Biological fixation of nitrogen and growth of bacteria of the genus <Emphasis Type="Italic">Azotobacter</Emphasis> in liquid media in the presence of perfluorocarbons

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).     

Biological fixation of nitrogen and growth of bacteria of the genus Azotobacter in liquid media in the presence of perfluorocarbons

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Efficacy of perfluorodecalin as an oxygen carrier for mouse and rat testes perfused in vitro

Perfluorodecalin is a superior artificial oxygen carrier because of its high oxygen dissolving capacity, low toxicity, and short retention times within tissues. However, the instability of perfluorodecalin emulsions has hindered its application in blood substitutes. The present study addressed two questions. First, is perfluorodecalin deleterious to the endocrine function of testes? This question was examined by comparing testosterone secretion by testes perfused in vitro with medium incorporating either perfluorodecalin or erythrocytes as oxygen carriers. Second, can stable emulsions of perfluorodecalin be attained with the new surfactant Butronic U-1? This question was approached by determining the stability of perfluorodecalin emulsions containing either Butronic U-1 or Pluronic F-68, a proven ineffective surfactant. The experimental results support the efficacy of perfluorodecalin emulsions as oxygen carriers for mouse and rat testes perfused in vitro. Perfluorodecalin emulsions formed with Butronic U-1 were stable during the 4-hr perfusions but not during long-term storage.     

Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68

Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of > 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P<0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.Abbreviations ATP adenosine triphosphate - PFCs perfluorochemicals - STP standard temperature and pressure     

Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second liquid phase

Oxygenation is an important parameter involved in the design and operation of mixing-sparging bioreactors and it can be analyzed by means of the oxygen mass transfer coefficient (k(L)a). The operational conditions of a stirred, submerged aerated 2-L bioreactor have been optimized by studying the influence of a second liquid phase with higher oxygen affinity (perfluorodecalin or olive oil) in the k(L)a. Using k(L)a measurements, the influence of the following parameters on the oxygen transfer rate was evaluated: the volume of working medium, the type of impellers and their position, the organic phase concentration, the aqueous phase composition, and the concentration of inactive biomass. This study shows that the best experimental conditions were achieved with a perfluorodecalin volume fraction of 0.20, mixing using two Rushton turbines with six vertical blades and in the presence of YPD medium as the aqueous phase, with a k(L)a value of 64.6 h(-1). The addition of 20% of perfluorodecalin in these conditions provided a k(L)a enhancement of 25% when pure water was the aqueous phase and a 230% enhancement when YPD medium was used in comparison to their respective controls (no perfluorodecalin). Furthermore it is shown that the presence of olive oil as a second liquid phase is not beneficial to the oxygen transfer rate enhancement, leading to a decrease in the k(L)a values for all the concentrations studied. It was also observed that the magnitude of the enhancement of the k(L)a values by perfluorodecalin depends on the biomass concentration present.     

Strategies for promoting division of cultured plant protoplasts: Beneficial effects of oxygenated perfluorocarbon

Summary Assessments have been made of physical and chemical options, alone and in combination, for gaseous manipulation of cassava (Manihot esculenta Crantz.) leaf protoplast cultures. Protoplasts were cultured for 25 d in liquid medium at an initial plating density of 4 × 10⁵ ml^–1 overlying (1) agar-solidified medium (control), (2) agar medium under an oxygen-enriched (10 mbar, 1 min) atmosphere, (3) agar medium supplemented with perfluorodecalin (Flutec ^® PP5), or (4) agar medium supplemented with oxygenated (10 mbar, 15 min) perfluorodecalin. Similar experimental treatments were also set up, with glass rods embedded in the agar medium. The mean initial plating efficiency (IPE) of protoplasts following culture with oxygenated perfluorodecalin without glass rods (6.7 ± 0.6%; n = 3) was over 2-fold greater (P < 0.05) than that of control cultures (2.6 ± 0.2%; n = 3). The mean IPE of protoplasts cultured with oxygenated perfluorodecalin in the presence of glass rods (5.8 ± 0.2%; n = 3) was also over 2-fold greater (P < 0.05) than controls. There was no significant difference between the IPE of protoplasts cultured under an increased oxygen atmosphere or with oxygenated perfluorodecalin, irrespective of the presence of glass rods.     

Effects of perfluorinated oxygen carrier application in yeast, fungi and plant cell suspension cultures

The growth of the yeast Saccharomyces cerevisiae, the fungus Rhizopus nigricans and Nicotiana tabacum cells with perfluorodecalin as an oxygen carrier has been studied. The volumetric mass transfer coefficient (k_La) measured by the dynamic method was higher for the perfluorodecalin oxygenation system than for the conventional aeration system. The results show that perfluorocarbon can be successfully used as an efficient gas carrier, especially for the culture of delicate plant cells. The increase in yeast biomass in the suspension culture aerated by perfluorodecalin was as much as 110% higher than in the culture aerated by air. The fungus R. nigricans grew better when the conventional aeration system was used due to the fact that growth of the mycelium is limited by the transport of oxygen by diffusion in the pellets rather than by interfacial oxygen transport. In the case of isolated tobacco cells, an increase of over 350% in biomass growth was observed for the PFC aeration system.     

Artificial carrier for oxygen supply in biological systems

Several poly (dimethylsiloxanes) (PDMS) copolymers of dimethylsiloxane (DMS) with ethylene or propylene oxide were tested as artificial carriers for the delivery of oxygen to biological systems. Copolymers with a DMS content of 33% or lower enhanced glucose oxidation by 200% in contrast to the 25% increase produced by the same concentration of perfluorodecalin. When 0.05% of the copolymer with 18% DMS was included in the growth media of Bacillus thuriginensis, the biomass (growth rate) increased 1.5-fold. With 0.1% of this copolymer, actinorhodin production by Streptomyces coelicolor A3 (2) occurred in half the normal time and with an increased yield. In conclusion, these PDMS copolymers are a good alternative to perfluorodecalin as oxygen carriers in biotechnological processes.     

Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the "mitochondrial" fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the "microsomal" fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase.     

Models for Cutaneous Gas Exchange and Transport

SYNOPSIS. Models suited for quantitative analysis of cutaneousgas exchange and transport with particular emphasis on diffusionlimitation are discussed. I. The diffusive transfer of gasesbetween body and environment can be compared to diffusion acrossa homogeneous flat tissue sheet characterized by a diffusionconductance or diffusing capacity (D). II. A flat barrier separatingthe medium from blood flow is a widely useful model. The characteristic parameter is theratio , where rß is the effective solubility of the gas in blood. A high value means predominant perfusionlimitation; low signifies diffusion limitation. III. In some conditions, as in still wateror at body-soil contact, a considerable diffusion resistancein the medium may become important. IV. Unequal distributionof D to may strongly reduce gas exchange efficiency. V. Diffusive arteriovenous shuntingof gases may lead to additional reduction of gas exchange efficiency.VI. The parallel arrangement of skin circulation with respectto tissue circulation (in contrast to the arrangement in seriespresent in fish gills and in tetrapod lungs) leads to mixingof arterialized with venous blood and reduced efficiency ofblood flow in gas transport.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Helicobacter pylori in Liquid Culture: Evaluation of Growth Rates and Ultrastructure

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order). Received: 10 November 1997 / Accepted: 29 January 1998     

Effect of perfluorodecalin, carbogal, and perfluoromethyldecalin on growth and ice-forming activity of bacteria

The isolates of pseudomonads (56) and of Plesiomonas shigelloides (7) exhibiting ice-forming activity were obtained from plant leaves and rhizosphere. The theoretical possibility of the application of perfluorocarbons (PFC) with a gas-transporting function (perfluorodecalin, carbogal, and perfluoromethyldecalin) for the cultivation of bacteria with different levels of ice-forming activity (IFA) in order to enhance their growth rates, biomass yields, and IFA was demonstrated. Introduction of 5% perfluorodecalin, carbogal, or perfluoromethyldecalin resulted for two strains in a 1.7–3.1-fold increase in biomass and a 3.2–24.5-fold increase in ice-forming activity compared to the control (without PFC).     

Mechanism of inhibiting the cytochrome P-450-dependent monooxygenase system in liver with fluorocarbons]

The inducer of the liver monooxygenase system perfluorodecalin added to microsomes as a submicron emulsion forms an enzyme-substrate complex with cytochrome P-450. The K(app) values for the perfluorodecalin binding to cytochrome P-450 in microsomes isolated from the livers of control and phenobarbital-treated rats are 5 x 10(-5) M and 2.3 x 10(-6) M, respectively. Perfluorodecalin competitively inhibits the binding of substrates to cytochrome P-450 and decreases the rates of monooxygenase reactions. Perfluorodecalin extrusion from the active center of cytochrome P-450 occurs when an excess of perfluorocarbons non-interacting with cytochrome P-450 is added to microsomes. There is a significant vagueness in the rates of various monooxygenase reactions because of simultaneous induction and inhibition of monooxygenase enzymes after perfluorodecalin administration to rats. The data obtained are consistent with the hypothesis that constitutive forms of cytochrome P-450 are primary receptors for xenobiotic-inducers of phenobarbital-type cytochrome P-450 isoforms.     

A survey of the statistical power of research in behavioral ecology and animal behavior

We estimated the statistical power of the first and last statisticaltest presented in 697 papers from 10 behavioral journals. Firsttests had significantly greater statistical power and reportedmore significant results (smaller p values) than did last tests.This trend was consistent across journals, taxa, and the typeof statistical test used. On average, statistical power was13–16% to detect a small effect and 40–47% to detecta medium effect. This is far lower than the general recommendationof a power of 80%. By this criterion, only 2–3%, 13–21%,and 37–50% of the tests examined had the requisite powerto detect a small, medium, or large effect, respectively. Neitherp values nor statistical power varied significantly across the10 journals or 11 taxa. However, mean p values of first andlast tests were significantly correlated across journals ( ), with a similar trend for mean power ( ). There is therefore some evidence that power and p values are repeatable among journals. Mean p valuesor power of first and last tests were, however, uncorrelatedacross taxa. Finally, there was a significant correlation betweenpower and reported p value for both first ( ) and last tests ( ). If true effect sizes are unrelated to study sample sizes, the averagetrue effect size must be nonzero for this pattern to emerge.This suggests that failure to observe significant relationshipsis partly owing to small sample sizes, as power increases withsample size.     

Image analysis assessments of perfluorocarbon- and surfactant-enhanced protoplast division

Image analysis has been used to assess the growth of cell suspension-derived protoplasts of Petunia hybrida cv. Comanche at an interface between aqueous culture medium (KM8P), supplemented with 0.01% (w/v) Pluronic F-68, and oxygenated (10 mbar; 10 min) perfluorodecalin. Protoplasts synthesised a new cell wall and entered normal mitotic division which was sustainable to the cell colony/callus stage. This process was accentuated by the collective and additive effects of oxygen, perfluorodecalin and surfactant media supplements. The mean area (mm³) of protoplast-derived cell colonies after 68 days of growth was increased 35 fold over control (media alone) in the presence of these combined treatments. The new cultural regime, leading to improved cell throughput from protoplasts, is discussed primarily in relation to the role of perfluorodecalin as a gas carrier and possible effects of Pluronic F-68 in stimulating cellular uptake of nutrients and/or growth regulators. Image analysis provides a novel and accurate approach to quantifying cell growth responses.Abbreviations dpi dots per inch - FPE final plating efficiency - IPE initial plating efficiency - KM Kao & Michayluk (1975) - PFC Perfluorocarbon - UM Uchimiya & Murashige (1974)     

Intracellular pH Regulation in Ricinus communis Grown with Ammonium or Nitrate as N Source: The Role of Long Distance Transport

Allen, S. and Raven, J. A. 1987. Intracellular pH regulationin Ricinus communis grown with ammonium or nitrate as N source:the role of long distance transport.—J. exp. Bot. 38:580–596. Charge balance and intracellular pH regulation were studiedin Ricinus communis grown in water culture with 1–0 molm^–3 or l-0 mol m ^–3 as sole N source. Seedling and 70-d-old plant parts were analysed for total P, C, organic N and S, and ash alkalinity;xylem and phloem saps were analysed for mineral ion content,and amino acids, amides, and dicarboxylates. Excreted H⁺ andbase were also measured. It was shown that in -N plants, H ⁺ produced and excreted directly bythe roots accounted for all net —COOH produced in theplant, but not for cation uptake by net H⁺ exchange. IntracellularpH perturbation in the shoot was regulated partly by reduction and partly by the transportof OH^- -generating dicarboxylates in the xylem. Phloem sap hadthe capacity to transport organic N and carboxylates excessto shoot requirements back to the root. In -N plants, 60% of total reduction occurred in the root, and 70% of all OH^- produced by root and reduction was excreted directly as base. Very little —COO^–from root reduction was stored in the root: most was transported to the shoot as xylem dicarboxylateand stored in the shoot. Of the OH^- produced from shoot and reduction, 40% was stored as shoot carboxylate: the phloem saphad the capacity to transport the rest back to the roots whereit was excreted as base. Key words: Intracellular pH, ammonium nutrition, nitrate nutrition, xylem dicarboxylates, phloem dicarboxylates, charge balance     

Intensification of microbial degradation of crude oil and oil products in the presence of perfluorodecalin

The possibility of using perfluorinated organic compounds for growing microorganisms and degrading xenobiotics has been demonstrated for the first time with perfluorodecalin (PFD), a gas-transporting component of the blood substitute Perftoran. In particular, this is promising for intensifying microbial degradation of oil and oil products and production of biodegrader biomass in synthetic mineral media. Addition of PFD to a mineral medium with crude oil and masut increased 4.5-10.2 times maximum concentrations and growth rates of all bacterial strains under study (Pseudomonas, Rhodococcus, and Bacillus genera). The degree of oil product consumption was increased 8.7-12.7 times.     

Use of Sodium Polyanethol Sulfonate in the Preparation of 5% Sheep Blood Agar Plates

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.