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 isolated 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).     

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).     

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.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

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.     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

Effect of perfluorodecalin on activities of hepatic phase II xenobiotic biotransformation enzymes.

The activity of the hepatic phase II enzymes of xenobiotic biotransformation after intravenous administration of perfluorodecalin emulsion to rats was measured. Perfluorodecalin was found to increase the microsomal glutathione S-transferase and UDP-glucuronosyltransferase activities 1.4- and 2.3-fold, respectively. The activity of sulphotransferase was decreased 2-fold. These results show that perfluorodecalin is an inducer of both the enzymes of cytochrome P-450-dependent monooxygenase system [Mishin V. et al (1989) Chem.-Biol. Interactions, 72, 143-155.] and those catalyzing conjugation reactions: microsomal glutathione S-transferase and UDP-glucuronosyltransferase.     

Hapten-Specific T Cell Response to Azobenzenearsonate-N-Acetyl-L-Tyrosine (ABA-tyr) in Lewis Rats: II. Induction and Suppression of ABA-Specific Helper Cell Activity for Antibody Production

Immunization of Lewis rats with azobenzenearsonate-N-acetyl-l-tyrosine (ABA-tyr) in complete Freund's adjuvant (CFA), produces a hapten-specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) prior to immunization with ABA-tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly l-glutamine-lysine-tyrosine (l-GLT); however, a d-amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA-tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA-tyr in IFA followed by boosting with ABA-tyr in CFA induced unresponsiveness to subsequent induction of hapten-specific help.     

Carbon dioxide-gassed fluorocarbon enhances micropropagation of rose (Rosa chinesis Jacq.)

The inert perfluorochemical (PFC) liquid, perfluorodecalin (Flutec PP6), has been used to increase the CO₂ supply to cultured shoots of Rosa chinensis Jacq. cv. Baby Love. Culture of shoots in semi-solid medium overlaying CO₂-gassed PFC (2 mbar; 5 min repeated every 7 days) for up to 42 days, increased biomass as reflected by significant (P<0.01) increases in shoot number, number of leaves per shoot and mean shoot fresh weight. Additionally, there were significant (P<0.01) increases in the number of roots and their fresh and dry weights following a further 10 days of culture on rooting medium prior to transfer of plants to the glasshouse. Treatment of cultured rose shoots with CO₂-gassed PFC also significantly reduced (P<0.01) the accumulation of phenolic compounds in roots. The total chlorophyll of aerial parts was unaffected, although total protein in shoots and roots was significantly (P<0.01) lower than in the control. The biotechnological implications of this novel cultural régime are discussed for the micropropagation of woody species. Received: 5 June 1996 / Revision received: 29 July 1996 / Accepted 19 August 1996     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. II. Induction of helper activity demonstrated by TNP-haptenated ABA-peptide-Ficoll

In an effort to study T cell functions in Lewis rats immunized with ABA-N-acetyl-L-tyrosine (ABA-tyr), we developed an antigen that provides a sensitive assay of ABA-specific helper function that is read as an increase in TNP-specific plaque-forming cells (PFC). This antigen has ABA coupled to AECM-Ficoll by virtue of a tripeptide (tyr-ala-ala) spacer and TNP coupled to the AECM side chains. At subimmunogenic doses, this antigen induced 400 anti-TNP PFC/10(6) spleen cells in ABA-tyr-immunized rats. As many as 8000 PFC/10(6) spleen cells were induced with larger doses of antigen (200 micrograms). By contrast, only 490 PFC/10(6) spleen cells could be induced with 1 mg of the conventional doubly haptenated protein carriers such as ABA-BSA-TNP. Both direct and indirect PFC were induced by this antigen in primed rats. The use of this antigen and passive transfer techniques to study ABA-specific helper activity revealed some differences from ABA-specific delayed-type hypersensitivity (DTH) and in vitro proliferation, which were studied previously. Cells responsible for helper activity appeared sooner after immunization and were found most prominently in peritoneal exudates but also significantly in spleen where the cells responsible for DTH or in vitro proliferative responses were never found. By contrast, helper cells were not seen in lymph nodes, where some proliferative activity could be found. Of these three ABA-specific T cell functions, helper activity was least easily suppressed by the previously used regimens of ABA-tyr in incomplete freunds adjuvant (IFA). Moreover, helper activity appears after injection of ABA-tyr in IFA, a method that has never in our hands yielded detectable DTH or in vitro proliferative responses. Despite these differences, phenotyping with monoclonal antibodies indicated that cells responsible for helper and proliferative activities were both W3/25+ and OX8-.     

Effect of perfluoroorganic compounds on growth of streptomycetes and biosynthesis of antibiotic daunorubicin]

The use of perfluoroorganic compounds, i.e. perfluorodecalin and "Perftoran", a blood substitute with the gas transfer function, known as "blue blood" (containing 3 vol. % of perfluoromethylcyclohexylpiperidin and 7 vol. % of perfluorodecalin as emulsion with the average particle size of 0.07 microns) in biotechnology for intensification of the Streptomyces purpurogeniscleroticus growth under submerged conditions was shown promising for the first time. An increase of the biomass output and higher yields of daunorubicin were also demonstrated.     

B-cell amplification by dibutyryl cyclic AMP in mice

Mice injected with N⁶-2′-O-dibutyryl-adenosine 3′,5′ monophosphate (DBcAMP) showed increased anti-sheep erythrocyte (anti-SE) antibody plaque-forming spleen cell (PFC) responses up to 7-fold greater than control mice. The amount of increase was related to the immunizing dose of SE and to the dose of DBcAMP, and it was more pronounced in 19S PFC than in 7S PFC. Mice thymectomized (Tx) within 16 hr after birth and injected with SE and DBcAMP at 40 days showed a 2.7-fold greater anti-SE PFC response than Tx controls injected with SE alone. DBcAMP restored the PFC response of Tx mice to 75% of that seen for sham Tx, DBcAMP-treated controls. These data suggest that a T cell-B cell interaction is not stringently required in mouse anti-SE antibody responses in vivo, since a T cell-like effect may be substituted or a minimal response can be enhanced with a soluble amplifier such as dibutyryl cyclic AMP.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Patterns of proliferation of antibody-forming cells after an intravenous immunization with hamster erythrocytes (HRBC) were compared in groups of mice possessing different activities of thymus-derived lymphocytes (T cells). 1) Marked differences in the numbers of hemolysin plaque-forming cells (PFC) after HRBC injection were found among the low- and high-responder normal mice and those pretreated with HRBC in complete Freund's adjuvant (CFA) or incomplete adjuvant (IFA), and they appeared to depend primarily upon the different rates of proliferation of antibody-forming cells rather than on the numbers of antigen-specific lymphocytes initiating the antibody response. 2) The numbers of hemolytic foci were slightly larger in mice with large numbers of PFC (normal SL mice, the pretreated SL and C57BL/6 mice) than in those with small numbers of PFC (normal C57BL/6 mice). The numbers of hemolytic foci increased at almost the same rate from day 2 to day 3 in both groups, while the numbers of PFC increased more efficiently in mice with large numbers of PFC than in those with small numbers of PFC from day 2 to day 3. Individual hemolytic foci appeared to contain larger numbers of PFC in mice with large total numbers of PFC than in those with small total numbers of PFC. 3) The numbers of rosette-forming cells (RFC) were increased by pretreatment with HRBC in CFA and by pretreatment with HRBC in IFA to almost the same extent. Rates of increases in PFC were, however, larger by pretreatment with HRBC in CFA than with HRBC in IFA. These results suggested that the activity of the T cell determined not only the rates of proliferation of antibody-forming cells but also the antibody-producing capacity of each cell.     

Catalytic properties of CYP1A isoforms in the liver of an agnathan (Lampetra fluviatilis) and two species of teleost (Pleuronectes flesus,Anguilla anguilla)

The catalytic activity of CYP1A isoforms and the effect of mammalian CYP1A-specific inhibitors in liver S9 fractions were studied in an agnathan (River lamprey, Lampetra fluviatilis, 30–33 cm) and in two species of teleost fish (European flounder, Pleuronectes flesus, 11–18 cm and common eel, Anguilla anguilla, 31–48 cm). Ethoxyresorufin O-deethylation (EROD), caffeine N-demethylation/C-oxidation and phenacetin O-deethylation (POD) activity increased 3–4-fold in flounders and 17–46-fold in eels, 5 days after fish were injected (i.p.) with 100 mg kg⁻¹ benzo(a)pyrene (B[a]P). In lampreys, basal EROD activity was very low and no increase in activity was observed following exposure to B[a]P. While the apparent Michaelis constant (K_m) for each assay showed only small changes after B[a]P injection, maximum reaction velocity (V_max) values increased by up to 19- and 84-fold for EROD activity, 4- and 35-fold for caffeine-related metabolism and 4- and 19-fold for POD activity in flounders and eels, respectively. The mammalian CYP1A2 inhibitor furafylline (50 μM–1 mM) reduced activity in the EROD, caffeine and POD assays to 65, 21 and 20% of control values in flounders and to 85, 10 and 5% of control values in eels, respectively. By contrast, low concentrations (0.025–0.050 μM) of the mammalian CYP1A1 inhibitor ellipticine completely abolished EROD activity, but had no effect (up to 1 mM) on caffeine metabolism or POD activity in either species. While the inhibitor studies strongly suggest that two separate enzymes are present in flounders and eels, the monophasic Michaelis–Menten kinetics obtained in all the assays imply that only a single CYP1A protein is present that has substrate and inhibitor specificities characteristic of both mammalian CYP1A1 and CYP1A2 isoforms.     

Differential effects of concanavalin A on T helper dependent and independent antibody responses

The in vivo immunosuppressive effects of Concanavalin A (Con A) on the thymus (T) helper dependent response to sheep erythrocytes (SRBC) and the T helper independent response to E. coli lipopolysaccharide 055: B5 have been investigated. Maximum suppression was observed in BALB/c mice treated with 3 successive ip injections of 100 μg each of Con A administered on Days ?1, 0, and +1 relative to the day of immunization (Day 0) with SRBC (splenic PFC on Day 4 reduced from 74,000 down to 1400). As little as 10 μg × 3 of Con A was capable of depressing both the PFC and serologic response while 2.5 μg × 3 was ineffective. A single ip injection of 300 μg of Con A administered simultaneously at the time of immunization with SRBC reduced splenic PFC from 74,000 down to 9990 and serum antibody titers by 3–4 log₂ units. Significant depression was noted if mice were treated 1, 2, or 3 days prior to but not following immunization. Immunosuppression was noted in mice which had been treated and immunized ip or iv or treated iv and immunized ip. Heat inactivation reduced if not abolished the immunosuppressive properties of Con A.Mice immunized with varying doses of a bacterial vaccine of E. coli 055: B5 (15–1500 × 10⁶ killed organisms) and treated with Con A on days ?1, 0, and +1 had no significant depression of splenic PFC when compared to nontreated controls. Mice treated with Con A and simultaneously immunized with both SRBC and E. coli had a 37-fold reduction in the PFC response to SRBC but only a 2-fold reduction in the response to E. coli. This differential immunosuppressive effect on T helper dependent and independent responses is consistent with the recently reported in vitro specificity which Con A has for theta antigen bearing lymphocytes.     

Effects of perfluorochemical distribution and elimination dynamics on cardiopulmonary function.

Based on a physicochemical property profile, we tested the hypothesis that different perfluorochemical (PFC) liquids may have distinct effects on intrapulmonary PFC distribution, lung function, and PFC elimination kinetics during partial liquid ventilation (PLV). Young rabbits were studied in five groups [healthy, PLV with perflubron (PFB) or with perfluorodecalin (DEC); saline lavage injury and conventional mechanical ventilation (CMV); saline lavage injury PLV with PFB or with DEC]. Arterial blood chemistry, respiratory compliance (Cr), quantitative computed tomography of PFC distribution, and PFC loss rate were assessed for 4 h. Initial distribution of PFB was more homogenous than that of DEC; over time, PFB redistributed to dependent regions whereas DEC distribution was relatively constant. PFC loss rate decreased over time in all groups, was higher with DEC than PFB, and was lower with injury. In healthy animals, arterial PO(2) (Pa(O(2))) and Cr decreased with either PFC; the decrease was greater and sustained with DEC. Lavaged animals treated with either PFC demonstrated increased Pa(O(2)), which was sustained with PFB but deteriorated with DEC. Lavaged animals treated with PFB demonstrated increased Cr, higher Pa(O(2)), and lower arterial PCO(2) than with CMV or PLV with DEC. The results indicate that 1) initial distribution and subsequent intrapulmonary redistribution of PFC are related to PFC properties; 2) PFC distribution influences PFC elimination, gas exchange, and Cr; and 3) PFC elimination, gas exchange, and Cr are influenced by PFC properties and lung condition.     

Heterogeneity of rabbit platelets. Effect of chronic blood loss on glycolytic and related enzyme activity

Rabbits were chronically bled, with Fe replacement, every 3–4 days for 21 days. Their platelet count and megathrombocyte number (large-heavy, young platelets) increased 1.7-and 2.3-fold, respectively. Eight of the 11 enzymes of the Embden-Meyerhof pathway increased 2–5-fold in activity per g wet weight during the period of blood letting. The five related enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, glutathione reductase, and α-glycerol-P dehydrogenase) as well as the three Embden-Meyerhof pathway enzymes (aldolase, enolase, and pyruvate kinase) did not increase in activity over basal values. It is concluded that chronic blood loss with Fe replacement leads to a specific increase in enzyme activity of 8 of the 11 Embden-Meyerhof pathway enzymes.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

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)