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PubMed2001

Green fluorescent protein as a novel tool to measure apoptosis and necrosis.

BACKGROUND: Several apoptosis-detecting methods are currently available. Many of them are work intensive and require the additional use of antibodies, dyes, specific substrates, or enzymatic reactions. A simple, fast, and reliable method was developed to test for apoptosis or necrosis using mouse and human cell lines (e.g., Jurkat, A20.2J, and PB3c cells) stably transfected with a vector coding for green fluorescent protein (GFP) as indicator cells. METHODS: Apoptosis in GFP-transfected cell lines was induced either by soluble Fas-Ligand (sFasL), recombinant human TRAIL (rhTRAIL), or interleukin-3 (IL-3) deprivation. Necrosis was induced by polyclonal anti-A20 and complement treatment of GFP-transfected A20. Cells were analyzed by flow cytometry for GFP fluorescence. Propidium iodide and Annexin V staining were used to confirm the results obtained with the GFP-method. RESULTS: Live GFP-transfected cells show a strong fluorescence intensity, which is significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lose their GFP-associated fluorescence. Apoptosis but not necrosis of GFP-transfected cells was blocked by the use of a caspase inhibitor. The results are highly comparable to conventional apoptosis-detecting methods. CONCLUSIONS: The advantage of our GFP-based assay compared with other methods is the analysis of apoptosis or necrosis without the necessity for additional staining or washing steps, making it an ideal tool for screening apoptotic or necrotic stimuli.

Animals↗
PubMed1997

A fluorescence polarization based Src-SH2 binding assay.

The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.

Amino Acid Sequence↗
PubMed1994

Differentiation of periodic from nonperiodic low-frequency heart rate fluctuations.

Periodic low-frequency (LF) fluctuations of heart rate (HR) may be of diagnostic and prognostic value in diverse pathologic cardiopulmonary conditions. Two principal components of LF HR rate variations may be distinguished: periodic fluctuations and nonperiodic, nonstationary changes. The frequency content of these two components may overlap considerably. In order to avoid a tedious work-intensive visual analysis an efficient computer-based method for detection, differentiation, and quantitation of these signals is required. Two methods for separating periodic from nonperiodic HR changes are presented, namely, detrending and bandwidth (BW) calculation. A group of healthy individuals was evaluated in order to assess these methods in individuals with significant LF periodic episodes (15 patients) contrasted to those without LF periodic episodes (94 patients). The commonly used method of detrending consists of a fitted polynomial which by subtraction removes low frequencies originating from nonstationary changes without affecting periodic fluctuations. We found, however, that the frequencies involved in nonstationary and periodic fluctuations often overlap and thus the detrending method may not be highly efficient. In a second method we postulated different shapes for power spectrum curves of periodic and nonperiodic episodes. This latter method is based on BW calculation of the LF component of the R-R power spectrum and proved to be more efficient in detecting periodic episodes. It showed higher significance levels for the difference between the periodic and nonperiodic groups when the BW or the ratio between peak power and BW in the LF range was used. This new, alternative detection method may be employed in further studies which seek to elucidate the clinical relevance of the LF range and, in particular, the mechanisms for such long-wavelength periodic fluctuations.

Adolescent↗
PubMed1997

A tool for analyzing and annotating genomic sequences.

We describe a tool for analyzing and annotating large genomic sequences containing introns. The analysis and annotation tool (AAT) includes two sets of programs, one for comparing the query sequence with a protein database and the other for comparing the query with a cDNA database. Each set contains a fast database search program and a rigorous alignment program. The database search program quickly identifies regions of the query sequence that are similar to a database sequence. Then the alignment program constructs an optimal alignment for each region and the database sequence. The alignment program also reports the coordinates of exons in the query sequence. Pairwise alignments of the query sequence with protein and cDNA database sequences are combined into multiple sequence alignments, which provide a view of all protein and cDNA sequences matching a query region. On a data set of 570 DNA sequences, AAT identified 94% of coding nucleotides correctly and 74% of exons exactly. Results of analyzing a human BAC sequence with the AAT tool are also presented. The AAT tool reduces the labor-intensive work of locating the exons of the query sequence and improves the process of defining intron-exon boundaries by using the wealth of available protein and cDNA data.

Amino Acid Sequence↗
PubMed1998

Lymphoid-specific transcription mediated by the conserved octamer site: who is doing what?

The conserved octamer site found in the promoters of Immunoglobulin (Ig) genes has been identified early on as a key element for B cell-specific Ig promoter activity and has also been associated with expression of other B cell-specific genes. Several transcription factors--ubiquitous or cell-restricted--interact specifically with the octamer motif in B cells: Oct-1 and Oct-2 are POU domain transcription factors that bind directly to the octamer site and thereby can recruit the coactivator OBF-1 to the promoters of various genes. Yet, in spite of intense work, the precise role played by each of these factors for transcription activation through the octamer motif is unclear. In particular, genetic inactivation in mice of the lymphoid cell-specific transcription factor Oct-2 or of the B cell-specific coactivator OBF-1 led to surprising findings that are being discussed.

Animals↗
PubMed1998

Muscle deoxygenation in aerobic and anaerobic exercise.

It has been generally accepted that the use of oxygen is a major contributor of ATP synthesis in endurance exercise but not in short sprints. In anaerobic exercise, muscle energy is thought to be initially supported by the PCr-ATP system followed by glycolysis, not through mitochondrial oxidative phosphorylation. However, in real exercise practice, we do not know how much of this notion is true when an athlete approaches his/her maximal capacity of aerobic and anaerobic exercise, such as during a graded VO2max test. This study investigates the use of oxygen in aerobic and anaerobic exercise by monitoring oxygen concentration of the vastus lateralis muscle at maximum intensity using Near Infra-red Spectroscopy (NIRS). We tested 14 sprinters from the University of Penn track team, whose competitive events are high jump, pole vault, 100 m, 200 m, 400 m, and 800 m. The Wingate anaerobic power test was performed on a cycle ergometer with 10% body weight resistance for 30 seconds. To compare oxygenation during aerobic exercise, a steady-state VO2max test with a cycle ergometer was used with 25 watt increments every 2 min. until exhaustion. Results showed that in the Wingate test, total power reached 774 +/- 86 watt, about 3 times greater than that in the VO2max test (270 +/- 43 watt). In the Wingate test, the deoxygenation reached approximately 80% of the established maximum value, while in the VO2max test resulted in approximately 36% deoxygenation. There was no delay in onset of deoxygenation in the Wingate test, while in the VO2max test, deoxygenation did not occur under low intensity work. The results indicate that oxygen was used from the beginning of sprint test, suggesting that the mitochondrial ATP synthesis was triggered after a surprisingly brief exercise duration. One explanation is that prior warm-up (unloaded exercise) was enough to provide the mitochondrial substrates; ADP and Pi to activate oxidative phosphorylation by the type II a and type I myocytes. In addition, transmural pressure created by the muscle contraction reduces blood flow, causing relative hypoxia.

Adult↗
PubMed1982

Blood metabolites during prolonged exercise in swimming and leg cycling.

This study was designed to compare the influence of two modes of exercise (swimming and leg cycling) on the blood concentrations of metabolic substrates and metabolites during a 45-min exercise period. Eight college students (mean age = 21.6 +/- 1.2 year) exercised at 70% of VO2 max, in water using the front crawl on one occasion, and on a cycle ergometer using the legs on another. Blood samples were drawn at 0, 15, 30, and 45 min and analyzed for free fatty acids, glycerol, glucose, pyruvate, and lactate concentrations. Mean oxygen uptakes (2.23 vs 2.12 l . min-1) and heart rates (152 vs 150 b . min-1) for cycling and swimming respectively were not significantly different. Lactate and pyruvate were significantly (p less than 0.01) higher during swimming as compared to cycling. Free fatty acids, glycerol, and glucose were not significantly different between the two modes of exercise (p greater than 0.05). Assuming venous blood concentrations provide some indication of metabolic events, these data are compatible with a tendency to a higher relative carbohydrate oxidation rate during swimming as compared to cycling during prolonged exercise at the same relative work intensities.

Adult↗
PubMed1979

Time course of O2-pulse during various tests of aerobic power.

The purpose of this study was to test the hypothesis that oxygen pulse typically reaches a maximum before maximal oxygen consumption by observing the time course of oxygen pulse throughout exercise to maximal stress and to discern those physiologic variables which might predispose an individual to reach a peak in oxygen pulse before achieving maximal oxygen consumption. Thirty male volunteers ranging in age from 18-25 (X = 20.5) years were recruited for this study. Maximal oxygen uptake was assessed on both bicycle ergometer and treadmill. Based upon the results of the exercise tests, subjects were classified into subgroups as a consequence of whether or not a maximal oxygen pulse or a plateau in oxygen pulse was demonstrated during submaximal exercise. The results indicate that submaximal peaking or at least the achieving of plateau values of oxygen pulse does in fact occur in some but not all indivuals. It was observed that this phenomenon occurs at a relatively high percentage of maximal heart rate and maximal oxygen consumption. It appeared that individuals who demonstrate low heart rates at low-work intensities, high maximal heart rates, and a disproportionate increase in R for a given ventilation are most likely to reach a submaximal peak in oxygen pulse. Oxygen pulse during submaximal exercise appears to provide a good indication of cardiorespiratory fitness.

Adolescent↗
PubMed1995

Heart rate and oxygen uptake relationship: a comparison of loaded marching and running in women.

Heart rate (beats.min-1; fc) measured during marching with a load is often used to predict the oxygen cost (l.min-1; VO2) of the activity. The prediction comes from the fc/VO2 relationship determined from laboratory measures of fc and VO2 during treadmill running. Studies in men have suggested that this may not be appropriate although this has yet to be examined in women. This study, therefore, compared the fc/VO2 relationship between loaded marching and maximal running protocols in women. Sixteen female subjects [mean (SD), age 21.9 (2.3) years, height 6 (0.06) m, weight 62.6 (7.6) kg] had their fc (from three-lead chest electrodes) and VO2 measured first during standard treadmill run protocols, and again 1 week later during loaded marching protocols. The slopes and intercepts determined from linear regression of fc on VO2 for each individual for each protocol were compared as were the maximal fc(fcmax), VO2 and ratings of perceived exertion (RPE) from the last work period of each protocol in paired t-tests. The fc/VO2 slopes (P < 0.01) and intercepts (P < 0.05) differed significantly between loaded marching and running. fcmax for loaded marching were 90% of fcmax for running (P < 0.01) and VO2peak for loaded marching were 80% of those for running (P < 0.01). However, RPE at the final levels for the two protocols were not significantly different. The data suggest that in women the fc/VO2 relationships for loaded marching and for running are different. This difference is similar to that found in men when speed is held constant and the load and gradient are varied. The results suggest that it would be erroneous to use fc and VO2 measured during running protocols in the laboratory to estimate energy expenditure and work intensity during loaded marching in the field.

Adult↗
PubMed1988

Slow upward drift of VO2 during constant-load cycling in untrained subjects.

The oxygen uptake kinetics during constant-load exercise when sitting on a bicycle ergometer were determined in 7 untrained subjects by measuring breath-by-breath VO2 during continuous exercise to volitional exhaustion (mean endurance time = 1160 +/- 172 s) at a pedal frequency of 70 revolutions.min-1. The power output, averaging 189.5 W, was set at 82.5% of that eliciting the individual VO2max during a 5 min incremental exercise test. Throughout the exercise period, the VO2 kinetics could be appropriately described by a two-component exponential equation of the form: VO2(t) = Ya[1 - exp(-kat)] + Yb[1 - exp(-kbt)] where VO2 is net oxygen consumption and t the time from work onset. VO2 measured at the end of exercise was close to VO2max (98% VO2max) and the mean values of Ya, ka, Yb and kb amounted to 1195 ml O2.min-1, 0.034 s-1, 1562 ml O2.min-1, and 0.005 s-1 respectively. The initial rate of increase in VO2 predicted from the above equation is slower than that calculated, for the same work intensity, on the basis of the data obtained by Morton (1985) in trained subjects. For t greater than 480 s, however, the two models yield substantially equal results.

Bicycling↗
PubMed1994

Plasma catecholamines during endurance exercise of different intensities as related to the individual anaerobic threshold.

The study investigated the concentrations of free plasma catecholamines (CAT), adrenaline and noradrenaline, in comparison to heart rate and lactic acid concentrations during endurance exercises (EE) of different intensities related to the individual anaerobic threshold (IAT). A group of 14 endurance trained male athletes took part in the tests on a treadmill. After an exhausting incremental graded test (increasing 0.5 m.s-1 every 3 min) to determine the IAT, the subjects performed EE of 45 min in randomized order with intensities of 85%, 95%, 100% and 105% (E85-E105) of the IAT. The heart rate and CAT increased continuously during all EE. The CAT reacted sensitively to EE above IAT (E105) and showed an overproportional increase in comparison to EE performed with an intensity at or below IAT. At the same time, at exercise intensities up to IAT (E85-E100) a lactate steady state was observed whereas mean lactate concentrations increased during E105. The changes of lactate concentration allowed a better differentiation between E85-E100 as CAT measurements. In E95, E100 and E105 there was a partial overlap of heart rate, which in contrast to lactate concentration only differed by about 5%, so that small variations in heart rate could have coincided with considerable differences of exercise intensity when working at intensities near or above IAT. It was concluded that the range of IAT seemed to represent a real physiological breakpoint which corresponded to the aerobic-anaerobic transition.

Adult↗
PubMed1994

Information systems vendor support and CQI.

As we providers of health care use information technology more comprehensively in the information-intensive and labor-intensive work of health care, we are finding that the quality of daily, on-the-spot support for use of that technology has become a critical success factor. And, since we use a great range of vendor products, the quality of vendor support is especially critical. This article describes our practical application of CQI indicators of vendor responsiveness and quality, and the vendor responses to this new strategy.

Hospital Information Systems↗
PubMed1994

Bone density and lifestyle characteristics in premenopausal and postmenopausal Chinese women.

The relationships between bone health and various lifestyle factors were examined in a cross-sectional study in 775 Chinese women aged 35-75 years. Bone mass was significantly positively associated with body weight, height, body mass index (BMI) and duration of breastfeeding, but was not significantly associated with other lifestyle variables, including alcohol consumption, parity, age at menarche and age at menopause after adjusting for age and body weight. A positive association was observed in premenopausal women (but not in postmenopausal women) for bone density with current cigarette smoking and plasma and urinary cotinine (the major metabolite of smoking). However, the confounding effects of other unknown factors present in this cross-sectional study may not be excluded. Daily physical activity, as indicated by time spent working in the fields, was found consistently to be positively associated with bone mass (p < 0.0001). When these women were grouped into three physical activity levels on the basis of occupation and daily work intensity, those undertaking heavy labor also had significantly higher radial bone mass than women undertaking light or medium labor. These results suggest a protective effect of daily physical activity on bone health in both pre- and postmenopausal women.

Adult↗
PubMed1989

The pattern of breathing and the ventilatory response to breathing through a tube and to physical exercise in sport divers.

Well-trained divers can be expected to differ from healthy controls in their ventilatory response to breathing through a tube and to physical exercise. Therefore, we measured their minute ventilation (VE) at rest and during breathing through a tube combined with two levels of physical exercise (1 or 2 W.kg body weight-1). For breathing through a tube an additional dead space of 600 ml was used. All divers were trained in the breath-hold technique and in the use of the breathing apparatus. Their mean period of training as divers was 9 +/- 6 years. The approximate age of the subjects was 25 years. The pattern of breathing and the oxygen uptake were measured by spirometer, the end-tidal concentration of CO2 was measured and all experiments were carried out above sea level. The ventilation of the divers at rest was comparable to that of the controls. During physical exercise it was smaller whether during breathing through a tube or not. The inadequate increase of VE during exercise in divers was associated with hypercapnia only at a higher physical work intensity (of 2 W.kg-1). This finding is interpreted as a lower chemoregulatory response to the combined stimuli of hypercapnia, hypoxia and physical exercise. In some situations significant bradypnoea and higher tidal volumes were found in the divers.

Adolescent↗
PubMed2002

Myocardial perfusion imaging using contrast echocardiography.

BACKGROUND: Intense work during the last two decades has brought forth the use of myocardial contrast echocardiography to the clinical threshold for the diagnosis and evaluation of coronary artery disease. CLINICAL USE: A number of ultrasound contrast agents have been developed that act as red blood cell tracers and display myocardial perfusion when imaged by dedicated ultrasound imaging modalities. A considerable amount of experimental and clinical research has shown that myocardial contrast echocardiography can aid in the recognition of acute and chronic myocardial infarction, viable myocardium, and functionally significant coronary stenoses. Comparison of this technique to nuclear imaging and coronary arteriography has demonstrated excellent diagnostic accuracy in the evaluation of various coronary syndromes. Optimal practice of perfusion imaging requires a thorough knowledge of microbubble characteristics and imaging modalities, as well as good experience in the method. PERSPECTIVES: The technique continues to evolve from intermittent gated examination to real-time perfusion imaging that allows evaluation of both perfusion and functional parameters. The opportunity to target sites of pathology with specially engineered microbubbles could also aid in many therapeutic applications besides diagnostic imaging.

Animals↗
PubMed2000

[Hodgkin lymphoma. Classification and pathogenesis].

In the last few years our understanding of Hodgkin's lymphoma (HL) has enormously progressed. Molecular analysis has revealed that almost all cases of this disease are clonal B cell neoplasms, therefore the term Hodgkin's lymphoma instead of Hodgkin's disease is being proposed in the new WHO classification. Lymphocyte predominance HL (LPHL) differs in respect to morphology, immunophenotype and clinical features from the other forms of HL and represents its own distinct entity. In addition to morphologic features (nodularity, presence of L&H cells) the immuno-phenotype of tumor cells is most important in establishing a diagnosis of LPHL, and particularly in differentiating LPHL from the other forms of HL. The remaining forms of HL (nodular sclerosis, mixed cellularity, lymphocyte depletion) display a mostly identical antigen profile and similar clinical characteristics, they are therefore grouped together in the REAL classification under the heading of classical HL. Recent immuno-histological analysis have revealed that one third of HL cases, which formerly were classified as LPHL, display the immuno-phenotype of classical HL. These cases are now considered to represent examples of classical HL and termed nodular lymphocyte rich classical HL. According to retrospective clinico-pathological analysis, the biological behaviour of this newly identified form of classical HL also differs from LPHL. Differences between classical HL and LPHL also occur on the molecular level. Thus LPHL often displays ongoing mutations of the immunoglobulin genes, and the tumor cells express immunoglobulin protein and transcripts, while these characteristics are absent in classical HL. Since peripheral B cells that do not express immunoglobulins die from apoptosis, these findings imply that the regulation of apoptosis is defective in Hodgkin and Sternberg Reed cells. Several laboratories are currently working intensely to clarify the defective apoptosis pathway in HL.

B-Lymphocytes↗
PubMed2004

Moving on land: an explanation of pedometer counts in children.

The pedometer has become a popular instrument to measure physical activity in humans. The pedometer records physical activity as a simple, raw measure of ambulatory movement: the number of steps taken. Two fundamental questions of the use of the pedometer in pediatric research are: How many steps do children take in a day? How many steps should children accumulate on a daily basis? This raises the question: is 13,000 steps the same amount of physical activity in a 6-year-old and a 12-year-old, given the difference in body size and stride length? Although previous authors have concluded that the number of steps taken is an equivalent amount of physical activity in children regardless of body size, the conclusion has not been explained by a scientific rationale or on an empirical basis depending on the energetics and mechanics of terrestrial locomotion and body size. One approach to explain this question is allometric scaling, which has long been utilized by comparative physiologists to show the relationships between body size and locomotion in animals from shrews to elephants. The aim of this paper was to use allometric equations, examples from comparative mammalian physiology, and results from previous papers that have examined the cost of locomotion in children to provide a better understanding of the interpretation of the pedometer in pediatric research. We show that empirically derived results confirm to allometric equations that consider the relationship between body size and terrestrial locomotion to cover a given distance at a given speed. Thus, the number of steps taken and the energy expended for a particular task is greater in smaller children. However, if the cost of locomotion is related to one step, animals tend to be equally economical, whether large or small. Therefore, the number of steps taken as measured by a pedometer may be interpreted as an equivalent level of physical activity between a 6-year-old and a 12-year-old, if taken at the same speed. However, the locomotor activities in daily life consist of walking and running up- and downhill, carrying loads, and at varying speeds or work intensities, and this exemplifies the limitations of the pedometer. In conclusion, this paper provides a better understanding of the interpretation of pedometer counts and highlights that the findings depend on the expression of physical activity.

Anthropometry↗
PubMed2000

Relationships between physical activity, physical fitness, muscle strength and nutritional state in 5- to 11-year-old children.

The purpose of the present study was to assess different aspects of physical activity and fitness in order to develop a basis for sport programmes for overweight and obese children. Eighty-eight prepubertal children (49 boys, 39 girls, 4.8-11.4 years old, 61% obese, 14% overweight and 25% normal weight) were examined. Body composition was assessed by combined use of anthropometrics and bioelectrical impedance analysis. Resting energy expenditure (REE) and total energy expenditure (TEE) were measured by indirect calorimetry (IC) and individually calibrated 24-h heart rate (HR) monitoring, respectively. Activity-related energy expenditure (AEE) and physical activity level (PAL) were calculated from TEE and REE. Fitness [assessed by O2-pulse, respiratory exchange ratio (RER) at submaximal work intensities] was determined by ergometry. The maximal isometric muscle strength of the legs (m. quadriceps, Fa max, m. ischiocruralis, Fb max) was measured by computer tensiometry. Children were grouped according to their nutritional state, AEE, O2-pulse and muscle strength. When compared with normal weight children, obese and overweight children had increased fat mass (FM), fat-free mass (FFM), waist-to-hip ratio and REE, but no group differences were observed for TEE, AEE, and PAL. Obese and overweight children spent more hours per day watching TV. After correction for body weight and FFM, no group differences in REE were observed, but normal weight children had a higher O2-pulse than overweight and obese children. By contrast, RER was increased in the latter group. The fittest group had the lowest body weight, BMI, FM and FFM. Children with a low O2-pulse spent more hours per day watching TV. Grouping children according to their degree of muscle strength, younger children (4-7.5 years) did not show group differences in nutritional state, energy expenditure, physical activity and fitness. However, in the group of 7.6- to 11-year-old children, those with the greatest muscle strength and FFM had reduced BMI, skin folds, FM and FFM. FM correlated inversely with O2-pulse, but was not associated with TEE, AEE, PAL or muscle strength. By contrast TV consumption was positively associated with FM. To summarize, overweight and obese children were less fit and watched more TV than their normal weight counterparts. FM did not correspond to TEE, AEE or PAL. Muscle strength was not associated with FM in young children, but was inversely associated with FM in older children. Our cross-sectional data are consistent with the idea that increased fitness and reduced physical inactivity may prevent children from being overweight.

Body Composition↗