Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

Exploring Parallels Between Molecular Changes Induced in PNS by Aging and Demyelinating Neuropathies

The peripheral nervous system (PNS) is involved in many age-dependent neurological deficits, including numbness, pain, restless legs, trouble with walking and balance that are commonly found in the elderly. These symptoms generally result from demyelination and/or loss of axonal integrity. However, the precise identity of age-regulated molecular changes in either neuronal or glial compartments of the nerve is unclear. Interestingly, these deficiencies are also present in inherited neuropathies, where the expressivity of the rapid and early onset phenotypes is undeniably more severe than in normal aging. Nevertheless, especially the molecular changes underlying loss of axonal integrity in neuropathy condition are also poorly understood. To unravel molecular mechanisms affected by PNS aging, we used wildtype mice at 17 time-points from day of birth until senescence (28 months-old). For the neuropathy study, we focused on 56 day-old Schwann cell-specific neuropathy-inducing mutants, MPZCre/1/ LpinfE2-3/fE2-3 and MPZCre/1/ScapfE1/fE1 mice, that have, at this age, already developed neuropathic symptoms. Transcriptomes of dissected Schwann cell-containing endoneurium or sensory neuron-containing dorsal root ganglia have been analyzed throughout time or genotypes, using Illumina Bead Chips. Following data validation, we identified groups of differentially expressed genes in the development, aging and in the neuropathic mutants, in both glial and neuronal compartments. We detected substantial differences in the dynamics of changes in gene expression during development and aging between these two compartments. Furthermore, considering the above-mentioned phenotypic similarities, we integrated aging and mutant data. Interestingly, we observed that there are some parallels at the molecular level between processes involved in aging, which leads to less severe and more progressive PNS alterations, and in the rapid onset peripheral neuropathies. Apart from helping the understanding of molecular alterations underlying age-related PNS phenotypes, this data should also contribute to the identification of pathways that could be used as targets for therapeutical approaches to prevent complications associated with both aging and inherited forms of neuropathies.

Central and peripheral metabolic changes induced by gamma-hydroxybutyrate.

STUDY OBJECTIVES: Gamma-hydroxybutyrate (GHB) was originally introduced as an anesthetic but was first abused by bodybuilders and then became a recreational or club drug.1 Sodium salt of GHB is currently used for the treatment of cataplexy in patients with narcolepsy. The mode of action and metabolism of GHB is not well understood. GHB stimulates growth hormone release in humans and induces weight loss in treated patients, suggesting an unexplored metabolic effect. In different experiments the effect of GHB administration on central (cerebral cortex) and peripheral (liver) biochemical processes involved in the metabolism of the drug, as well as the effects of the drug on metabolism, were evaluated in mice. DESIGN: C57BL/6J, gamma-aminobutyric acid B (GABAB) knockout and obese (ob/ob) mice were acutely or chronically treated with GHB at 300 mg/kg. MEASUREMENTS AND RESULTS: Respiratory ratio decreased under GHB treatment, independent of food intake, suggesting a shift in energy substrate from carbohydrates to lipids. GHB-treated C57BL/6J and GABAB null mice but not ob/ob mice gained less weight than matched controls. GHB dramatically increased the corticosterone level but did not affect growth hormone or prolactin. Metabolome profiling showed that an acute high dose of GHB did not increase the brain GABA level. In the brain and the liver, GHB was metabolized into succinic semialdehyde by hydroxyacid-oxoacid transhydrogenase. Chronic administration decreased glutamate, s-adenosylhomocysteine, and oxidized gluthathione, and increased omega-3 fatty acids. CONCLUSIONS: Our findings indicate large central and peripheral metabolic changes induced by GHB with important relevance to its therapeutic use.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Changes in running mechanics and spring-mass behaviour induced by a 5-hour hilly running bout.

Abstract The aim of this study was to investigate changes in running mechanics and spring-mass behaviour with fatigue induced by 5-hour hilly running (5HHR). Running mechanics were measured pre- and post-5HHR at 10, 12 and 14 km · h(-1) on an instrumented treadmill in eight ultramarathon runners, and sampled at 1000 Hz for 10 consecutive steps. Contact (t(c) ) and aerial (t(a) ) times were determined from ground reaction force (GRF) signals and used to compute step frequency (f). Maximal GRF, loading rate, downward displacement of the centre of mass (Δz), and leg length change (ΔL) during the support phase were determined and used to compute both vertical (K(vert) ) and leg (K(leg) ) stiffness. A significant decrease in t(c) was observed at 12 and 14 km · h(-1) resulting in an increase of f at all speeds. Duty factor and F(max) significantly decreased at 10 km · h(-1). A significant increase in K(vert) and K(leg) was observed at all running speeds with significant decreases in Δz and ΔL. Despite the shorter duration, the changes in running mechanics appeared to be in the same direction (increased f and K(vert) , decrease in Δz and F(max) ) but of lower amplitude compared with those obtained after an ultra-trail or an ultramarathon.

The alarmin HMGB1 enhances CXCR4-induced activities by binding CXCL12

A variety of chemokines and inflammatory molecules are concomitantly produced at target sites of leukocyte trafficking and homing, accounting for the complex cellular responses occurring in homeostasis and inflammation. The chemokine CXCL12 plays an essential and unique role in homeostatic regulation of leukocyte traffic and tissue regeneration. The chromatin protein HMGB1 is released by dying and distressed cells, and acts as a Damage Associated Molecular Pattern or alarmin, promoting cell migration towards the site of tissue damage. We show here that HMGB1 synergises with CXCL12 by forming a heterocomplex that we characterized by NMR chemical shift mapping. The heterocomplex enhances CXCR4-induced responses on cells of the immune system, acting exclusively through the CXCL12 receptor CXCR4, and not through the HMGB1 receptors RAGE, TLR2 and TLR4. FRET analysis show that CXCL12 and CXCL12+HMGB1 promote a different conformational change in the homodimer CXCR4. The enhancement induced by HMGB1 on CXCL12-induced migration is selective, since little changes in migration of neutrophils and PreB 300.19-CCR2+ or -CCR7+ are observed towards CXCL8 and CCR2 or CCR7 agonists. HMGB1 also promotes CXCL 12 release, which is ultimately responsible for the chemoattractant activities of HMGB1. This study highlights the role of HMGB1 in promoting CXCL12-dependent cell migration, and suggests a cooperative role of these two molecules in tissue repair as well as in pathological conditions, such as rheumatoid arthritis.

Changes in leg spring behaviour, plantar loading and foot mobility magnitude induced by an exhaustive treadmill run in adolescent middle-distance runners.

OBJECTIVES: This study aimed to determine adjustments in spring-mass model characteristics, plantar loading and foot mobility induced by an exhaustive run. DESIGN: Within-participants repeated measures. METHODS: Eleven highly-trained adolescent middle-distance runners ran to exhaustion on a treadmill at a constant velocity corresponding to 95% of velocity associated with VO₂max (17.8 ± 1.4 kmh(-1), time to exhaustion=8.8 ± 3.4 min). Contact time obtained from plantar pressure sensors was used to estimate spring-mass model characteristics, which were recorded (during 30 s) 1 min after the start and prior to exhaustion using pressure insoles. Foot mobility magnitude (a composite measure of vertical and medial-lateral mobility of the midfoot) was measured before and after the run. RESULTS: Mean contact area (foot to ground), contact time, peak vertical ground reaction force, centre of mass vertical displacement and leg compression increased significantly with fatigue, while flight time, leg stiffness and mean pressure decreased. Leg stiffness decreased because leg compression increased to a larger extent than peak vertical ground reaction forces. Step length, step frequency and foot mobility magnitude did not change at exhaustion. CONCLUSIONS: The stride pattern of adolescents when running on a treadmill at high constant velocity deteriorates near exhaustion, as evidenced by impaired leg-spring behaviour (leg stiffness) and altered plantar loading.

Changes in sleeping and basal energy expenditure and substrate oxidation induced by short term thyroxin administration in man.

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.

Changes in insulin secretion and glucose metabolism induced by dexamethasone in lean and obese females

Résumé But: Chez les individus sveltes et en bonne santé, les modifications de la sensibilité à l'insuline secondaires à l'administration de dexaméthasone pendant deux jours sont compensées par une modification de la sécrétion d'insuline, permettant le maintien de l'homéostasie glucidique. Cette étude évalue les modifications du métabolisme glucidique et de la sécrétion d'insuline induites par une administration limitée de dexaméthasone chez les femmes obèses. Méthode de recherche: Onze femmes obèses ayant une tolérance au glucose normale ont été étudiées à deux reprises, 1° sans dexaméthasone et 2° après deux jours d'administration de dexaméthasone à faible dose. Un clamp hyperglycémique comportant deux plateaux (taux plasmatique de glucose à 7.5, respectivement 10 mM) avec du glucose marqué (6.6 ²H2 glc) a été utilisé pour déterminer la sécrétion d'insuline et le métabolisme du glucose du corps entier. Les résultats ont été comparés à ceux d'un groupe de huit femmes sveltes. Résultats : Sans dexaméthasone, les femmes obèses avaient un taux d'insuline plasmatique supérieur à jeun, durant le premier pic de sécrétion d'insuline, et aux deux plateaux hyperglycémiques. Elles avaient toutefois un métabolisme glucidique normal comparé à celui des femmes sveltes, ce qui indique une compensation adéquate. Après administration de la dexaméthasone, les femmes obèses avaient une augmentation du taux d'insuline plasmatique de 66 à 92%, mais une baisse de stockage du glucose de 15.4%. Ceci contrastait avec l'augmentation du taux d'insuline plasmatique de 91 à 113% chez les femmes sveltes et l'absence de changement de stockage du glucose du corps entier. Discussion : L'administration de dexaméthasone conduit à une baisse significative du stockage du glucose du corps entier pour une glycémie fixée chez les femmes obèses mais non chez les femmes sveltes. Ceci indique que les femmes obèses sont incapables d'accroître adéquatement leur sécrétion d'insuline. Abstract: Objective: In healthy lean individuals, changes in insulin sensitivity occurring as a consequence of a 2-day dexamethasone administration are compensated for by changes in insulin secretion, allowing glucose homeostasis to be maintained. This study evaluated the changes in glucose metabolism and insulin secretion induced by short-term dexamethasone administration in obese women. Research Methods and Procedures: Eleven obese women with normal glucose tolerance were studied on two occasions, without and after 2 days of low-dose dexamethasone administration. A two-step hyperglycemic clamp (7.5 and 10 mr1/1 glucose) with 6,6 2H2 glucose was used to assess insulin secretion and whole body glucose metabolism. Results were compared with those obtained in a group of eight lean women. Results: Without dexamethasone, obese women had higher plasma insulin concentrations in the fasting state, during the first phase of insulin secretion, and at the two hyperglycemic plateaus. However, they had normal whole body glucose metabolism compared with lean women, indicating adequate compensation. After dexamethasone, obese women had a 66% to 92% increase in plasma insulin concentrations but a 15.4% decrease in whole body glucose disposal. This contrasted with lean women, who had a 91% to 113% increase in plasma insulin concentrations, with no change in whole body glucose disposal. Discussion: Dexamethasone administration led to a significant reduction in whole body glucose disposal at fixed glycemia in obese but not lean women. This indicates that obese women are unable to increase their insulin secretion appropriately.

Role of reactive hyperreninemia in blood pressure changes induced by sodium depletion in patients with refractory hypertension

Sixteen patients with refractory hypertension were submitted to vigorous sodium depletion while cardiovascular homeostasis was monitored with measurements of hormonal and hemodynamic parameters and repeat saralasin tests. This regimen resulted in a negative sodium balance by an average of 300 mEq. The loss of sodium closely correlated to the decrease of body weight (r = 0.70, p less than 0.005). Blood pressure (BP) decreased from 176/166 +/- 8/3 to 155/109 +/-6/3 mm Hg. There was a significant correlation between percent increments in plasma renin activity (PRA) and the rise in plasma norepinephrine (r = 0.68, p less than 0.05) and a close negative correlation between percent increase in PRA and the ratio of fall in mean blood pressure (MAP) per unit of weight loss (r = -0.73, p less than 0.005). Thus, patients with the least percent increase in PRA demonstrated the greatest fall in BP per unit of weight loss, indicating that relative rather than absolute elevation of renin may be the factor limiting antihypertensive efficacy of sodium depletion. Sodium depletion induced increase in peripheral resistance and decrease in cardiac output, both mostly attributable to relative hyperreninemia. Indeed, the adverse hemodynamic changes were reversed by angiotensin inhibition, during which BP normalized. It is concluded that vigorous sodium depletion complemented by angiotensin blockade or suppression with sympatholytic agents improves management of otherwise refractory hypertension.

Impact of controlled changes in grain size and pore space characteristics on the hydraulic conductivity and spectral induced polarization response of "proxies" of saturated alluvial sediments

Understanding the influence of pore space characteristics on the hydraulic conductivity and spectral induced polarization (SIP) response is critical for establishing relationships between the electrical and hydrological properties of surficial unconsolidated sedimentary deposits, which host the bulk of the world's readily accessible groundwater resources. Here, we present the results of laboratory SIP measurements on industrial-grade, saturated quartz samples with granulometric characteristics ranging from fine sand to fine gravel, which can be regarded as proxies for widespread alluvial deposits. We altered the pore space characteristics by changing (i) the grain size spectra, (ii) the degree of compaction, and (iii) the level of sorting. We then examined how these changes affect the SIP response, the hydraulic conductivity, and the specific surface area of the considered samples. In general, the results indicate a clear connection between the SIP response and the granulometric as well as pore space characteristics. In particular, we observe a systematic correlation between the hydraulic conductivity and the relaxation time of the Cole-Cole model describing the observed SIP effect for the entire range of considered grain sizes. The results do, however, also indicate that the detailed nature of these relations depends strongly on variations in the pore space characteristics, such as, for example, the degree of compaction. The results of this study underline the complexity of the origin of the SIP signal as well as the difficulty to relate it to a single structural factor of a studied sample, and hence raise some fundamental questions with regard to the practical use of SIP measurements as site- and/or sample-independent predictors of the hydraulic conductivity.

Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

T cell receptor-ligand interactions: a conformational preequilibrium or an induced fit.

Kinetic parameters of T cell receptor (TCR) interactions with its ligand have been proposed to control T cell activation. Analysis of kinetic data obtained has so far produced conflicting insights; here, we offer a consideration of this problem. As a model system, association and dissociation of a soluble TCR (sT1) and its specific ligand, an azidobenzoic acid derivative of the peptide SYIPSAEK-(ABA)I (residues 252-260 from Plasmodium berghei circumsporozoite protein), bound to class I MHC H-2K(d)-encoded molecule (MHCp) were studied by surface plasmon resonance. The association time courses exhibited biphasic patterns. The fast and dominant phase was assigned to ligand association with the major fraction of TCR molecules, whereas the slow component was attributed to the presence of traces of TCR dimers. The association rate constant derived for the fast phase, assuming a reversible, single-step reaction mechanism, was relatively slow and markedly temperature-dependent, decreasing from 7.0 x 10(3) at 25 degrees C to 1.8 x 10(2) M(-1).s(-1) at 4 degrees C. Hence, it is suggested that these observed slow rate constants are the result of unresolved elementary steps of the process. Indeed, our analysis of the kinetic data shows that the time courses of TCR-MHCp interaction fit well to two different, yet closely related mechanisms, where an induced fit or a preequilibrium of two unbound TCR conformers are operational. These mechanisms may provide a rationale for the reported conformational flexibility of the TCR and its unusual ligand recognition properties, which combine high specificity with considerable crossreactivity.

Oviposition-induced changes in Arabidopsis genome expression: anticipating your enemy?

Plants have evolved exquisite ways to detect their enemies and are able to induce defenses responses tailored to their specific aggressors. Insect eggs deposited on a leaf represent a future threat as larvae hatching from the egg will ultimately feed on the plant. Although direct and indirect defenses towards oviposition have been documented, our knowledge of the molecular changes triggered by egg deposition is limited. Using a whole-genome microarray, we recently analyzed the expression profile of Arabidopsis thaliana leaves after oviposition by two pierid butterflies. Eggs laid by the large white Pieris brassicae modified the expression of hundreds of genes. The transcript signature included defense and stress-related genes that were also induced in plants experiencing localized cell death. Further analyses revealed that cellular changes associated with a hypersensitive response occur at the site of egg deposition and that they are triggered by egg-derived elicitors. Our study brings molecular evidence for previous observations of oviposition-induced necrosis in other plant species and might illustrate a direct defense of the plant against the egg. In this addendum, we discuss the relevance of the oviposition-induced gene expression changes and the possibility that plants use eggs as cues to anticipate their enemies.