Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

A cladistic analysis of Nassauvia Comm. ex Juss. (Asteraceae, Mutisieae) and related genera

FREIRE, S. E., CRISCI, J. V. & KATINAS, L., 1993. A cladistic analysis of Nassauvia Comm. ex Juss. (Asteraceae, Mutisieae) and related genera. Nassauvia and the most closely related genera Calopappus and Triptilion from the southern Andes and Patagonia of South America, form a monophyletic group diagnosed by the following synapomorphies: cypsela trichomes single two-celled, cypsela testa with strengthened cells, pollen grains spheroidal to spheroidal-oblate, colpi membrane with sexine processes, pappus bristles two to six, and pappus deciduous. Furthermore, Nassauvia, Triptilion, and Calopappus form a group with two other Andean genera, Moscharia and Polyachyrus, diagnosed by occurrence of pseudocephalia and a reduction in the number of flowers to five, three or one. A cladistic analysis of the group was undertaken using 35 characters from morphology, anatomy, and palynology. The monophyletic terminal taxa were the 38 species of Nassauvia, the genus Triptilion, the monotypic genus Calopappus, the genus Polyachyrus, and the genus Moscharia. Character polarity was based on outgroup comparison using Cephalopappus. The analysis resulted in 223 equally parsimonious cladograms, each with 70 steps and a consistency index of 0.57. A successive weighting procedure was applied, resulting in 15 cladograms with a consistency index of 0.82. Results of the cladistic analysis support most of the current systematic classification of Nassauvia, with three exceptions: (1) Nassauvia (excluding Calopappus) is paraphyletic; (2) section Masligophorus appears to be a polyphyletic group (N. pygmaea does not cluster with the remaining species of the section); (3) section Panargyrum (without N. lagascae= section Caloptilium) appears to be a paraphyletic group. The capitula arranged in cymose conflorescences in Triptilion are regarded as a primitive condition which gave rise to all stages present in Nassauvia (conflorescence spicate, pseudocephalium, capitula solitary). The capitula arranged in pseudocephalia in Moscharia and Polyachyrus are regarded as a parallel development to the pseudocephalium found in Nassauvia. Nassauvia, subgenus Strongyloma appears as the most primitive taxon, with its spicate conflorescence, whereas section Masligophorus with its solitary capitulum is thought-derived. These results correspond well with cytological data where species of the subgenus Strongyloma have n= 11 and the species of section Masligophorus are tetraploids (n = 22).     

Diatom assemblages in superficial sediments from the Gulf of Riga,eastern Baltic Sea

Twenty one superficial sediment samples from areas of high and moderate eutrophication in the Gulf of Riga were studied with respect to siliceous microfossils, mainly diatoms.The results seem to imply that the number of taxa and the abundance of the frustules are affected by runoff from rivers and the degree of eutrophication in different parts of the Gulf. Areas with high eutrophication, e.g. the river estuaries, have diatom assemblages of varying composition, while in areas with moderate eutrophication the composition is almost constant and the influx of freshwater diatoms and littoral periphyton small. The high abundance and low diversity of brackish-marine and brackish, planktonic diatoms seem to be a result of an influx of nutrients and pollutants in the water and bottom sediments. The sea ice diatoms occurring in the Baltic waters and also in the Gulf of Riga tend to be resistant to eutrophication or are even favoured by it.     

Purification and characterization of catalase from goat (Capra capra) lung

Catalase plays a major role in the protection of tissues from toxic effects of H₂O₂ and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127±2 Å. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with K_i values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.     

Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH₂-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4 n-octyl tetraoxyethylene - M_r apparent molecular weight - Octyl-POE n-octyl polyoxyethylene - LDAO N,N-dimethyl dodecyl aminoxide - LPS lipopolysaccharide - PAGE polyacrylamide gel-electrophoresis - PEG polyethylene glycol     

Behaviour of suspended and sedimentary organic matter in the deltaic areas of the Gulf of Lions (Mediterranean Sea)

The behaviour of organic matter has been approached in two fluvio-marine areas of the Gulf of Lions in the Northwestern Mediterranean Sea: deltas of the Rhône and Têt rivers. Elemental analysis, pyrolysis-gas chromatography-mass spectrometry (main classes of organic compounds) and high performance liquid chromatography (phenolic compounds) enabled to show the fractioning processes and the differentiation of water layers from the river mouth to the open sea. Transitory horizontal and vertical geochemical gradients appeared in the water column. They were more marked in surficial sediments. They underlined the effects of the river inputs which were characterized by high phenol contents. The increase of the marine character towards the open sea was shown by increasing nitrogen-containing compounds. In the Rhône delta, the distribution of the suspended material and organic compounds emphasized the occurrence of a multi-layered water system. In the surficial layers the suspended organic matter was in a little degraded state. On the contrary, the bottom nepheloid contained altered organo-mineral complexes enriched in resistant organic compounds. In the deeper areas of the Lacaze-Duthiers Canyon, in front of the Têt prodelta, the abundance of phytoplanktonic material in the euphotic zone was emphasized by the increase of both nitrogen-containing compounds and aminosugars. In the deeper layers, the suspended material transported by advective currents showed a detrital characteri.e. enriched in clays and in degraded organic matter. The observations carried out in the Rhône delta, the Têt prodelta and the Lacaze-Duthiers Canyon underlined the importance of the general transfer system of suspended material from East to West enhanced by the Liguro-Provençal current.