Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade.

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 µg/106 cells) or belatacept (40 µg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Vascular characteristics in young women-Effect of extensive endurance training or a sedentary lifestyle.

AIM: To explore whether high-level endurance training in early age has an influence on the arterial wall properties in young women. METHODS: Forty-seven athletes (ATH) and 52 controls (CTR), all 17-25 years of age, were further divided into runners (RUN), whole-body endurance athletes (WBA), sedentary controls (SC) and normally active controls (AC). Two-dimensional ultrasound scanning of the carotid arteries was conducted to determine local common carotid artery (CCA) geometry and wall distensibility. Pulse waves were recorded with a tonometer to determine regional pulse wave velocity (PWV) and pulse pressure waveform. RESULTS: Carotid-radial PWV was lower in WBA than in RUN (P < .05), indicating higher arterial distensibility along the arm. Mean arterial pressure was lower in ATH than in CTR and in RUN than in WBA (P < .05). Synthesized aortic augmentation index (AI@75) was lower among ATH than among CTR (-12.8 ± 1.6 vs -2.6 ± 1.2%, P < .001) and in WBA than in RUN (-16.4 ± 2.5 vs -10.7 ± 2.0%, P < .05), suggesting a diminished return of reflection waves to the aorta during systole. Carotid-femoral PWV and intima-media thickness (IMT), lumen diameter and radial distensibility of the CCA were similar in ATH and CTR. CONCLUSION: Elastic artery distensibility and carotid artery IMT are not different in young women with extensive endurance training over several years and in those with sedentary lifestyle. On the other hand, our data suggest that long-term endurance training is associated with potentially favourable peripheral artery adaptation, especially in sports where upper body work is added. This adaptation, if persisting later in life, could contribute to lower cardiovascular risk.

The fat cell epigenetic signature in post-obese women is characterized by global hypomethylation and differential DNA methylation of adipogenesis genes.

BACKGROUND/OBJECTIVES: Obese subjects have increased number of enlarged fat cells that are reduced in size but not in number in post-obesity. We performed DNA methylation profiling in fat cells with the aim of identifying differentially methylated DNA sites (DMS) linked to adipose hyperplasia (many small fat cells) in post-obesity. SUBJECTS/METHODS: Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells from 16 women examined 2 years after gastric bypass surgery at a post-obese state (body mass index (BMI) 26±2 kg m(-2), mean±s.d.) and from 14 never-obese women (BMI 25±2 kg m(-2)). Gene expression was analyzed in subcutaneous adipose tissue from nine women in each group. In a secondary analysis, we examined DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. RESULTS: The average degree of DNA methylation of all analyzed CpG sites was lower in fat cells from post-obese as compared with never-obese women (P=0.014). A total of 8504 CpG sites were differentially methylated in fat cells from post-obese versus never-obese women (false discovery rate 1%). DMS were under-represented in CpG islands and surrounding shores. The 8504 DMS mapped to 3717 unique genes; these genes were over-represented in cell differentiation pathways. Notably, 27% of the genes linked to adipogenesis (that is, 35 of 130) displayed DMS (adjusted P=10(-8)) in post-obese versus never-obese women. Next, we explored DNA methylation and expression of genes linked to adipogenesis in more detail in adipose tissue samples. DMS annotated to adipogenesis genes were not accompanied by differential gene expression in post-obese compared with never-obese women. In contrast, adipogenesis genes displayed differential DNA methylation accompanied by altered expression in obese women. CONCLUSIONS: Global CpG hypomethylation and over-representation of DMS in adipogenesis genes in fat cells may contribute to adipose hyperplasia in post-obese women.

Female athlete's heart: Systolic and diastolic function related to circulatory dimensions.

There are relatively few studies on female athletes examining cardiac size and function and how these measures relate to maximal oxygen uptake (VO2max). When determining sports eligibility, it is important to know what physiological adaptations and characteristics may be expected in female athletes, taking body and cardiac size into account. The purposes of this study were (a) to compare right and left heart dimensions and function in female endurance athletes (ATH) and in non-athletic female controls of similar age (CON); and (b) to explore how these measures related to VO2max. Forty-six ATH and 48 CON underwent a maximal bicycle exercise test and an echocardiographic examination at rest, including standard and color tissue Doppler investigation. All heart dimensions indexed for body size were larger in ATH (all P < 0.01). The diastolic mitral E/A ratio was 27% higher in ATH (P < 0.001) while systolic left and right atrio-ventricular longitudinal displacement was 7% (P = 0.002) and 15% (P < 0.001) larger in ATH, respectively. Half (50.3%) of the variability in VO2max could be explained by left ventricular end-diastolic volume. Our results could be useful in evaluating female endurance athletes with suspected cardiac disease and contribute to understanding differences between female athletes and non-athletes.

The expression of heat shock protein in human skeletal muscle: effects of muscle fibre phenotype and training background.

AIM: Exercise-induced adaptations of skeletal muscle are related to training mode and can be muscle fibre type specific. This study aimed to investigate heat shock protein expression in type I and type II muscle fibres in resting skeletal muscle of subjects with different training backgrounds. METHODS: Three groups of subjects were included: healthy active not engaged in any training programme (ACT, n = 12), resistance trained (RES, n = 6) and endurance trained (END, n = 8). Biopsies were obtained from vastus lateralis, and immunohistochemistry was performed using monoclonal antibodies against myosin heavy chain I and IIA, αB-crystallin, HSP27, HSP60 and HSP70. RESULTS: In ACT and RES, but not in END, a fibre type-specific expression with higher staining intensity in type I than type II fibres was seen for αB-crystallin. The opposite (II > I) was found for HSP27 in subjects from ACT (6 of 12 subjects) and RES (3 of 6), whereas all subjects from END displayed uniform staining. HSP60 showed no fibre-specific expression. HSP70 displayed a fibre-specific expression pattern (I > II) in ACT (4 of 12), but not in END or RES. CONCLUSION: This study shows that the level of expression of the different HSPs in human skeletal muscle is influenced by muscle fibre phenotype. The fibre type-specific expression of HSP70 is influenced by resistance and endurance training, whereas those of αB-crystallin and HSP27 is influenced only by endurance training, suggesting the existence of a training-modality-specific action on the adaptive processes including heat shock proteins in human skeletal muscle.

Population pharmacokinetic analysis of ropivacaine and its metabolite 2',6'-pipecoloxylidide from pooled data in neonates, infants, and children.

BACKGROUND: The aim was to characterize ropivacaine and 2',6'-pipecoloxylidide (PPX) pharmacokinetics and factors affecting them in paediatric anaesthesia. METHODS: Population pharmacokinetics of ropivacaine and its active metabolite PPX were estimated after single and continuous ropivacaine blocks in 192 patients aged 0-12 yr from six pooled published studies. Unbound and total ropivacaine and PPX plasma concentration and PPX urinary excretion data were used for non-linear mixed-effects modelling by NONMEM. Covariates included age, body weight, gender, ethnic origin, ASA, site and method of administration, and total dose. RESULTS: One-compartment first-order pharmacokinetic models incorporating linear binding of ropivacaine and PPX to α(1)-acid glycoprotein were used. After accounting for the effect of body weight, clearance of unbound ropivacaine and PPX reached 41% and 89% of their mature values, respectively, at the age of 6 months. Ropivacaine half-life decreased with age from 13 h in the newborn to 3 h beyond 1 yr. PPX half-life differed from 19 h in the newborn to 8-11 h between 1 and 12 months to 17 h after 1 yr. Simulations indicate that for a single caudal block, the recommended dose could be increased by a factor of 2.9 (0-1 month group) and 6.3 (1-12 yr group) before the unbound plasma concentrations would cross the threshold for systemic toxicity. Corresponding factors for continuous epidural infusion are 1.8 and 4.9. CONCLUSIONS: Ropivacaine and PPX unbound clearance depends on body weight and age. The results support approved dose recommendations of ropivacaine for the paediatric population.

Pharmacokinetics of ropivacaine in patients with chronic renal failure.

BACKGROUND: As ropivacaine and its metabolites are excreted by the kidneys, we studied their disposition in subjects with renal dysfunction. METHODS: Twenty patients with moderate or severe renal insufficiency and 10 healthy volunteers received ropivacaine 1 mg kg(-1) i.v. over 30 min. The concentrations of ropivacaine and its main metabolites, pipecoloxylidide (PPX) and 3-hydroxy-ropivacaine, were measured in plasma and urine for 16-48 h. The relationship between pharmacokinetic parameters and creatinine clearance (CL(CR)) was assessed. A model for estimating non-renal clearance of a metabolite of ropivacaine is described. RESULTS: Renal dysfunction had little or no influence on the pharmacokinetics of ropivacaine. The median plasma concentrations of unbound ropivacaine were similar in uraemic and non-uraemic subjects. Renal clearance of PPX correlated significantly with CL(CR) (R(2)=0.81). Lack of correlation between total PPX exposure, expressed as area under the total plasma concentration-time curve from zero to infinity, and CL(CR) suggests that the clearance of PPX also includes non-renal elimination. However, in two uraemic patients, there was increased exposure to PPX resulting from low non-renal elimination. CONCLUSIONS: The pharmacokinetics of ropivacaine is not affected by renal failure. Although the renal clearance of PPX correlates with CL(CR), non-renal elimination seems to compensate for reduced renal clearance in most patients. PPX may accumulate in plasma during long-term postoperative infusions, in particular in patients with co-existing low non-renal elimination. Systemic toxicity is still unlikely because PPX is markedly less toxic than ropivacaine.

Prolonged exercise-induced stimulation of skeletal muscle glucose uptake is due to sustained increases in tissue perfusion and fractional glucose extraction.

CONTEXT: The mechanisms behind the positive effects of physical activity on glucose metabolism in skeletal muscle and the time course of the effects need to be more elucidated. OBJECTIVE: The aim was to examine the prolonged effects of an acute bout of one-legged exercise on local skeletal muscle glucose utilization and tissue perfusion. DESIGN AND SETTING: Interstitial glucose concentration, local tissue perfusion, glucose uptake, and effects of insulin infusion were studied 12 h after an acute bout of exercise and without prior exercise. PARTICIPANTS: Ten healthy subjects, five women and five men, participated in the study. INTERVENTION: Microdialysis measurements, (133)Xe clearance, and a 2-h hyperinsulinemic euglycemic clamp were performed on two occasions. MAIN OUTCOME MEASURES: We measured interstitial glucose concentration and tissue perfusion in the quadriceps femoris muscle of both legs. RESULTS: Tissue perfusion (3.3 ± 0.6 ml × 100 g(-1) × min(-1) vs. 1.4 ± 0.2 ml × 100 g(-1) × min(-1); P = 0.007) and basal glucose uptake (2.3 ± 0.5 µmol × 100 g(-1) × min(-1) vs. 0.9 ± 0.2 µmol × 100 g(-1) × min(-1); P = 0.006) were increased in the leg that had exercised compared to the resting leg; the findings in the resting leg were comparable to those in the control experiment without prior exercise. The relative effect of insulin on fractional skeletal muscle glucose uptake was the same in all experimental settings, and insulin did not affect tissue perfusion. CONCLUSIONS: The prolonged stimulatory effect of physical exercise on skeletal muscle glucose uptake was mediated via vascular effects combined with an increase in basal glucose transport independent of enhancement of insulin responses.

Muscle blood flow response to mental stress and adrenaline infusion in man: microdialysis ethanol technique compared to (133)Xe clearance and venous occlusion plethysmography.

BACKGROUND AND AIM: Adrenaline, administered locally by microdialysis in skeletal muscle, causes vasoconstriction around the microdialysis catheter. This is contrary to the vasodilation that normally occurs when adrenaline is infused intravenously or intra-arterially. The hypothesis was tested that vasoconstriction, measured by microdialysis, would not occur with two interventions causing increased plasma levels of adrenaline, mental stress and intravenous adrenaline infusion (0.1 nmol kg(-1) min(-1)). METHODS: Twenty-four men (27 +/- 1.6 years) underwent these interventions. Blood flow was determined by the microdialysis ethanol technique and (133)Xe clearance (gastrocnemius muscle, medial head) and by venous occlusion plethysmography (calf). RESULTS: The ethanol outflow/inflow ratio, which is inversely related to blood flow, decreased to 92.0 +/- 3.4% of basal, P = 0.014 (mean +/- SEM, n = 16) during the mental stress test, but increased to 108.3 +/- 2.2% of basal, P = 0.001 (n = 16) during the adrenaline infusion. The latter increase was abolished when adrenaline was infused during alpha-receptor blockade by phentolamine. On the contrary, by (133)Xe clearance and venous occlusion plethysmography, blood flow increased during both interventions; 2.0-1.7-fold (mental stress) and 1.3-1.4-fold (adrenaline infusion), respectively, P<0.05. CONCLUSION: Adrenaline causes vasoconstriction in skeletal muscle when blood flow is measured with the microdialysis ethanol technique, irrespective of the mode of administration. The discrepant blood flow result obtained with the microdialysis ethanol technique might, at least partly, be explained by differential diffusion properties of ethanol and (133)Xe. An additional or alternative explanation might be that an inserted microdialysis catheter shifts the balance of vasoconstrictor and vasodilator effects of adrenaline in skeletal muscle.

Urea clearance: a new method to register local changes in blood flow in rat skeletal muscle based on microdialysis.

SUMMARY: Increasing evidence suggests that local blood flow should be monitored during microdialysis (MD) as the recovery of analytes is affected by local blood flow. At present ethanol clearance is the standard technique for this purpose, but it is not functional at very low perfusion velocities. Here, we introduce a technique for MD whereby local tissue blood flow is recorded by the use of urea clearance (changes inflow/outflow concentration), in conjunction with measurements of tissue metabolism (glucose, lactate and puruvate). MD probes were inserted into the gracilis muscle of 15 rats and perfused with a medium containing urea (20 mmol l(-1)). Changes in muscle blood flow were made by addition of noradrenaline (5 microg ml(-1)) to the perfusion medium at two perfusion velocities (0.6 and 0.4 microl min(-1)). The clearance of urea from the perfusion medium was then calculated and examined in relation to the dose of noradrenaline and to the coexisting changes in extracellular metabolites. The results showed reproducible and dose-dependent changes in blood flow that were induced by noradrenaline. These were characterized by dose-dependent changes in the urea clearance as well as blood-flow-specific changes in the MD metabolic markers (reduction in glucose and increase in lactate). The sensitivity for blood flow changes as assessed by urea clearance (MD) was increased at 0.4 compared with the 0.6 microl min(-1) perfusion speed. The results indicate that inclusion of urea to the perfusion medium may be used to monitor changes in skeletal muscle blood flow at low perfusion velocities and in parallel assess metabolic variables with a high recovery (>90%).

Immunohistochemical changes in the expression of HSP27 in exercised human vastus lateralis muscle.

AIM: The role of HSP27 in the adaptive process of skeletal muscle to exercise, especially in humans, is not well understood. The objective of this study was to investigate immunohistochemical changes in HSP27 expression in human vastus lateralis muscle following resistance and endurance exercises. METHODS: Two different exercise protocols were used: (1) one-leg ergometer cycling (EC, n = 6) consisting of two 30-min bouts at 40% and 75% of peak oxygen uptake, respectively, and (2) leg extension resistance exercise (RE, n = 9) including 10 sets of eight repetitions at a load corresponding to 70% of one maximal repetition (1RM). Immunohistochemistry using specific monoclonal antibodies was used to determine the location of HSP27 protein in muscle biopsies from human vastus lateralis. RESULTS: Our results show that RE, but not EC, induced a significant appearance of scattered accumulations of HSP27 protein in muscle fibres from five of nine subjects. The number of fibres with accumulation of HSP27 in RE ranged from 0% to 32% with a mean of 6.3% of the total number of fibres. CONCLUSION: We conclude that this rapid HSP27 protein relocation after RE is an important player in the cellular remodelling of human muscle fibres in response to exercise involving high-force contractions, but not in response to endurance exercises.

Interstitial glucose concentration in insulin-resistant human skeletal muscle: influence of one bout of exercise and of local perfusion with insulin or vanadate.

The present study investigated the changes occurring in interstitial metabolite concentrations and blood flow in insulin-resistant human skeletal muscle during the post-exercise recovery period following a single 2-h bout of one-legged exercise. In addition, the effect of microdialysis perfusion with insulin or the insulin-mimetic trace element vanadate was explored. Eight microdialysis catheters, four in each leg, were inserted in the quadriceps femoris muscle of nine insulin-resistant obese male subjects 2 h following exercise. Two catheters in each leg were perfused at 0.2 microl/min for metabolite determinations and two at 1.33 microl/min for the determination of blood flow. Samples were collected until 9 h after the end of exercise had passed. The interstitial glucose concentration (mean +/- SD) was significantly lower in the exercised (2.8 +/- 1.3 mM) than in the rested leg (3.7 +/- 0.9 mM), P = 0.001, a difference that lasted at least 8 h after the exercise bout. On the other hand, blood flow was not different in the two legs. Microdialysis perfusion with insulin (14 mU/ml) or sodium metavanadate (100 mM) decreased the interstitial glucose concentration (P = 0.001) in both the exercised and rested leg. With vanadate, this decrease was similar in the exercised (-69%) and the rested leg (-71%), whereas insulin had a larger effect in the exercised leg (-29 vs. -6.9%), P = 0.05. This study shows that the interstitial glucose concentration in insulin-resistant skeletal muscle is markedly decreased for several hours following a single exercise session. This is in accordance with recent findings in healthy subjects. This change is accompanied by an increased insulin effect on the interstitial glucose concentration. The effect of vanadate was not decreased in insulin-resistant human skeletal muscle and was not augmented by exercise.

A single bout of exercise is followed by a prolonged decrease in the interstitial glucose concentration in skeletal muscle.

AIM: The present study was performed to test the hypothesis that the interstitial glucose concentration in human skeletal muscle is decreased for a prolonged period following a single bout of exercise, while blood flow has returned to resting levels. METHODS: Muscle interstitial concentrations of glucose, lactate, pyruvate and urea were monitored in six healthy individuals during 8 h following a 2-h one-leg exercise session by microdialysis at low perfusion flow rate. Simultaneously the blood flow was measured by the microdialysis ethanol technique. RESULTS: The blood glucose and the control leg interstitial glucose concentrations were stable during the experiment averaging 5.7 +/- 0.1 and 4.1 +/- 0.3 mm, respectively. In contrast, the interstitial glucose concentration in the exercise leg was markedly decreased, averaging 1.9 +/- 0.5 mm, during the first 5.5 h following exercise (P < 0.01), after which it returned towards normal values. Muscle blood flow at the site of the microdialysis catheter, measured as the ethanol outflow-to-inflow ratio, did not change significantly over time in the control or exercise leg and did not differ significantly between the two legs. Interstitial concentrations of lactate, pyruvate and urea were not significantly different between the control and exercise leg. CONCLUSION: The study shows that the interstitial glucose concentration in skeletal muscle is markedly decreased for several hours following a single exercise session. The decreased interstitial glucose concentration may serve to limit the rate of post-exercise muscle glucose uptake to a rate compatible with normal blood glucose levels and may also be speculated to have a positive long-term health implication by augmenting muscle insulin sensitivity.

Acidosis in muscle tissue distal to vascular contusion despite unchanged global blood flow in rats: an uncoupling of microvascular blood flow and metabolism?

Studies using a contusion trauma model have shown that the femoral artery of the rat remains patent in 85% despite a severe vessel injury. A significant increase in tissue oxygenation (PtO2) has been found despite only a minor effect on blood flow (<20% decrease) on the muscle surface distal to the injury indicating a disturbed relationship between microvascular blood flow and metabolism. The aim of the present study was to further study the interplay between microvascular blood flow and metabolism within the distal muscle using an ethanol clearance technique (blood flow) in conjunction to the determination of an ischemia marker (lactate) by use of microdialysis. Although skeletal muscle blood flow remained unaltered as assessed by ethanol clearance, skeletal muscle lactate levels increased significantly (P<0.001) post-trauma in both legs. The increase was initially higher, faster and the increase over time larger in the trauma leg as compared to the control leg (P<0.001). These findings indicate a systemic effect of the trauma. Further, it suggests a functional impairment of the relationship between microvascular blood flow and/or muscle metabolic processes when the trauma is directed towards the supplying blood vessel. The reason for this anaerobic insult as found in this study compared to the presence of a local increase in PtO2 in the trauma leg as shown in our previous study is suggestive of an microvascular blood flow and tissue metabolism uncoupling.

Local effect of vanadate on interstitial glucose and lactate concentrations in human skeletal muscle.

The aim of this study was to investigate the local effect of the insulin-mimetic agent vanadate on glucose metabolism in human skeletal muscle in vivo. Interstitial concentrations of glucose and lactate were determined by microdialysis at a low flow rate in the quadriceps femoris muscle of 18 men. In the same leg two microdialysis catheters were inserted. In one catheter, the perfusion medium was supplemented with sodium metavanadate (10-100 mM) after a basal period, the other catheter served as control. In the catheter perfused with metavanadate, the interstitial glucose concentration was decreased by 13-50% compared to the control catheter (p<0.05). The lactate concentration was higher in the 50 mM and 100 mM metavanadate catheters compared to control (39-89%, p<0.05). There was no difference between control and metavanadate catheters in urea concentrations. Five of the subjects were insulin-resistant and for them the results were similar, although the effect was somewhat smaller. The decreased interstitial glucose concentration, and the increased lactate concentration, in the vicinity of the microdialysis catheter most likely reflects an increased cellular glucose uptake. The present study thus indicates that vanadate mimics the effect of insulin in human skeletal muscle in vivo.

Chromosomal size variation in Trypanosoma cruzi is mainly progressive and is evolutionarily informative.

The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruz II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P < 0.001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.

Microdialysis in human skeletal muscle: effects of adding a colloid to the perfusate.

Microdialysis catheters (CMA-60 with a polyamide dialysis membrane; 20,000-molecular wt cutoff) were either immersed in an external medium or were inserted in the quadriceps femoris muscle of healthy subjects, using perfusate with or without dextran 70. Varying the position of the outflow tubing induced changes in hydrostatic pressure. The sample volumes were significantly smaller in catheters perfused without a colloid compared with those perfused with a colloid [11-50% (in vitro) and 8-59% (in vivo) lower than in colloid-perfused catheters with the same position of the outflow tubing]. The sample volumes were also significantly smaller when the dialysis membrane was influenced by maximal hydrostatic pressure (above position) compared with minimal hydrostatic pressure (below position) [7-38% (in vitro) and 3-46% (in vivo) lower than in catheters in the below position with the same perfusion fluid]. In vivo, glucose concentration at a perfusion flow rate of 0.33 microl/min was higher when the catheters were perfused without a colloid [18-28% higher than in colloid-perfused catheters with the same position of the outflow tubing (P < 0.001)] than with a colloid. A corresponding difference also tended to occur with lactate, glycerol, and urea. At 0.16 microl/min, the glucose concentration was the same irrespective of whether fluid loss had been counteracted by colloid inclusion or by lowering of outlet tubing. The mechanism behind the observed concentration difference is thought to be a higher effective perfusion flow rate when fluid loss is prevented at low-perfusion flows. This study shows that fluid imbalances can have important implications for microdialysis results at low-perfusion flow rates.

Bacterial senescence: protein oxidation in non-proliferating cells is dictated by the accuracy of the ribosomes.

We have investigated the causal factors behind the age-related oxidation of proteins during arrest of cell proliferation. A proteomic approach demonstrated that protein oxidation in non-proliferating cells is observed primarily for proteins being produced in a number of aberrant isoforms. Also, these cells exhibited a reduced translational fidelity as demonstrated by both proteomic analysis and genetic measurements of nonsense suppression. Mutants harboring hyperaccurate ribosomes exhibited a drastically attenuated protein oxidation during growth arrest. In contrast, oxidation was augmented in mutants with error-prone ribosomes. Oxidation increased concomitantly with a reduced rate of translation, indicating that the production of aberrant, and oxidized proteins, is not the result of titration of the co-translational folding machinery. The age-related accumulation of the chaperones, DnaK and GroEL, was drastically attenuated in the hyperaccurate rpsL mutant, demonstrating that the reduced translational fidelity in growth-arrested cells may also be a primary cause for the induction of the heat shock regulon. The data point to an alternative way of approaching the causal factors involved in protein oxidation in eukaryotic G(0) cells.

Effects of concentric and eccentric contractions on phosphorylation of MAPK(erk1/2) and MAPK(p38) in isolated rat skeletal muscle.

1. Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen-activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise-related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. 2. Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal-regulated kinase 1 and 2 (MAPK(erk1/2)) and stress-activated protein kinase p38 (MAPK(p38)). 3. Concentric contractions induced a 5-fold increase in MAPK(erk1/2) phosphorylation. Application of the antioxidants N-acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction-induced increase in MAPK(erk1/2) phosphorylation. Mild passive stretches of the muscle increased MAPK(erk1/2) phosphorylation by 1.8-fold, whereas the combination of acidosis and passive stretches resulted in a 2.8-fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPK(p38). 4. High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7- and 9.5-fold increases of phosphorylated MAPK(erk1/2), respectively, whereas phosphorylation of MAPK(p38) increased by 7.6- and 1.9-fold (not significant), respectively. 5. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPK(erk1/2) and MAPK(p38) is induced by mechanical alterations, whereas contraction-related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPK(erk1/2) only. Thus, contraction-induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPK(erk1/2) and by high mechanical stress for MAPK(p38).