Aphotic N2 Fixation in the Eastern Tropical South Pacific Ocean

We examined rates of N₂ fixation from the surface to 2000 m depth in the Eastern Tropical South Pacific (ETSP) during El Niño (2010) and La Niña (2011). Replicated vertical profiles performed under oxygen-free conditions show that N₂ fixation takes place both in euphotic and aphotic waters, with rates reaching 155 to 509 µmol N m⁻² d⁻¹ in 2010 and 24±14 to 118±87 µmol N m⁻² d⁻¹ in 2011. In the aphotic layers, volumetric N₂ fixation rates were relatively low (<1.00 nmol N L⁻¹ d⁻¹), but when integrated over the whole aphotic layer, they accounted for 87–90% of total rates (euphotic+aphotic) for the two cruises. Phylogenetic studies performed in microcosms experiments confirm the presence of diazotrophs in the deep waters of the Oxygen Minimum Zone (OMZ), which were comprised of non-cyanobacterial diazotrophs affiliated with nifH clusters 1K (predominantly comprised of α-proteobacteria), 1G (predominantly comprised of γ-proteobacteria), and 3 (sulfate reducing genera of the δ-proteobacteria and Clostridium spp., Vibrio spp.). Organic and inorganic nutrient addition bioassays revealed that amino acids significantly stimulated N₂ fixation in the core of the OMZ at all stations tested and as did simple carbohydrates at stations located nearest the coast of Peru/Chile. The episodic supply of these substrates from upper layers are hypothesized to explain the observed variability of N₂ fixation in the ETSP.     

Patterns of Ecosystem Metabolism in the Tonle Sap Lake,Cambodia with Links to Capture Fisheries

The Tonle Sap Lake in Cambodia is a dynamic flood-pulsed ecosystem that annually increases its surface area from roughly 2,500 km² to over 12,500 km² driven by seasonal flooding from the Mekong River. This flooding is thought to structure many of the critical ecological processes, including aquatic primary and secondary productivity. The lake also has a large fishery that supports the livelihoods of nearly 2 million people. We used a state-space oxygen mass balance model and continuous dissolved oxygen measurements from four locations to provide the first estimates of gross primary productivity (GPP) and ecosystem respiration (ER) for the Tonle Sap. GPP averaged 4.1±2.3 g O₂ m⁻³ d⁻¹ with minimal differences among sites. There was a negative correlation between monthly GPP and lake level (r = 0.45) and positive correlation with turbidity (r = 0.65). ER averaged 24.9±20.0 g O₂ m⁻³ d⁻¹ but had greater than six-fold variation among sites and minimal seasonal change. Repeated hypoxia was observed at most sampling sites along with persistent net heterotrophy (GPP<ER), indicating significant bacterial metabolism of organic matter that is likely incorporated into the larger food web. Using our measurements of GPP, we calibrated a hydrodynamic-productivity model and predicted aquatic net primary production (aNPP) of 2.0±0.2 g C m⁻² d⁻¹ (2.4±0.2 million tonnes C y⁻¹). Considering a range of plausible values for the total fisheries catch, we estimate that fisheries harvest is an equivalent of 7–69% of total aNPP, which is substantially larger than global average for marine and freshwater systems. This is likely due to relatively efficient carbon transfer through the food web and support of fish production from terrestrial NPP. These analyses are an important first-step in quantifying the resource pathways that support this important ecosystem.     

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10⁶ m⁻¹ s⁻¹ (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s⁻¹. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s⁻¹. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent k_cat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s⁻¹. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s⁻¹) and hydroxylation (36 ± 2 s⁻¹) partially control the overall turnover.     

Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Intra- specific variation in response of Jatropha (Jatropha curcas L.) to elevated CO2 conditions

Twenty genotypes of Jatropha collected from diverse eco-geographic regions from the states of Chhattisgarh (3), Andhra Pradesh (12), Rajasthan (4) and Uttarakhand (1) of India were subjected to elevated CO₂ conditions. All the genotypes showed significant difference (p < 0.05 and 0.01) in the phenotypic traits in both the environments (elevated and ambient) and genotype x environment interaction. Among the physiological traits recorded, maximum photosynthetic rate was observed in IC565048 (48.8 μmol m⁻² s⁻¹) under ambient controlled conditions while under elevated conditions maximum photosynthetic rate was observed in IC544678 (41.3 μmol m⁻² s⁻¹), and there was no significant difference in the genotype x environment interaction. Stomatal conductance (Gs) emerged as the key factor as it recorded significant difference among the genotypes, between the environments and also genotype x environment interaction. The Gs and transpiration (E) recorded a significant decline in the genotypes under the elevated CO₂ condition over the ambient control. Under elevated CO₂ conditions, the minimum values recorded for Gs and E were 0.03 mmol m⁻² s⁻¹ and 0.59 mmol m⁻² s⁻¹ respectively in accession IC565039, while the maximum values for Gs and E were 1.8 mmol m⁻² s⁻¹ and 11.5 mmol m⁻² s⁻¹ as recorded in accession IC544678. The study resulted in the identification of potential climate ready genotypes viz. IC471314, IC544654, IC541634, IC544313, and IC471333 for future use.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Quantification of Methylated Selenium,Sulfur, and Arsenic in the Environment

Biomethylation and volatilization of trace elements may contribute to their redistribution in the environment. However, quantification of volatile, methylated species in the environment is complicated by a lack of straightforward and field-deployable air sampling methods that preserve element speciation. This paper presents a robust and versatile gas trapping method for the simultaneous preconcentration of volatile selenium (Se), sulfur (S), and arsenic (As) species. Using HPLC-HR-ICP-MS and ESI-MS/MS analyses, we demonstrate that volatile Se and S species efficiently transform into specific non-volatile compounds during trapping, which enables the deduction of the original gaseous speciation. With minor adaptations, the presented HPLC-HR-ICP-MS method also allows for the quantification of 13 non-volatile methylated species and oxyanions of Se, S, and As in natural waters. Application of these methods in a peatland indicated that, at the selected sites, fluxes varied between 190–210 ng Se·m⁻²·d⁻¹, 90–270 ng As·m⁻²·d⁻¹, and 4–14 µg S·m⁻²·d⁻¹, and contained at least 70% methylated Se and S species. In the surface water, methylated species were particularly abundant for As (>50% of total As). Our results indicate that methylation plays a significant role in the biogeochemical cycles of these elements.     

Influence of a Decaying Cyclonic Eddy on Biogenic Silica and Particulate Organic Carbon in the Tropical South China Sea Based on 234Th-238U Disequilibrium

Eddies play a critical role in regulating the biological pump by pumping new nutrients to the euphotic zone. However, the effects of cyclonic eddies on particle export are not well understood. Here, biogenic silica (BSi) and particulate organic carbon (POC) exports were examined inside and outside a decaying cyclonic eddy using ²³⁴Th-²³⁸U disequilibria in the tropical South China Sea. For the eddy and outside stations, the average concentrations of BSi in the euphotic zone were 0.17±0.09 μmol L^-1 (mean±sd, n = 20) and 0.21±0.06 μmol L^-1 (n = 34). The POC concentrations were 1.42±0.56 μmol L^-1 (n = 34) and 1.30±0.46 μmol L^-1 (n = 51). Both BSi and POC abundances did not show change at the 95% confidence level. Based on the ²³⁴Th-²³⁸U model, BSi export fluxes in the eddy averaged 0.18±0.15 mmol Si m^-2 d^-1, which was comparable with the 0.40±0.20 mmol Si m^-2 d^-1 outside the eddy. Similarly, the average POC export fluxes were 1.5±1.4 mmol C m^-2 d^-1 and 1.9±1.3 mmol C m^-2 d^-1 for the eddy and outside stations. From these results we concluded that cyclonic eddies in their decaying phase have little effect on the abundance and export of biogenic particles.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

In Situ Coral Reef Oxygen Metabolism: An Eddy Correlation Study

Quantitative studies of coral reefs are challenged by the three-dimensional hard structure of reefs and the high spatial variability and temporal dynamics of their metabolism. We used the non-invasive eddy correlation technique to examine respiration and photosynthesis rates, through O₂ fluxes, from reef crests and reef slopes in the Florida Keys, USA. We assessed how the photosynthesis and respiration of different reef habitats is controlled by light and hydrodynamics. Numerous fluxes (over a 0.25 h period) were as high as 4500 mmol O₂ m⁻² d⁻¹, which can only be explained by efficient light utilization by the phototrophic community and the complex canopy structure of the reef, having a many-fold larger surface area than its horizontal projection. Over diel cycles, the reef crest was net autotrophic, whereas on the reef slope oxygen production and respiration were balanced. The autotrophic nature of the shallow reef crests implies that the export of organics is an important source of primary production for the larger area. Net oxygen production on the reef crest was proportional to the light intensity, up to 1750 µmol photons m⁻² s⁻¹ and decreased thereafter as respiration was stimulated by high current velocities coincident with peak light levels. Nighttime respiration rates were also stimulated by the current velocity, through enhanced ventilation of the porous framework of the reef. Respiration rates were the highest directly after sunset, and then decreased during the night suggesting that highly labile photosynthates produced during the day fueled early-night respiration. The reef framework was also important to the acquisition of nutrients as the ambient nitrogen stock in the water had sufficient capacity to support these high production rates across the entire reef width. These direct measurements of complex reefs systems yielded high metabolic rates and dynamics that can only be determined through in situ, high temporal resolution measurements.     

Whole-Body Insulin Sensitivity Rather than Body-Mass-Index Determines Fasting and Post-Glucose-Load Growth Hormone Concentrations

Background

Obese, non-acromegalic persons show lower growth hormone (GH) concentrations at fasting and reduced GH nadir during an oral glucose tolerance test (OGTT). However, this finding has never been studied with regard to whole-body insulin-sensitivity as a possible regulator.

Methods

In this retrospective analysis, non-acromegalic (NonACRO, n = 161) and acromegalic (ACRO, n = 35), non-diabetic subjects were subdivided into insulin-sensitive (IS) and –resistant (IR) groups according to the Clamp-like Index (CLIX)-threshold of 5 mg·kg⁻¹·min⁻¹ from the OGTT.

Results

Non-acromegalic IS (CLIX: 8.8±0.4 mg·kg⁻¹·min⁻¹) persons with similar age and sex distribution, but lower (p<0.001) body-mass-index (BMI = 25±0 kg/m², 84% females, 56±1 years) had 59% and 70%, respectively, higher (p<0.03) fasting GH and OGTT GH area under the curve concentrations than IR (CLIX: 3.5±0.1 mg·kg⁻¹·min⁻¹, p<0.001) subjects (BMI = 29±1 kg/m², 73% females, 58±1 years). When comparing on average overweight non-acromegalic IS and IR with similar anthropometry (IS: BMI: 27±0 kg/m², 82% females, 58±2 years; IR: BMI: 27±0 kg/m², 71% females, 60±1 years), but different CLIX (IS: 8.7±0.9 vs. IR: 3.8±0.1 mg·kg⁻¹·min⁻¹, p<0.001), the results remained almost the same. In addition, when adjusted for OGTT-mediated glucose rise, GH fall was less pronounced in IR. In contrast, in acromegalic subjects, no difference was found between IS and IR patients with regard to fasting and post-glucose-load GH concentrations.

Conclusions

Circulating GH concentrations at fasting and during the OGTT are lower in non-acromegalic insulin-resistant subjects. This study seems the first to demonstrate that insulin sensitivity rather than body-mass modulates fasting and post-glucose-load GH concentrations in non-diabetic non–acromegalic subjects.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Calcification by Reef-Building Sclerobionts

It is widely accepted that deteriorating water quality associated with increased sediment stress has reduced calcification rates on coral reefs. However, there is limited information regarding the growth and development of reef building organisms, aside from the corals themselves. This study investigated encruster calcification on five fore-reefs in Tobago subjected to a range of sedimentation rates (1.2 to 15.9 mg cm⁻² d⁻¹). Experimental substrates were used to assess rates of calcification in sclerobionts (e.g. crustose coralline algae, bryozoans and barnacles) across key reef microhabitats: cryptic (low-light), exposed (open-horizontal) and vertical topographic settings. Sedimentation negatively impacted calcification by photosynthesising crustose coralline algae in exposed microhabitats and encrusting foram cover (%) in exposed and cryptic substrates. Heterotrophs were not affected by sedimentation. Fore-reef, turbid water encruster assemblages calcified at a mean rate of 757 (SD ±317) g m⁻² y⁻¹. Different microhabitats were characterised by distinct calcareous encruster assemblages with different rates of calcification. Taxa with rapid lateral growth dominated areal cover but were not responsible for the majority of CaCO₃ production. Cryptobiont assemblages were composed of a suite of calcifying taxa which included sciaphilic cheilostome bryozoans and suspension feeding barnacles. These calcified at mean rates of 20.1 (SD ±27) and 4.0 (SD ±3.6) g m⁻² y⁻¹ respectively. Encruster cover (%) on exposed and vertical substrates was dominated by crustose coralline algae which calcified at rates of 105.3 (SD ±67.7) g m⁻² y⁻¹ and 56.3 (SD ±8.3) g m⁻² y⁻¹ respectively. Globally, encrusting organisms contribute significant amounts of carbonate to the reef framework. These results provide experimental evidence that calcification rates, and the importance of different encrusting organisms, vary significantly according to topography and sediment impacts. These findings also highlight the need for caution when modelling reef framework accretion and interpreting results which extrapolate information from limited data.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Metabolic and Behavioral Compensations in Response to Caloric Restriction: Implications for the Maintenance of Weight Loss

Background

Metabolic and behavioral adaptations to caloric restriction (CR) in free-living conditions have not yet been objectively measured.

Methodology and Principal Findings

Forty-eight (36.8±1.0 y), overweight (BMI 27.8±0.7 kg/m²) participants were randomized to four groups for 6-months; Control: energy intake at 100% of energy requirements; CR: 25% calorie restriction; CR+EX: 12.5% CR plus 12.5% increase in energy expenditure by structured exercise; LCD: low calorie diet (890 kcal/d) until 15% weight reduction followed by weight maintenance. Body composition (DXA) and total daily energy expenditure (TDEE) over 14-days by doubly labeled water (DLW) and activity related energy activity (AREE) were measured after 3 (M3) and 6 (M6) months of intervention. Weight changes at M6 were −1.0±1.1% (Control), −10.4±0.9% (CR), −10.0±0.8% (CR+EX) and −13.9±0.8% (LCD). At M3, absolute TDEE was significantly reduced in CR (−454±76 kcal/d) and LCD (−633±66 kcal/d) but not in CR+EX or controls. At M6 the reduction in TDEE remained lower than baseline in CR (−316±118 kcal/d) and LCD (−389±124 kcal/d) but reached significance only when CR and LCD were combined (−351±83 kcal/d). In response to caloric restriction (CR/LCD combined), TDEE adjusted for body composition, was significantly lower by −431±51 and −240±83 kcal/d at M3 and M6, respectively, indicating a metabolic adaptation. Likewise, physical activity (TDEE adjusted for sleeping metabolic rate) was significantly reduced from baseline at both time points. For control and CR+EX, adjusted TDEE (body composition or sleeping metabolic rate) was not changed at either M3 or M6.

Conclusions

For the first time we show that in free-living conditions, CR results in a metabolic adaptation and a behavioral adaptation with decreased physical activity levels. These data also suggest potential mechanisms by which CR causes large inter-individual variability in the rates of weight loss and how exercise may influence weight loss and weight loss maintenance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00099151","term_id":"NCT00099151"}}NCT00099151     

Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses

Using molecular techniques and microsensors for H₂S and CH₄, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S²⁻ m⁻³ s⁻¹ or 2 × 10⁻⁹ mmol s⁻¹ per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S²⁻ m⁻³ s⁻¹ or 7 × 10⁻⁹ mol s⁻¹ per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH₄ m⁻³ s⁻¹ or 10⁻⁹ mmol s⁻¹ per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH₄ m⁻³ s⁻¹ or 5 × 10⁻⁹ mmol s⁻¹ per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H₂), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.