birth records of those cases born within the area, and select controls from birth records within the same area. Bias can occur in these studies if the disease risk factors are related to migration from the area. The effects of this bias were examined in a study in Oxfordshire, England. Cases (n = 218) of diabetes in children and young adults born during 1965-1986 were identified from hospital discharges during 1965-1986; controls (n = 753) were selected from livebirths during 1965-1986. By 1987, 219 controls (29.1%) had migrated from Oxfordshire or died. Low maternal parity and high social class were strongly related to migration, more than the other perinatal factors studied. Migration, therefore, could lead to apparent associations of diabetes risk with parity or social class. For a general instance, the authors show how much bias is caused by different degrees of migration and of association between migration and a perinatal risk factor. Examples are given of how migration can produce apparent trends in risk as well as increased or decreased individual relative risks. If more than 25% of controls migrate, bias may be appreciable.

Adult↗
PubMed1996

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation.

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT-3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross-sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.

Animals↗
PubMed1995

Human uridine monophosphate synthase: baculovirus expression, immunoaffinity column purification and characterization of the acetylated amino terminus.

Human uridine monophosphate (UMP) synthase, a bifunctional protein containing orotate phsophoribosyltransferase (OPRTase, EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (ODCase, EC 4.1.1.23) activities, has been overproduced by construction and use of a recombinant baculovirus containing the cDNA for this protein. Expression of the virus in cabbage looper larvae produces a crude larval homogenate having UMP synthase enriched about 180-fold over human placental homogenates and allows larger quantities of this human protein as well as analog proteins to be prepared for structure/function studies. A vastly improved purification procedure using a monoclonal immunoaffinity column was developed. Human UMP synthase purified from larval extracts yielded a product which comigrates in SDS gel electrophoresis with UMP synthase purified from human placenta; pure proteins prepared from these two tissue sources have the same specific activities. We found that OPRTase requires Pi ions in the assay buffers for optimal OPRTase activity; BSA in the assay vessel increases to a lesser degree both OPRTase and ODCase activities. These changes in the assay are essential to observe a parallel enrichment of the two enzyme activities. The baculovirus system was used to express human UMP synthase because it usually yields a product with posttranslational modifications that reflect those of the organism that provided the cDNA. We report data to show that human UMP synthase derived from either human placenta or larval extracts both have a sequence in which the N-terminal methionine has been removed and the formerly penultimate alanine has been acetylated.

Acetylation↗
PubMed1995

The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.

Amino Acid Sequence↗
PubMed1995

Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.

Amino Acid Sequence↗
PubMed1995

Leadership development: educating nurse practitioners for the future.

The development of leadership potential is recognized to be an essential component of nurse practitioner education to prepare graduates for a dynamic health care system. Leadership potential refers to those skills that enable a nurse practitioner to function as a change agent and to effectively resolve conflict. The purpose of the study was to examine personal variables (personality type, self-esteem, and learning style) with respect to the leadership potential of pediatric nurse practitioner graduates over a 3-year period. The research study was descriptive, and data were analyzed using descriptive statistics. The subjects consisted of 21 female students who completed five instruments used to measure the relationships between variables and leadership potential. Significant results were found between those students who learned abstractly and the ability to function effectively as a change agent post-program.

Adult↗
PubMed1994

Left ventricular dilatation and multivessel coronary artery disease on thallium-201 SPECT are important prognostic indicators in patients with large defects in the left anterior descending distribution.

This study examines the importance of left ventricular (LV) dilatation, and evidence of multivessel coronary artery disease identified on thallium-201 (TI-201) single-photon emission computed tomographic (SPECT) scintigrams, for predicting long-term outcome in patients with an extensive left anterior descending (LAD) perfusion deficit. Impaired contractility of the left ventricle determined by low ejection fraction, elevated LV end-systolic volume, and dilatation of the left ventricle are known as major predictors of mortality after myocardial infarction. TI-201 single-photon emission computed tomography primarily reveals status of perfusion/redistribution, but also contains indirect information on LV function. To date, there are no TI-201 SPECT data on impaired function of the left ventricle (LV dilatation) and extent of perfusion deficits, discussed together as correlates of survival. Patient data were prospectively collected in the computer data base at Emory University. A large perfusion defect involving more than one third of the LAD territory was identified in 291 of 2,652 consecutive patients examined with TI-201 SPECT initial and 3-hour redistribution studies. Follow-up data were obtained for 284 patients (98%) at 38 +/- 14 months. Of the 291 patients, 58 died. The most powerful multivariate correlates of death were LV dilatation, multivessel disease, and the ratio of the LAD severity stress score to total severity of SDs stress score. Cox model analysis was used to determine correlates of survival. Three-year survival for patients with LV dilatation was 73% versus 89% without LV dilatation (p < 0.001). Three-year survival in patients with 1-vessel disease ("LAD only") was 94% versus 78% for multivessel disease (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary Disease↗
PubMed1994

Luria-Delbrück fluctuation experiments; accounting simultaneously for plating efficiency and differential growth rate.

Cells growing in culture are subject to mutation, and as mutation is a random event, the number of mutants in a culture will be a random variable. The size of the clone of mutants arising from a single mutational event depends on the timing of the mutation; the earlier the mutation the larger the corresponding clone of mutants. The frequency with which new mutations arise may be estimated from examining the number of mutants found in a number of parallel cultures, each culture arising from a single cell. An efficient estimator of mutation rate in such an experiment is a maximum likelihood estimator. The use of such an estimator presupposes knowledge of the probability distribution for the number of mutants to be detected in a culture, given the mutation rate. In turn this depends on the probability distribution for the size of the clone of detected mutants arising from any single mutation. This latter distribution depends on the relative cell cycle time, tau, of the mutants, and on the probability, s, that a mutant which exists will be detected. This paper develops the required probability distribution.

Cell Division↗
PubMed1994

Luria-Delbrück fluctuation experiments: design and analysis.

Luria and Delbrück, in a seminal paper, introduced fluctuation analysis primarily as a means to elucidate the timing of mutation in relation to the imposition of selective conditions. Their work, and subsequently that of LEA and COULSON, established also a basis for measuring the frequency of mutational events. The several estimators proposed by these authors differ both in complexity and in efficiency, and the published literature relies mainly on the less efficient but computationally trivial estimators. The estimators as originally proposed assume that all mutants occurring in culture will be counted in the subsequent assay, but a relaxation of this assumption suggests an alternative experimental design and alternative estimators which offer advantages over those currently in common use.

Biometry↗
PubMed1994

The relation between climatic temperature and sudden infant death syndrome differs among communities: results from an ecologic analysis.

We examined the negative relation between temperature and the sudden infant death syndrome (SIDS) in 22 communities in seven countries. We estimated the percentage increase in SIDS rate for a 1 degree C drop in climatic temperature. The relation differed substantially among communities. In New Zealand and Australia (10 communities), the association was consistently strong; in Europe (seven communities), it varied from strong to weak; and in the USA (five communities), it was moderate or weak. We postulate that low climatic temperature indirectly increases the incidence of SIDS, particularly in countries where outdoor climatic temperature modifies the indoor temperature and clothing habits.

Australia↗
PubMed1994

Heterogeneity at the beta-lactamase structural gene ampC amongst Citrobacter spp. assessed by polymerase chain reaction analysis: potential for typing at a molecular level.

Considerable biochemical diversity and polynucleotide sequence variation have been reported amongst strains of Citrobacter spp. However, sequence heterogeneity has not been investigated at gene loci of clinical relevance. In this study, sequence heterogeneity in the beta-lactamase structural gene, ampC, amongst 91 clinical isolates of Citrobacter spp. that showed resistance to various third-generation cephalosporins was investigated. Variation was examined by high-stringency polymerase chain reactions (PCR) with primers homologous to the known ampC sequences of C. freundii strains OS60 and I113, and C. diversus NF85. If an isolate contained an ampC gene homologous to one of these three characterised ampC genes, a single PCR band of a predictable size was generated with the appropriate primer set; 50 (60%) of isolates gave a PCR product of the expected size with the OS60 primer set and nine (10%) gave a product with the I113 primer set. All these 59 isolates were identified as C. freundii by API-20E strips. Six isolates (7%) gave a product with the C. diversus NF85 primer set but only four of these were identified as C. diversus in API-20E tests; the other two isolates were identified as C. freundii. Of the 91 isolates, 28 (31%), were identified as either C. freundii or C. diversus, but gave no PCR product with any primer set tested. Five of these showed no homology to any of the reference strain ampC PCR products in hybridisation tests. Nevertheless, all showed beta-lactamase activity. Overall, this method allowed the identification of novel ampC gene loci, which may serve as a basis for the identification of Citrobacter spp. rapidly at a molecular level.

Base Sequence↗
PubMed1994

Composition of human pulmonary surfactant varies with exercise and level of fitness.

We have tested the hypothesis that the composition of alveolar surfactant varies with pattern of breathing and level of fitness. We examined three major components of surfactant, surfactant protein A (SP-A), disaturated phospholipids (DSP), and cholesterol (CHOL) in bronchoalveolar lavage (BAL) fluid from 12 healthy men before and after exercise. Fitness was assessed as work load/heart rate ([kpm.min-1]/[HR.HRmax-1]) achieved during cycling for 30 min at 90% theoretical maximal heart rate. Using a bronchoscope, four 20-ml vols of 0.15 M NaCl at 37 degrees C were instilled and then recovered from first a right upper and then a right lower lobe segmental bronchus. As we found no differences in the BAL from upper and lower lobes, the fluid was combined. We found a direct relationship between CHOL and DSP (rs = 0.84, p < 0.001), SP-A and CHOL (rs = 0.40, p < 0.025), and between SP-A and DSP (rs = 0.44, p < 0.025). The change in the ratios CHOL/DSP, SP-A/CHOL, and SP-A/DSP immediately after exercise was correlated with fitness (rs = -0.56, p < 0.025; rs = 0.75, p < 0.005; rs = 0.62, p < 0.025, respectively). We conclude that the composition of surfactant can change rapidly with exercise in a manner related to fitness, and we suggest that this is consistent with the existence of at least two pools of tissue surfactant of different composition supplying the alveolar compartment.

Adult↗
PubMed1994

Age-related changes in pharyngeal and supraglottic sensation.

As one ages, sensory discrimination in the oral cavity progressively diminishes, and dysphagia and aspiration are more likely to occur. Whether similar age-related laryngeal and pharyngeal sensory abnormalities exist and contribute to dysphagia and aspiration is unknown. The purpose of this study was to determine if sensory discrimination in the area innervated by the superior laryngeal nerve diminishes with increasing age. By applying a previously described new device and technique that utilizes brief air pulse stimulation of the anterior wall of the pyriform sinus, sensory discrimination can be reliably determined. We carried out 672 trials in 56 healthy adults divided into three age groups: 20 to 40, 41 to 60, and 61 to 90 years of age. Overall, the average sensory discrimination was 2.30 +/- 0.50 mm Hg. In subjects 20 to 40 years of age, sensory discrimination was 2.07 +/- 0.20 mm Hg, while in subjects 61 to 90 years of age, sensory discrimination was 2.68 +/- 0.63 mm Hg (p < .05). There also was a statistically significant difference between the 41- to 60-year and 61- to 90-year age groups (p < .05). Progressive diminution in pharyngeal and supraglottic sensitivity with increasing age might be a contributing factor in the development of dysphagia and aspiration in the elderly.

Adult↗
PubMed1994

Meniscal suturing.

There is a popular misconception that the meniscus is avascular and that tears of the meniscus do not heal. Over the years this notion has gradually been dispelled as being incorrect and the fact that the periphery of the meniscus is in fact vascular has been accepted. Attempts to resuture this particular structure in the knee have, in some cases, proved to be very successful.

Arthroscopy↗