Further evidence for local osmotic coupling in the rabbit cornea

1. 1. The present experiments measure net fluxes of fluid, Cl⁻ and HCO₃⁻ across de-epithelialised rabbit corneas clamped between half chambers and bathed in Ringer solutions.

2. 2. Net fluxes of HCO₃⁻ and fluid occurred together across the cornea from stroma to aqueous when HCO₃⁻ and CO₂ were present in the bathing solution.

3. 3. No net trans-corneal Cl⁻ flux was found

4. 4. The initiation of fluid flow in the presence of HCO₃⁻ and CO₂ cannot be accounted for by bulk-phase osmotic flow across the cornea.

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO⁻₃ together with a decrease in pCO₂, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of ⁴⁵Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca²⁺ and abolished at low extracellular Na⁺ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca²⁺. The removal of HCO⁻₃ and decrease in the pCO₂ also reduced the magnitude of both the secondary rise in ⁴⁵Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca²⁺ from islet cells as mediated by Na⁺-Ca²⁺ countertransport and the inflow of Ca²⁺ by gated Ca²⁺ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.     

A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen fluxes in plants

 A simple procedure is described for the fabrication of micrometer to nanometer-scale platinum electrodes to be used in a vibrating oxygen-selective system. The electrode was prepared by etching a fine platinum wire and insulating it with an electrophoretic paint. The dimensions allowed this electrode to be used with the “vibrating probe technique” in exploratory studies aimed at mapping and measuring the patterns of net influxes as well as effluxes of oxygen in Olea europaea L. leaves and roots with spatial and temporal resolutions of a few microns and a few seconds, respectively. The magnitude and spatial localisation of O₂ influxes in roots was characterised by two distinct peaks. The first, in the division zone, averaged 38 ± 5 nmol m⁻² s⁻¹; the second, in the elongation region, averaged 68 ± 6 nmol m⁻² s⁻¹. Long-term records of oxygen influx in the elongation region of the root showed an oscillatory regime characterised by a fast oscillation with periods of about 8–9 min. In leaves, the system allowed the measurement of real-time changes in O₂ evolution following changes in light. Furthermore, it was possible to obtain “topographical” images of the photosynthetically generated oxygen diffusing through different stomata from a region of the leaf of 120 μm × 120 μm. The combination of topographic and electrochemical information at the micrometer scale makes the system an efficient tool for studying biological phenomena involving oxygen diffusion. Received: 12 November 1999 / Accepted: 1 February 2000     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Endocardial endothelium in the rat: junctional organization and permeability

Selective permeability of endocardial endothelium has been suggested as a mechanism underlying the modulation of the performance of subjacent myocardium. In this study, we characterized the organization and permeability of junctional complexes in ventricular endocardial endothelium in rat heart. The length of intercellular clefts viewed en face per unit endothelial cell surface area was lower, and intercellular clefts were deeper in endocardial endothelium than in myocardial vascular endothelium, whereas tight junctions had a similar structure in both endothelia. On this basis, endocardia endothelium. might be less permeable than capillary endothelium. However, confocal scanning laser microscopy showed that intravenously injected dextran 10000 coupled to Lucifer Yellow penetrated first the endocardial endothelium and later the myocardial capillary endothelium. Penetration of dextran 10000 in myocardium occurred earlier through subepicardial capillary endothelium than through subendocardial capillary endothelium. Penetration of tracer might thus be influenced by hydrostatic pressure. Dextran of MW 40000 did not diffuse through either endocardial endothelium or capilary endothelium. The ultrastructure of endocardial endothelium may constitute an adaptation to limit diffusion driven by high hydrostatic pressure in the heart. Differences in paracellular diffusion of dextran 10000 between endocardial endothelium and myocardial vessels, may result from differing permeability properties of the endocardium and underlying myocardium.     

Interactions of photosystem II with bicarbonate, formate and acetate

In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO₂ by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (β₁), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40–50 mM formate or acetate results in a significant increase in the miss parameter and to an ∼50% (formate) and ∼10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased β₁ parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2–10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S₀, S₂, and S₃ states were measured, and no differences were found between the two sample types. (2) The S₁, S₀, S₋₁, and S₋₂ states were probed by incubation with small concentrations of NH₂OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent S_i/S_−i state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO₂ concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).     

Coordinating responses to iron and oxygen stress with DNA and mRNA promoters: The ferritin story

Combinations of DNA antioxidant response element and mRNA iron responsive element regulate ferritin expression in animals in response to oxidant and iron stress, or normal developmental signals. Ferritins are protein nanocages, found in animals, plants, bacteria, and archaea, that convert iron and oxygen to ferric oxy biominerals in the protein central cavity; the mineral traps potentially toxic reactants and concentrates iron for the future synthesis of other iron/heme proteins. Regulatory signals and the nanocage gene products are the same throughout biology, but the genetic mechanisms, DNA versus DNA + mRNA, vary. The number of genes, temporal regulation, tissue distribution in multi-cellular organisms, and gene product size (maxi-ferritins have 24 subunits and mini-ferritins, or Dps proteins, have 12 subunits and are restricted to bacteria and archaea) suggest an overwhelming diversity and variability. However, common themes of regulation and function are described which indicate not only that the three-dimensional protein structure and the functions of the ferritins are conserved, but also that broad features of genetic regulation are conserved relative to organismal and/or community needs. The analysis illustrates the centrality of the ferritins to life with iron and oxygen and models how Nature harnesses potentially dangerous chemistry for biology.     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

Relation between dissolved oxygen concentration and ajmalicine production rate in high-density cultures of Catharanthus roseus

The relation between dissolved oxygen (DO) and the ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the ajmalicine production rate was less than 0.06 mumol/g/d. Above a DO of 43% the ajmalicine production rate was constant at 0.21 mumol/g/d. Between a DO of 29% and 43% there was a strong relation between the ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO /=57%. A kinetic equation is proposed for the relation between DO and the specific ajmalicine production rate. (c) 1995 John Wiley & Sons, Inc.     

Response of Alvinella pompejana to variable oxygen stress: A proteomic approach

Alvinella pompejana is one of the most emblematic species of the animal communities colonizing the deep‐sea hydrothermal vents of the East Pacific Rise. This extreme environment is characterized by high temporal variability of its physical–chemical parameters. Among these, the variation in concentration of available oxygen should lead to a specific physiological adaptive response of the animal. To evaluate the mechanisms of this response at a molecular level, a classical 2‐DE‐based proteomic approach has been implemented. After collection (Garrett‐18S and ‐17S vent sites) animals were reconditioned in a high‐pressure chamber pressurized at 260 bar and then the oxygen concentration was regulated to a constant value corresponding to hypoxia, normoxia or hyperoxia for 7 h. The soluble proteins from gills were then analyzed by 2‐DE. The protein content of spots showing specific changes following oxygen concentration variation was determined based on comparison of MS/MS sequence data with a recently established A. pompejana ESTs database. Fifteen proteins, belonging mainly to three families, cytoskeleton protein, enzymes of energetic metabolism and heat shock proteins, have been identified as potentially involved in the response to the change in oxygen concentration. The significance of the relatively small set of proteins modulated by oxygen variations is discussed in the context of a potential universal cellular response to stress.     

Protoplast ion fluxes: their measurement and variation with time, position and osmoticum

The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating cell wall. In this study net H⁺ and Ca²⁺ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a non-invasive microelectrode technique for ion flux measurements. For Ca²⁺, the flux distribution was almost symmetrical, ranging ±30 nmol · m⁻² · s⁻¹ around zero. For H⁺ it was skewed towards efflux ranging from −100 to +10 nmol · m⁻² · s⁻¹. The distribution of H⁺ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H⁺ and Ca²⁺ fluxes increased, which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange. Received: 11 June 1997 / Accepted: 10 July 1997     

The effect of oxygen concentration on photosynthetic biomass production by algae

The effect of decreased oxygen concentration on photosynthetic biomass production was determined for Euglena gracilis Klebs strain z and Chlamydomonas reinhardtii Dangeard. At a constant carbon dioxide concentration of 0.03% (v/v), decreasing the oxygen concentration from 21% to 2% (v/v) gave a two-fold increase in dry-weight yield for E. gracilis; a result consistent with the operation of a functional glycollate pathway in this alga. A similar effect of oxygen concentration on dry-weight yield was not observed with C. reinhardtii.     

The endothelium in health and disease: A discussion of the contribution of non-nitric oxide endothelium-derived vasoactive mediators to vascular homeostasis in normal vessels and in type II diabetes

Molecular and Cellular Biochemistry - Endothelial dysfunction is considered as a major risk factor of cardiovascular complications of type I and types II diabetes. Impaired endothelium-dependent...     

Ethanol production from cheese whey permeate by Kluyveromyces marxianus UFV-3: A flux analysis of oxido-reductive metabolism as a function of lactose concentration and oxygen levels

In order to investigate the effect of lactose concentration and oxygen level on the growth and metabolism of Kluyveromyces marxianus UFV-3 in cheese whey permeate, batch cultures were conducted under aerobic, hypoxic, and anoxic conditions, with lactose at initial concentration ranging from 1 to 240 g L⁻¹. The increase in lactose concentration increased ethanol yield and ethanol volumetric productivity, and has reduced cell yield. When lactose concentration was equal or above 50 g L⁻¹ and the oxygen levels were low, the ethanol yield was close to its theoretical value. Maximum ethanol concentrations attained in this study were 76 and 80 g L⁻¹ in hipoxia and anoxia, respectively. The lactose consumption rate in anoxia was greater than in aerobiosis and hipoxia. However, under anoxia, the lactose consumption rate of K. marxianus followed a saturation kinetics, which was not observed in hypoxia and aerobiosis. All oxygen levels investigated, showed a tendency for saturation of the ethanol production rate above 65 g L⁻¹ lactose. Ethanol production rate was also higher on anoxia.     

The passage of macrophages and lymphocytes from the interstitium across the lymphatic endothelium of rat lacteals

Summary The passage of cells across the lymphatic endothelium of rat lacteals in both normal and non-pathological experimental conditions (fasting, lymphatic stasis) was studied by means of serial thin sections and three-dimensional models. Two different pathways of transendothelial migration were observed: (1) macrophages enter the lymphatic lumen via the cytoplasm of endothelial cells, without involvement of intercellular junctions, whereas (2) lymphocytes migrate through intraendothelial channels, dynamic structures organized by the lymphatic endothelium under physiological conditions.     

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements

The photoacoustic signal from an intact leaf was analyzed as a vectorial summation of photothermal and photosynthetic oxygen-evolution contributions. A method is outlined to estimate each contribution separately. The amplitude of the oxygen-evolution component relative to that of the photothermal singnal decreases as the modulation frequency increases due to two processes which specifically damp the oxygen-evolution modulation: (1) diffusion of oxygen from the chloroplasts to the cell boundary, and (2) electron-transfer reactions occurring between the photochemical act and oxygen evolution. The effects of the two processes are well separated and are observed over different ranges of modulation frequency. Analysis of the data leads to a consistent estimation of the oxygen diffusion coefficient and also to a preliminary idea on the limiting time constant on the donor side of Photosystem II. The dependence of the photoacoustic oxygen-evolution signal on the intensity of added nonmodulated background light is used to construct the light saturation curve of (gross) Photsynthesis, with an estimation of the ratio maximal rate / maximal quantum yield. The photoacoustic method is distinguished by its sensitivity and rapidity (a single measurement takes approx. 1 s), far better than any other method to measure gross photosynthesis. The only disadvantage is in the fact that the quantum yield of oxygen evolution is determined in a relative basis only. Attempts to calibrate the photoacoustic measurements in an absolute sense are underway.