Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm² to 6, 10, or 20 dyn/cm² elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.     

Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca²⁺]_i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm² for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity^TM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca²⁺]_i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca²⁺]_i compared to statically cultured cells. The maximum increases of PI uptake and [Ca²⁺]_i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca²⁺]_i waves in shear cultured HUVECs was reduced compared to the statically cultured cells.     

Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations

Bone marrow mesenchymal stem cells (MSCs) have multi-differentiation capability. Their endothelial cell (EC) oriented differentiation is the key to vasculogenesis, in which both mechanical and chemical stimulations play important roles. Most previous studies reported individual effects of VEGF or fluid shear stress (SS), when MSCs were subjected to shear stress of 10–15 dyn/cm² over 24 hr. In this paper, we investigated responses of MSCs from young Sprague Dawley rats to shear stress, VEGF and the combination of the two stimuli. Our study showed that the combined stimulation of shear stress and VEGF resulted in more profound EC oriented differentiation of MSCs in comparison to any individual stimulation. Furthermore, we subjected MSCs to prolonged period of fluid shear stimulation, i.e. 48 hr rather than 24 hr, and increased the magnitude of the shear stress from 10 dyn/cm² to 15, 20 and 25 dyn/cm². We found that without VEGF, the endothelium oriented differentiation of MSCs that was seen following 24 hr of shear stimulation was largely abolished if we extended the shear stimulation to 48 hr. A similar sharp decrease in MSC differentiation was also observed when the magnitude of the shear stress was increased from 10–15 dyn/cm² to 20–25 dyn/cm² in 24 hr shear stimulation studies. However, with combined VEGF and fluid shear stimulation, most of the endothelial differentiation was retained following an extended period, i.e. at 48 hr, of shear stimulation. Our study demonstrates that chemical and mechanical stimulations work together in determining MSC differentiation dynamics.     

Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Mechanical regulation of cancer cell apoptosis and autophagy: Roles of bone morphogenetic protein receptor,Smad1/5, and p38 MAPK

Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G₂/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12 dyn/cm² induced death of these four tumor cell lines. In contrast to LSS at 0.5 dyn/cm², oscillatory shear stress (OSS) at 0.5 ± 4 dyn/cm² cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V–FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.     

Optimization of intravascular shear stress assessment in vivo

The advent of microelectromechanical systems (MEMS) sensors has enabled real-time wall shear stress (WSS) measurements with high spatial and temporal resolution in a 3-D bifurcation model. To optimize intravascular shear stress assessment, we evaluated the feasibility of catheter/coaxial wire-based MEMS sensors in the abdominal aorta of the New Zealand white (NZW) rabbits. Theoretical and computational fluid dynamics (CFD) analyses were performed. Fluoroscope and angiogram provided the geometry of aorta, and the Doppler ultrasound system provided the pulsatile flow velocity for the boundary conditions. The physical parameters governing the shear stress assessment in NZW rabbits included (1) the position and distance from which the MEMS sensors were mounted to the terminal end of coaxial wire or the entrance length, (L_e), (2) diameter ratios of aorta to the coaxial wire (D_aorta /D_{coaxial wire}=1.5–9.5), and (3) the range of Reynolds numbers (116–1550). At an aortic diameter of 2.4 mm and a maximum Reynolds number of 212 (a mean Reynolds number of 64.2), the time-averaged shear stress (τ_ave) was computed to be 10.06 dyn cm^?2 with a systolic peak at 33.18 dyn cm^?2. In the presence of a coaxial wire (D_aorta /D_{coaxial wire}=6 and L_e=1.18 cm), the τ_ave value increased to 15.54 dyn cm^?2 with a systolic peak at 51.25 dyn cm^?2. Real-time intravascular shear stress assessment by the MEMS sensor revealed an τ_ave value of 11.92 dyn cm^?2 with a systolic peak at 47.04 dyn cm^?2. The difference between CFD and experimental τ_ave was 18.5%. These findings provided important insights into packaging the MEMS sensors to optimize in vivo shear stress assessment.     

Laminar shear stress induces the expression of aquaporin 1 in endothelial cells involved in wound healing

Laminar shear stress (LSS) due to blood flow contributes to the maintenance of endothelial health by multiple mechanisms including promotion of wound healing. The present study examined the hypothesis that the induction of water channel aquaporin 1 (AQP1) expression by LSS might be functionally associated with endothelial wound healing. When human umbilical vein endothelial cells were exposed to LSS at 12 dyn cm^?2 for 24 h, significant increases in AQP1 expression were observed at the mRNA and protein levels as compared with static control. In the in vitro scratch wound healing assay, LSS treatments before and after wound creation enhanced endothelial wound healing and this effect was significantly attenuated by selective suppression of AQP1 expression using small interfering RNA. Ectopic expression of AQP1 enhanced wound healing in the absence of LSS. This study demonstrated that LSS stimulates the endothelial expression of AQP1 that plays a role in wound healing.     

Wall shear over high degree stenoses pertinent to atherothrombosis

Atherothrombosis can induce acute myocardial infarction and stroke by progressive stenosis of a blood vessel lumen to full occlusion. Since thrombus formation and embolization may be shear-dependent, we quantify the magnitude of shear rates in idealized severely stenotic coronary arteries (≥75% by diameter) using computational fluid dynamics to characterize the shear environment that may exist during atherothrombosis. Maximum shear rates in severe short stenoses were found to exceed 250,000 s^?1 (9500 dynes/cm²) and can reach a peak value of 425,000 s^?1 for a 98% stenosis. These high shear rates exceed typical shear used for in vitro blood flow experiments by an order of magnitude, indicating the need to examine thrombosis at very high shear rates. Pulsatility and stenosis eccentricity were found to have minor effects on the maximum wall shear rates in severe stenoses. In contrast, increases in the stenosis length reduced the maximum shear to 107,000 s^?1 (98% stenosis), while surface roughness could increase focal wall shear rates to a value reaching 610,000 s^?1 (90% stenosis). The “shear histories” of circulating platelets in these stenoses are far below reported activation thresholds. Platelets may be required to form bonds in 5 μs and resist shear forces reaching 8000 pN per platelet. Arterial thrombosis occurs in the face of pathological high shear stress, creating rapid and strong bonds without prior activation of circulating platelets.     

Effects of irradiance and water flow on formation and growth of spongy and filamentous thalli of Codium fragile

Effects of irradiance and water flow on formation and growth of filamentous and spongy thalli of Codium fragile (Suringar) Hariot growing on vinylon threads were investigated at the laboratory culture. They showed clear differences in their irradiance and water flow requirements for their formation and growth. Spongy thalli were formed from the cultured filamentous thalli only at the high water flow velocity (10 cm s⁻¹). Number of the spongy thalli remarkably increased with increasing irradiance because those at 10, 50 and 100 μmol m⁻² s⁻¹ reached 0, 2 and 76 thalli m⁻¹, respectively, by 10 weeks of culture. In contrast, filamentous thalli were formed from the cultured spongy thalli at 0 and 3 cm s⁻¹, and difference in irradiance had no effect on their formation. Growth of the spongy thalli greatly accelerated under the combination of the high irradiance and high water velocity (200 μmol m⁻² s⁻¹ and 10 cm s⁻¹) because their relative growth rate in wet weight under the condition was two–four times higher than those at the other examined irradiances and water velocities. On the other hand, difference in water velocity had no effect on growth of the filamentous thalli under flowing water, and their growth decelerated at the high irradiance (200 μmol m⁻² s⁻¹). This demonstrates that water flow is a major factor controlling the formation of the spongy and filamentous thalli. The formation and growth of the spongy thalli surely occur under the combination of the high irradiance and fast flowing water. In contrast, the formation of the filamentous thalli occurs in the calm water, and their growth is inhibited under the high irradiance.     

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm²) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H₂O₂). Physiological concentrations of H₂O₂ (flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H₂O₂- and LSS-induced eNOS phosphorylation and reduced ^?NO levels. We propose a model in which LSS promotes the formation of signaling levels of H₂O₂, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased ^?NO generation and protection of endothelial function.     

The effect of water temperature and velocity on barnacle growth: Quantifying the impact of multiple environmental stressors

Organisms employ a wide array of physiological and behavioral responses in an effort to endure stressful environmental conditions. For many marine invertebrates, physiological and/or behavioral performance is dependent on physical conditions in the fluid environment. Although factors such as water temperature and velocity can elicit changes in respiration and feeding, the manner in which these processes integrate to shape growth remains unclear. In a growth experiment, juvenile barnacles (Balanus glandula) were raised in dockside, once-through flow chambers at water velocities of 2 versus 19 cm s⁻¹ and temperatures of 11.5 versus 14 °C. Over 37 days, growth rates (i.e., shell basal area) increased with faster water velocities and higher temperatures. Barnacles at high flows had shorter feeding appendages (i.e., cirri), suggesting that growth patterns are unlikely related to plastic responses in cirral length. A separate experiment in the field confirmed patterns of temperature- and flow-dependent growth over 41 days. Outplanted juvenile barnacles exposed to the faster water velocities (32±1 and 34±1 cm s⁻¹; mean±SE) and warm temperatures (16.81±0.05 °C) experienced higher growth compared to individuals at low velocities (1±1 cm s⁻¹) and temperatures (13.67±0.02 °C). Growth data were consistent with estimates from a simple energy budget model based on previously measured feeding and respiration response curves that predicted peak growth at moderate temperatures (15 °C) and velocities (20–30 cm s⁻¹). Low growth is expected at both low and high velocities due to lower encounter rates with suspended food particles and lower capture efficiencies respectively. At high temperatures, growth is likely limited by high metabolic costs, whereas slow growth at low temperatures may be a consequence of low oxygen availability and/or slow cirral beating and low feeding rates. Moreover, these results advocate for approaches that consider the combined effects of multiple stressors and suggest that both increases and decreases in temperature or flow impact barnacle growth, but through different physiological and behavioral mechanisms.     

Shear stress-induced Ca2+ mobilization in MDCK cells is ATP dependent,no matter the primary cilium

Primary cilium has emerged as mechanosensor to subtle flow variations in epithelial cells, but its role in shear stress detection remains controversial. To probe the function of this non-motile organelle in shear stress detection by cells, we compared calcium signalling responses induced by shear stress in ciliated and unciliated MDCK cells. Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured using Fura-PE3 video imaging fluorescence microscopy in response to shear stress due to laminar flow (385 μl s^?1). Our results show that both unciliated and ciliated MDCK cells are shear stress sensitive via ATP release and autocrine feedback through purinergic receptors. However, purinergic calcium signals differed in response intensity and receptor subtypes. In unciliated cells, shear stress-induced elevation in [Ca²⁺]_i was predominantly mediated through P2X receptors (P2XR). In contrast, calcium mobilization in ciliated MDCK cells resulted from P2YRs and store-operated Ca²⁺-permeable channels besides P2XRs. These findings lend support to the hypothesis that ATP release in response to shear stress is independent of the primary cilium and that transduction of mechanical strain into a specific biochemical responses stems on the mobilization of different sets of purinergic receptors.     

Removal of chromium (VI) using poly(methylacrylate) functionalized guar gum

Using persulfate/ascorbic acid redox pair, poly(methylacrylate) was grafted on to guar gum and the conditions for the grafting were optimized. The copolymer sample having maximum %G was evaluated for the removal of Cr(VI) and the sorption conditions were optimized. The sorption was found pH dependent, pH 1.0 being the optimum value. Sorption data at pH 1.0 were modeled using both the Langmuir and Freundlich isotherms where the data fitted better to Freundlich isotherm. The equilibrium sorption capacity of 29.67 mg/g was determined from the Langmuir isotherm. The sorption followed a pseudo-second-order kinetics with a rate constant 2.5 × 10^?4 g mg^?1 min^?1. The grafted product was also evaluated for Cr(VI) removal from local electroplating industrial waste water. The regeneration experiments revealed that the guar-graft-poly(methylacrylate) could be successfully reused for five cycles. In the present study conductivity measurements were used instead of conventional photometric method for determining Cr(VI) concentration in the equilibrium solutions and the results obtained have been compared with photometric method. Optimum Cr(VI) binding under highly acidic conditions indicated significant contribution of non electrostatic forces in the adsorption process.     

Differential response of leaf gas exchange to enhanced ultraviolet-B (UV-B) radiation in three species of herbaceous climbingplants

We measured diurnal changes in photosynthetic rate, transpiration rate, stomatal conductance and water use efficiency in three species of herbaceous climbing plants (Luffa cylindrica, Trichosanthes kirilowii and Dioscorea opposita) exposed to two intensities of UV-B radiation: 3.0 μw cm^?2 (R1) and 8.0 μw cm^?2 UV-B (R2) radiation under ambient growth conditions. Responses differed per species and per treatment. In Luffa all values increased compared to the Control in both treatments, except for stomatal conductance in R2. In Trichosanthes photosynthetic rates and water use efficiency increased, while the transpiration rates decreased under both treatments, and stomatal conductance was lower in R1. In Dioscorea photosynthetic rates and water use efficiency decreased under both treatments, while the transpiration rates and stomatal conductance increased. The results suggested that to some extent increased UV-B radiation was beneficial to the growth of L. cylindrica and T. kirilowii, but detrimental to D. opposita.     

Colonization dynamics of biofilm-associated ciliate morphotypes at different flow velocities

The impact of flow velocity on initial ciliate colonization dynamics on surfaces were studied in the third order Ilm stream (Thuringia, Germany) at a slow flowing site (0.09 m s^?1) and two faster flowing sites (0.31 m s^?1) and in flow channels at 0.05, 0.4, and 0.8 m s^?1. At the slow flowing stream site, surfaces were rapidly colonized by ciliates with up to 60 cells cm^?2 after 24 h. In flow channels, the majority of suspended ciliates and inorganic matter accumulated at the surface within 4.5 h at 0.05 m s^?1. At 0.4 m s^?1 the increase in ciliate abundance in the biofilm was highest between 72 and 168 h at about 3 cells cm^?2 h^?1. Faster flow velocities were tolerated by vagile flattened ciliates that live in close contact to the surface. Vagile flattened and round filter feeders preferred biofilms at slow flow velocities. Addition of inorganic particles (0, 0.6, and 7.3 mg cm^?2) did not affect ciliate abundance in flow channel biofilms, but small ciliate species dominated and number of species was lowest (16 species cm^?2) in biofilms at high sediment content. Although different morphotypes dominated the communities at contrasting flow velocities, all functional groups contributed to initial biofilm communities implementing all trophic links within the microbial loop.     

In vitro enzymatic conversion of γ-aminobutyric acid immobilization of glutamate decarboxylase with bacterial cellulose membrane (BCM) and non-linear model establishment

The work investigated the properties and feasibility of using bacterial cellulose membrane (BCM) as a new and environmental friendly support carrier to immobilize glutamate decarboxylase (GAD) (a unique enzyme in the conversion of γ-aminobutyric acid (GABA) production). During cultivation, the porosities of BCM decreased successively with more extended fibrils piling above one another in a criss-crossing manner thus forming condensed and spatial structure. The BCM with this ultrafine network structure was found to immobilize GAD best via covalent binding because of the highest efficiency of immobilization (87.56% of the enzyme was bonded) and a good operational stability. And the covalent binding efficiency (amount of enzyme immobilized versus lost) was closely related to the porosity or the inner network of the BCM, not to the surface area. The capacity per surface area (mg/cm²) increased from 1.267 mg/cm² to 3.683 mg/cm² when the porosity of BCM ranged from 49% to 73.80%, while a declining trend of the loss of GAD specific activity (from 29.30%/cm² to 7.38%/cm²) was observed when the porosity increased from 49.9% to 72.30%. Two non-linear regression relationships, between the porosity and loading capacity and between porosity and enzyme activity loss, were empirically modeled with the determination of coefficient R² of 0.980 and 0.977, respectively. Finally, the established in vitro enzymatic conversion process demonstrated 6.03 g/L of GABA at 0.10 mol/L Glu, 60 min of retention time and 160 mL of suspension volume after the 1st run and a loss of 4.15% after the 4th run. The productivity of GABA was 6.03 g L^?1 h^?1, higher than that from other reported processes.     

Responses to climate change in radial growth of Picea schrenkiana along elevations of the eastern Tianshan Mountains,northwest China

Tree growth is largely driven by climate conditions in arid and alpine areas. A strong change in climate from warm-dry to warm-wet has already been observed in northwest China. However, little is known about the impacts of regional climate variability on the radial growth of trees along elevations of the eastern Tianshan Mountains. Consequently, we developed three tree-ring width chronologies of Schrenk spruce (Picea schrenkiana Fisch. et Mey.) ranging in elevation from 2159 to 2552 m above sea level (a.s.l.), which play an important role in the forestry ecosystem, agriculture, and local economy of Central Asia. In our study, the correlation analyses of growth-drought using the monthly standardized precipitation-evapotranspiration index (SPEI) at different temporal scales demonstrated that drought in growing season was the main factor limiting tree growth, regardless of elevation. The relationships between radial growth of Schrenk spruce and main climate factors were relatively stable by moving correlation function, and the trend of STD chronologies and basal area increment (BAI) also showed a synchronous decline across the three elevations in recent decades. And meanwhile, slight differences in responses to climate change in radial growth along elevations were examined. The drought stress increased as elevations decreased. Radial growth at the higher elevation depended on moisture availability due to high temperature, as indicated by the significant negative correlation with mean temperature in the late growing season of the previous year (August-September, p < 0.001). However, radial growth at the lower elevation were restricted by drought stress due to less precipitation and higher temperatures, as demonstrated by the significant negative correlation with mean temperature but positive with total precipitation in the early growing season of the current year (April-May, p < 0.05). In addition, the decline of radial growth (BAI) at the higher elevation (3.710 cm² yr⁻¹/decade, p < 0.001) was faster than that of the middle elevation (2.344 cm² yr⁻¹/decade, p < 0.001) and the lower elevation (3.005 cm² yr⁻¹/decade, p < 0.001) since 2000, indicating that the trees at higher elevation of a relatively humid environment were more susceptible to the effects of climate change due to their poor adaptability to water deficit. Therefore, the forest ecosystems would be suppressed as a result of increasing drought stress in the future, especially in the high-elevation forests of arid and semi-arid areas.     

Decomposition processes in soil of a healthy and a declining Phragmites australis stand

Decomposition processes were investigated in the soil of a declining, more eutrophic and a healthy, less eutrophic freshwater reed (Phragmites australis (Cav.) Trin. ex Steudel) stand in the littoral zone of Rožmberk fishpond, Czech Republic. Soil and pore water were sampled five times from April to October 1998. Chemical properties, CO₂ production in oxic and anoxic conditions, CH₄ production, denitrifying enzyme activity (DEA) and bacterial biomass were measured under laboratory conditions in suspensions prepared from homogenised soil samples. The more eutrophic West stand was more anaerobic than the East stand, with lower redox potential, lower pH and with a higher amount of organic acids, mainly acetic and lactic acid. Mean seasonal concentrations of total nitrogen in pore water, nitrogen of amino acids and proteins, and reducing sugars were all higher in the soil at the more eutrophic stand. Higher nutrient status and more reduced conditions at the more eutrophic stand were accompanied by (i) a limitation of aerobic microbial activities (CO₂ production in oxic conditions: 0.35 versus 0.54 μmol CO₂ cm⁻³ h⁻¹); lower DEA (4.0 versus 20.2 nmol N₂O cm⁻³ h⁻¹) and a lower proportion of bacteria that were active in aerobic conditions; (ii) by a prevalence of anaerobic over aerobic microbial processes; (iii) by a higher rate of methanogenesis (15.0 versus 11.5 nmol CH₄ cm⁻³ h⁻¹) and (iv) by an overall lower rate of microbial processes as compared to less eutrophied stand. The shift from aerobic to anaerobic microbial metabolism, and a coinciding restriction of metabolic activities at the more eutrophic stand are indicative of an elevated oxygen stress in the soil, associated with accumulation of metabolites toxic to both the micro-organisms and the reed. Possible links between eutrophication, decomposition processes in the soil and reed decline are discussed.