Insect cells respiratory activity in bioreactor

Specific respiration rate ( ) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure . Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 10⁶ cells/mL and maximum specific respiration rate () of 7.3 × 10⁻¹⁷ molO₂/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 10⁶ cells/mL and of 3.1 × 10^–18 molO₂/cell s. S2AcGPV (expressing with rabies virus glycoprotein) reached 24.9 × 10⁶ cells/mL and of 1.7 × 10^–17 molO₂/cell s, while S2MtEGFP (expressing green fluorescent protein) achieved 15.5 × 10⁶ cells/mL and  = 1.9 × 10⁻¹⁷ molO₂/cell s. Relating to the Sf9, S2 cells reached higher maximum cell concentrations and lower specific respiration rate, which can be explained by its smaller size. These results presented useful information for scale-up and process control of insect cells.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Structure of an abequose-containing O-polysaccharide from Citrobacter freundii O22 strain PCM 1555

The lipopolysaccharide of Citrobacter freundii O22 (strain PCM 1555) was degraded under mild acidic conditions and the O-polysaccharide released was isolated by gel chromatography. Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H ROESY and ¹H,¹³C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure:

Full-size image (4K)

where Abe is abequose (3,6-dideoxy-d-xylo-hexose). SDS–PAGE and immunoblotting revealed that the O-antigen of C. freundii O22 is serologically indistinguishable from those of Salmonella group B serovars (Typhimurium, Brandenburg, Sandiego, Paratyphi B) but not related to other abequose-containing O-antigens tested (Citrobacter werkmanii O38 and Salmonella Kentucky) or colitose (l enantiomer of abequose)-containing O-antigen of Escherichia coli O111.     

The ganglioside GD1α, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer,is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2

The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7×10¹¹ cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the G_M1a-pathway (G_M2, G_M1a and G_D1a) and G_M1b (IV³Neu5Ac-GgOse₄Cer) and GalNAc-G_M1b of the G_M1b-pathway, the dis8aloganglioside G_D1 (IV³Neu5Ac, III⁶Neu5Ac-GgOse₄Cer) was found in equal amounts compared to G_D1a (IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer). All gangliosides were substituted with C_24:0,24:1 and C_16:0 fatty acids, sphingosine andN-acetylneuraminic acid as the sole sialic acid. Abbreviations: FAB-MS, fast atom bombardment-mass spectrometry; GC-MS, gas chromatography-mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycoloylneuraminic acid [57]. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [58] and the nomenclature of Svennerholm [59]. Gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer gangliopentaosylceramide or GgOse₅Cer, GalNAc1-4Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1a, II³Neu5Ac-GgOse₄Cer; G_M1b, IV³Neu5Ac-GgOse₄Cer; GalNAc-G_M1b, IV³Neu5Ac-GgOse₅Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_D1 or G_D1e, IV³Neu5Ac, III⁶Neu5AcGgOse₄Cer; G_D1e, IV³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer.     

AgNO3 increases type II callus production from immature embryos of maize inbred B73 and its derivatives

Incorporating 10 to 100 M AgNO₃ into Phytagel (0.2%) solidified N6 medium containing 1 mg/L 2,4-D, 100 mg/L casamino acids and 25 mM praline (N6 1-100-25) promoted type II callus production from cultured Zea mays L. immature embryos of FRB73, B73 X A188 and a proprietary B73 BC6 genotype. Under these conditions, approximately 15, 80 and 80% of the respective FRB73, B73 X A188 and B73 BC6 explants produced type II calli after 2 to 3 weeks incubation in the dark at 28 C. In the absence of AgNO₃, the type II culture response from B73BC6 immature embryos was 25% on N6 1 100-25 solidified with Phytagel (0.2%) as compared to 0% for that solidified with 0.8% agar. Duncan's medium was tested using 10 to 100 m AgNO₃ and generally promoted type I callus initiation, although up to 6% of the explants produced type II cultures in the presence of 0.2% Phytagel. Ethylene emanation rates of up to 370 and 115 nL g-1 h-1 were detected from B73 X A188 immature embryos and calli, respectively, cultured on N6 1-100-25.     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Probing protein structural requirements for formation of the core light-harvesting complex of photosynthetic bacteria using hybrid reconstitution methodology

The - and -polypeptides of LH1 isolated from four different photosynthetic bacteria (Rhodospirillum rubrum, Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas viridis) were used for homologous and hybrid reconstitution experiments with bacteriochlorophyll a. Formation of B820-type subunit complexes and LH1-type complexes were evaluated. The -polypeptides of R. rubrum, Rb. sphaeroides and Rb. capsulatus behaved similarly and formed B820-type subunit complexes in the absence of an -polypeptide. The - and -polypeptides were both required to form a LH1-type complex with each of these three homologous systems. In hybrid experiments where the -polypeptides were tested for reconstitution with -polypeptides other than their homologous partners, half of the twelve possible combinations resulted in formation of both B820- and LH1-type complexes. Three of the combinations that did not result in formation of a LH1-type complex involved the -polypeptide of R. rubrum. It is suggested that these latter results can be explained by charge repulsion between the Lys at position-17 (assigning the conserved His located nearest to the C-terminus as position 0) in the -polypeptide of R. rubrum and each of the heterologous -polypeptides tested, all of which have an Arg at this location. Conclusions that can be derived from these experimental results include: (1) the experimental data support the idea that a central core region of approximately 40 amino acids exists in each of the polypeptides, which contains sufficient information to allow formation of both the B820- and LH1-type complexes and (2) a specific portion of the N-terminal hydrophilic region of each polypeptide was found in which ion pairs between oppositely charged groups on the - and -polypeptides seem to stabilize complex formation.Abbreviations BChl a bacteriochlorophyll a - BChl BChl a is implied - BChl a _P BChl a containing phytol as the esterifying alcohol - BChl a _gg BChl a containing geranylgeraniol as the esterifying alcohol - LH1 the core light-harvesting complex - B873 the core light-harvesting complex of the G-9 mutant (carotenoidless) of R. rubrum or of the wild-type light-harvesting complex after benzene extraction (both with absorption maxima at 873 nm) - B820 the subunit form of B873 consisting of native - and -polypeptides with the same stoichiometry of ₁₁·2BChl as LH1 - B820-type complex a complex exhibiting absorption and CD spectra indistinguishable from B820 but composed of either the -polypeptide only, or of a heterologous mixture of - and -polypeptides - RC reaction center - PRC photoreceptor complex consisting of the RC and LH1 - CD circular dichroism - OG n-octyl -d-glucopyranoside - HFA hexafluoroacetone trihydrate     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)     

Comparison of three granular support materials for anaerobic fluidized bed systems

Summary Three different materials, kaolin, pozzolana and biolite (a material used in a commercial anaerobic fluidized bed treatment process) when tested as supports for an anaerobic fluidized bed system had similar physical and fluidization properties but behaved differently towards the biomass hold-up. However, all three systems attained similar removal efficiency rates.Nomenclature U Fluidization velocity (m/s) - U₁ Terminal fluidization velocity (m/s) - g Local acceleration due to gravity (m/s²) - _s Solid density (kg/m³) - _f Fluid density (kg/m³) - P Pressure drop (Pa) - HRT Hydraulic retention time (days) - H_mf Height of bed at minimum fluidization (m) - H Height of bed (m) - C_d Drag coefficient (dimensionless) - W Mass of solids in bed (kg) - dp Particle diameter (m) - A Cross-sectional area of column (m²) - h column height (m) - Rc_t Terminal Reynolds no. - Voidagc (fractional free volume, dimensionless) - _mf Voidage (fractional free volume) at minimum of fluidization (dimensionless)     

Host-determined T cell fine specificity for self-H-2 in radiation bone-marrow chimeras of standard C57BL/6 (H-2b) mutantHz1 (H-2ba), and F1 mice

Reciprocal radiation bone-marrow chimeras were produced between the standard C57BL/6 (=B6) and the mutant B6.C-H-2 ^ba (=Hz1) strain. When infected with vaccinia virus, these chimeras, as well as an (Hz1 × B6)= Hz1 chimera, produced cytotoxic cells that killed vaccinia-infected H-2K^kH-2D^b target cells but failed to kill virus-infected H-2K^bH-2D^d cells. Virus-infected (Hz1 × B6)F₁ B6 chimeras, however, killed both types of target. These experiments demonstrate strict T-cell specificity capable of differentiating between two molecules that apparently differ by a single amino acid substitution.     

Structure and expression of glycoproteins controlled by the Qa-1 a allele

The alloantigen controlled by the Qa-1 ^a allele is a glycoprotein that exists in two forms. The first, an intracellular molecule of apparent M_r of 44 000 daltons, appears to be a kinetic precursor of the second, a cell-surface molecule with an apparent size of 47 000 daltons. The intracellular form of Qa-1 is distinct from that of the TL glycoprotein in two ways: (1) its polypeptide backbone is approximately 5000 daltons shorter, and (2) it possesses three sites of high-mannose carbohydrate attachment, while TL has only one. In the cell-surface form of Qa-1, all three carbohydrate chains are processed to structures that resist endoglycosidase H digestion, presumably complex-type oligosaccharides. Concomitant with these late carbohydrate-processing steps is the formation of stable complexes between Qa-1 and ₂-microglobulin. The timing of this association provides a further contrast between Qa-1 and TL, which is associated with ₂-microglobulin shortly after its synthesis. The Qa-1 glycoproteins have been identified genetically by their synthesis in B6-TL⁺ (Qa-1 ^a /Tla ^a ) splenocytes but not in splenocytes of congenic 136K1 and B6.K2 (Qa-I ^b /Tla ^b ) mice, and by their absence from the products of BALB/c (Qa-I ^vb /Tla ^c ) splenocytes. The cells synthesizing Qa-1 are at least as prevalent in Ig⁺ spleen-cell populations as in T-cell-enriched splenic Ig^– populations. Thus, active Qa-1 synthesis appears to take place at a high rate in normal splenic B cells without mitogenic stimulation.Abbreviations used in this paper EDTA disodium ethylenediaminetetraacetate - Endo H endo--N-acetylglucosaminidase H - FCS heat-inactivated fetal bovine serum - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Ig immunoglobulin - PBS Dulbecco's phosphate-buffered saline - SDS sodium dodecyl sulfate - TL thymus leukemia antigen     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

Ventilation and oxygen consumption inAmazona viridigenalis

Summary The BMR (6.00 ml O₂·min^–1) and thermal conductance (0.235 ml O₂·min^–1·°C^–1) ofAmazona viridigenalis, a medium sized parrot, are close to allometrically predicted values for nonpasserine birds, but theT _1c of 26.5 °C is 8.5 °C higher than predicted (Fig. 1). Minimal respiratory frequencies measured in four species of birds average 60% of the rate predicted by a previous equation and yield the relationship, breaths·min^–1= 10.3 kg^–0.32. Frequencies are very dependent upon the methods used to obtain the data (Fig. 3). Resting values of respiratory parameters are poorly defined in the existing literature, and there are no single resting values within the TNZ analogous to a BMR. Rather values change within, as well as below and above, the TNZ. Minimal values of different parameters occur at differentT _a's, not necessarily within the TNZ (Figs. 2, 4, 5). For clarity, resting respiratory parameters should be reported as standard values, analogous to standard metabolic rates, withT _a specified. In birds the pattern of ventilation (f andV _T) changes asT _a changes resulting in different extraction efficiencies at a given minute volume (Figs. 6, 7). This facilitates adjustment to both changing oxygen demands and changing thermoregulatory needs.Abbreviations and symbols BMR basal metabolic rate - TNZ thermoneutral zone - T _a ambient temperature - SMR standard metabolic rate - R.H. relative humidity - f respiratory frequency - br breath - T _b body temperature - T _lc lower critical temperature - Tuc upper critical temperature - T _Rlc respiratory lower critical temperature - RQ respiratory quotient - extraction efficiency - V _T tidal volume - minute volume (=V _T xf)     

Modes of proton translocation across the cell membrane of respiring cyanobacteria

Acidification of weakly buffered suspensions of the cyanobacteria Anacystis nidulans, Nostoc sp. strain MAC, Dermocarpa sp. and Anabaena variabilis was observed after the application of oxygen pulses to anaerobic cells. The acidification was caused by proton extrusion from the oxygen pulsed cells since it was eliminated by the uncoupler (H⁺ ionophore) carbonyl cyanide m-chlorophenylhydrazone. Results with the inhibitors dicyclohexylcarbodiimide or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, orthovanadate and cyanide indicated the association of various fractions of the observed proton extrusion with different activities of the cell membrane, viz. a H⁺-translocating reversible F₀F₁-ATPase, a unidirectional H⁺-translocating ATP hydrolase, and a respiratory electron transport system, respectively. Further parameters investigated were the pH dependence and the H⁺/O stoichiometry of the H⁺ extrusion from oxygen pulsed cyanobacteria. H⁺/O ratios at neutral pH were between 4 (Anacystis nidulans) and 0.3 (Dermocarpa) with uninhibited, actively phosphorylating cells and between 2 (Anacystis nidulans) and 0.4 (Dermocarpa) with ATPase-inhibited (ATP-depleted) cells, respectively. It is significant that with all four cyanobacteria tested a major fraction of the observed H⁺ ejection remained unaffected by ATPase inhibitors even at concentration which completely abolished all oxidative phosphorylation. Vanadate had a major effect on the H⁺ extrusion from Anabaena only. From this it is concluded that in the cyanobacterial species investigated part of the H⁺ extrusion from oxygen pulsed cells is directly linked to some H⁺-translocating respiratory electron transport chain present in the cell membrane.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N, N-dicyclohexylcarbodiimide - DCMU N-(3,4-dichlorophenyl-)N,N-dimethylurea - NBD 7-chloro-4-nitrobenzoxa-1,3-diazole - TPP⁺ tetraphenylphosphonium - Mes 2-(N-morpholino)ethanesulfonic acid - Pipes piperazine-N,N-bis-(2-ethanesulfonic acid) - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Taps tris (hydroxymethyl)-methyl-aminopropanesulfonic acid - Ches 2-(N-cyclohexylamino)-ethanesulfonic acid - Caps 3-cyclohexylamino)-1-propanesulfonic acid; according to most textbooks (e.g. Nicholls 1982) the terms proton electrochemical potential ( ) and protonmotive force (pmf, p), both of which equivalently describe the energetic state of energy-transducing membranes, were used synonymously and expressed in mV units throughout this article (however, cf. Lowe and Jones 1984) Dedicated to Prof. G. Drews on the occasion of his 60th birthday     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The ecology of Lake Nakuru (Kenya)

Summary Consumer biomass and spatial distribution in the equatorial alkaline-saline Lake Nakuru were studied from 1972–1976. These data will provide the basis for estimating feeding and production rates and for quantifying energy flow at the consumer level. Two of the main consumers, the Lesser Flamingo (Phoeniconaias minor) and the Soda Tilapia (Sarotherodon alcalicus grahami), were covered by earlier papers. The biomass of the only planktonic crustacean, the copepod Lovenula (=Paradiaptomus) africana was very high (1.5 gDW (dry weight)·m^-3, mean in 1972/73) in comparison with other tropical lakes. Lovenula was absent in 1974 and 1975, and at very low levels (0.1–0.4 gDW·m^-3) in part of 1976. Among the rotifers Brachionus dimidiatus dominated in 1972/73 (0.2 gDW· m^-3), but was outnumbered by B. plicatilis throughout most of 1974 to 1976 (mean total rotifer biomass 1.4 gDW· m^-3, peak densities 7 gDW·m^-3); during high salinity periods (>20) Hexarthra jenkinae occurred in very low numbers. For short periods rotifers can be the dominant species of L. Nakuru. Aquatic heteroptera (four species) played a minor role: they contributed 0.4% to total consumer biomass in 1972/73; in 1974–1976 the lake had no aquatic heteroptera. Benthic biomass (0.4 gDW·m^-2) was within the range of other tropical lakes, it consisted almost exclusively of Leptochironomus deribae. Bird counts of the twelve most important species are given for the years 1972–1974: Pelecanus onocrotalus accounts for 90% of the biomass (0.44 gDW·m^-3, mean 1972/73) with peak densities of almost 20,000 birds.-The consumer organisms covered by this and the two preceding papers represent >99% of L. Nakuru's consumer biomass. Population dynamics of various consumer species are discussed.     

The ecology of Lake Nakuru (Kenya)

Summary Abiotic factors, standing crop and photosynthetic production were studied in the equatorial alkaline-saline closed-basin Lake Nakuru (cond. 10,000–160,000 S). Meteorological conditions and abiotic factors offer suppositions for a high primary productivity: mean solar radiation is 450–550 kerg·cm^-2·s^-1, with little seasonal variation, regular winds circulate the lake every day and nutrient concentrations are usually high (>100 g P–PO₄·l^-1). Oxygen concentrations near sediments were <1 gO₂·m^-3 for at least 6 h·d^-1 in 1972/73, resulting in a release of 45 mg P–PO₄·m^-2·d^-1. Attenuation coefficients vary from 3.6–16.5 according to algal densities and mean depth from 0–400 cm. Algal biomass was 200 g·m^-3 (d.w.) in 1972/73, due to a lasting Spirulina platensis bloom (98.5% of algal biomass). In 1974 algal biomass suddenly dropped to 50 g·m^-3 (d.w.). Spirulina and several consumer organisms almost vanished, but coccoid cyanobacteria, Anabaenopsis and diatoms increased. Several causes for this change in ecosystem structure are discussed. The use of the light/dark bottle method to measure photosynthetic production in eutrophic alkaline lakes is discussed and relevant experiments were done. Oxygen tensions of 2–35 gO₂·m^-3 do not influence primary production rates. Net photosynthetic rates (mgO₂·m^-3·h^-1; photosynthetic quotient=1.18) reached 12–17.7 in 1972/73 and 2–3 in 1974, but vertically integrated rates were only 1–1.4 in 1972/73 and 0.8 in 1974, and daily net photosynthetic rates (gO₂·m^-3·24 h^-1) 3.5 in 1972/73 and 1 in 1974. 50% of areal rates were produced within the 10 most productive cm of the depth profile. The disproportion between high algal standing crops and relatively low production rates is due to self-shading of the algae, reducing the euphotic zone to 35 cm in 1972/73 and 77 cm in 1974. Efficiency of light utilization is 0.4–2%, varying with time of day and phytoplankton density. In situ efficiencies show an inverse relationship to light intensities. Photosynthetic rates of L. Nakuru remain within the range of other African lakes (0.1–3 gO₂·m^-2·h^-1). The relation of O₂ produced/Chl a of the euphotic zone is 50% lower then in tropical African freshwater lakes and conforms to lakes of temperate regions.     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium

A gene coding for endo--1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the endo--1,3-1,4-glucanase. The enzyme was produced during active growth with maximum activity. The B. subtilis (pLLS920) enzyme was 83 times (8522 mU ml^–1) more active than that of the gene donor cells (103 mU ml^–1). The B. megaterium (pLLS920) enzyme was 7 times (735 mU ml^–1) more active than that of the gene donor cells. While E. coli secreted only about 10% of the produced enzyme, B. subtilis excreted the enzyme completely into the medium and B. megaterium by about 98%. The plasmid pLLS920 was stable in B. megaterium (98%), and in B. subtilis (51%) but not in E. coli (29%).