Experimental hyperoxia (O2 supersaturation) reveals a gill diffusion limitation of maximum aerobic performance in fish

Several studies have demonstrated that hyperoxia increases the maximal O₂ consumption rate (ṀO_2max) in fish, but exactly how this occurs remains to be explained. Here, we tested the hypothesis that hyperoxia improves arterial oxygenation in rainbow trout during exhaustive exercise. We demonstrate a 35% higher ṀO_2max in hyperoxia (200% air saturation) relative to normoxia, which was achieved through a combined 15% increase in cardiac output due to elevated peak heart rate, and a 19% increase of the arterial–venous (A-V) O₂ content difference. While arterial O₂ partial pressure (PaO₂) and O₂ saturation of haemoglobin declined post-exhaustive exercise in normoxia, this did not occur in hyperoxia. This protective effect of hyperoxia on arterial oxygenation led to a 22% higher arterial O₂ content post-exhaustive exercise, thereby allowing a higher A-V O₂ content difference. These findings indicate that ṀO_2max is gill diffusion limited in exhaustively exercised rainbow trout. Moreover, as previous studies in salmonids have demonstrated collapsing PaO₂ in normoxia at maximal swimming speed and at acutely high temperatures, a diffusion limitation may constrain ṀO₂ in other situations eliciting peak metabolic demand. These findings, along with the fact that hyperoxia increases ṀO_2max in several other fishes, suggest that gill diffusion limitations of ṀO_2max may be widespread in fishes.     

Extracellular hemoglobin combined with an O2-generating material overcomes O2 limitation in the bioartificial pancreas

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O₂ supply after transplantation remains a major challenge. In this study, we address the O₂ limitation by combining silicone-encapsulated CaO₂ (silicone-CaO₂) to generate O₂ with an extracellular hemoglobin O₂-carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O₂-diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O₂ species produced by silicone-CaO₂. While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO₂ alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO₂ alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O₂ provider with hemoglobin as an effective strategy to overcome O₂ limitations in tissue engineering.     

Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Muscle maximal O2 uptake at constant O2 delivery with and without CO in the blood

In the present study we investigated the effects of carboxyhemoglobinemia (HbCO) on muscle maximal O2 uptake (VO2max) during hypoxia. O2 uptake (VO2) was measured in isolated in situ canine gastrocnemius (n = 12) working maximally (isometric twitch contractions at 5 Hz for 3 min). The muscles were pump perfused at identical blood flow, arterial PO2 (PaO2) and total hemoglobin concentration [( Hb]) with blood containing either 1% (control) or 30% HbCO. In both conditions PaO2 was set at 30 Torr, which produced the same arterial O2 contents, and muscle blood flow was set at 120 ml.100 g-1.min-1, so that O2 delivery in both conditions was the same. To minimize CO diffusion into the tissues, perfusion with HbCO-containing blood was limited to the time of the contraction period. VO2max was 8.8 +/- 0.6 (SE) ml.min-1.100 g-1 (n = 12) with hypoxemia alone and was reduced by 26% to 6.5 +/- 0.4 ml.min-1.100 g-1 when HbCO was present (n = 12; P less than 0.01). In both cases, mean muscle effluent venous PO2 (PVO2) was the same (16 +/- 1 Torr). Because PaO2 and PVO2 were the same for both conditions, the mean capillary PO2 (estimate of mean O2 driving pressure) was probably not much different for the two conditions, even though the O2 dissociation curve was shifted to the left by HbCO. Consequently the blood-to-mitochondria O2 diffusive conductance was likely reduced by HbCO.(ABSTRACT TRUNCATED AT 250 WORDS)     

流感病毒在无动物细胞的细菌共生培养基里的培养研究

流行性感冒病毒流行时,在一些病人的上呼吸道里,常可以见到流感病毒和一些细菌(如甲型链球菌、葡萄球菌、流感杆菌等)共存,以往的实验也证明它们的关系比较密切。此试验发现:流感病毒在仅有甲型链球菌生长的培养基中有自我复制的现象,共生培养的病毒经过一系列的病毒学实验方法,证明确实是流感病毒。流感病毒在共生培养基里与它在鸡胚里的生长曲线基本相同,共生培养基里,只要细菌生长,流感病毒就能生长,在不同的温度(15℃、22℃、37℃)条件下,流感病毒和细菌的生长趋势出现密切的平行关系。甲型链球菌以外的其它细菌,如乙型链球菌、肠球菌、葡萄球菌、酵母菌、革兰氏阳性杆菌等菌株也能与流感病毒共生,只有个别的菌株不共生。共生培养的流感病毒能在较低的温度(22℃)环境下保持它的活性达2个月之久;在8℃的环境里,流感病毒也能共生繁殖,经过长期低温共生培养,其致病性减弱;流感病毒和其它两种病毒能在同一共生培养基里共生繁殖。实验研究中还简要讨论了共生的机理和实际应用等问题。     

Damage mechanisms of suspended animal cells in agitated bioreactors with and without bubble entrainment

We show that when freely suspended hybridoma cells are cultured in an agitated bioreactor, two fluid-mechanical mechanisms can cause cell damage and growth retardation. The first is present only when there is a gas phase, and is associated with vortex formation accompanied by bubble entrainment and breakup. In the absence of a vortex and bubble entrainment, cells can be damaged only at very high agitation rates, above approximately 700 rpm, by stresses in the bulk turbulent liquid. Cell damage then correlates with Kolmogorov eddy sizes similar to or smaller than the cell size. In the absence of a vortex, the entrainment and motion of very fine bubbles cause no growth retardation even at agitation rates as high as 600 rpm.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.     

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.     

Appearance of beta-lactamase activity in animal cells upon liposome-mediated gene transfer

A restriction fragment, 875 bp, which encodes for a beta-lactamase activity, was isolated from the Escherichia coli plasmid pBR322 DNA and entrapped in liposomes. The incubation of the DNA-liposomes with avian, murine, and human cultured cells results in the uptake of the DNA with the efficiency of around 2000 molecules per cell. Extracts of the recipient cells show a beta-lactamase activity as demonstrated by spectroscopic and microbiological methods. These results indicate the expression of a prokaryotic gene in eukaryotic cells.     

Mate limitation does not explain the lack of capsules in a Pseudocalliergon trifarium population

Abstract

(1) Net assimilation and respiration rates were measured at intervals after re-moistening, following various periods of desiccation, in Hookeria lucens, Hylocomium splendens, Neckera crispa, Plagiochila spinulosa, Plagiothecium undulatum, Rhacomitrium lanuginosum, Rhytidiadelphus loreus, Saccogyna viticulosa, Scorpiurium circinatum and Tortula ruraliformis.

(2) Rhacomitrium lanuginosum was extremely resistant, recovering apparently normally after 239 days' desiccation at 32% R.H.; Plagiochila spinulosa and Hookeria lucens were the most sensitive.

(3) Rhacomitrium lanuginosum and Tortula ruraliformis were most quickly damaged at the highest humidity (76%) and Plagiothecium undulatum at the lowest humidity tested (32%).

(4) Saccogyna viticulosa and Scorpiurium circinatum combined relatively rapid impairment and slow recovery of assimilation with the capacity to survive long dry periods.

(5) Dark respiration was relatively slow (commonly c. 5–20% of net assimilation). It usually showed a slight initial stimulation and a longer-term build-up following moderate or prolonged desiccation.

(6) Desiccation responses of bryophytes can be characterized in terms of parameters defining rate of loss of photosynthetic capacity with desiccationtime, rate of recovery after short periods of desiccation, and survival.     

Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin (PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot was rectilinear over the range of 0.01–0.3 µg mL^?1 with a lower detection limit of 0.0017 µg mL^?1 and limit of quantitation of 0.005 µg mL^?1. The developed method was successfully applied to the analysis of the drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3). Statistical comparison of the results with those of the comparison method revealed good agreement and proved that there was no significant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated. Copyright © 2010 John Wiley & Sons, Ltd.