Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Genetics and gibberellin production inGibberella fujikuroi

Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development.The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.Abbreviations AMO 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenylpiperidine-1-carboxylate - hydroxykaurenoic acid ent-kaur-16-en-7-ol-19-oic acid - kaurenal ent-kaur-16-en-19-al - kaurene ent-kaur-16-ene - kaurenoic acid ent-kaur-16-en-19-oic acid - kaurenol ent-kaur-16-en-19-ol - paclobutrazol 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - pefurazoate pent-4-enyl-N-furfuryl-N-imidazol-1-ylcarbonyl-DL-homoa laninate - tetcyclacis 5-(4-chlorophenyl)-3,4,5,9,10-pentaazatetracyclo-5,4,10^2.6,O^8.11-dodeca-3,9-diene - triarimol -(2,4-dichlorophenyl)--phenyl-5-pyrimidine methyl alcohol     

Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.     

Overcoming seed dormancy in ex situ plant germplasm conservation programmes; an example in the endemic Argyranthemum (Asteraceae: Anthemideae) species from the Canary Islands

Germplasm of 21 diverse Argyranthemum taxa was collected from contrasting ecological zones in the Canary Islands. Seed dormancy was considerable in the majority of taxa. Extensive investigations, based on a germination test procedure algorithm for Asteraceae, with achenes from ray and disc florets of five contrasting taxa identified a procedure to promote full (85%) germination of the seeds from both ray and disc florets of all five taxa; viz, excision of the seeds from the achenes, followed by testing at 15°C with 2.6×10^-3 m GA₃ co-applied. Subsequent tests showed that this regime was effective in promoting full germination in seeds from both ray and disc florets of the remaining 16 taxa. The results are discussed in the context of ex situ plant germplasm conservation.     

Bats of the Gulf of Guinea islands: faunal composition and origins

The present study compares the bat faunas of the islands of the Gulf of Guinea. Species composition. endemism and hypothetical origins are discussed. All families present in the mainland region are found in Bioko, a typical landbridge island. Foliage gleaning guild species (Nycteridae) show limited colonization abilities. This is also true of the family Rhinolophidae, but not for the closely related family Hipposideridae. The majority of the oceanic island species are African bats which show a widespread distribution and, therefore, have a high ecological plasticity. The continental relatives of the two endemic species Myonycteris brachycephala and Chaerephon tomensis are restricted to relatively small forested areas. Bioko's bat fauna is the result of the recent isolation from a formerly land-connected community. The oceanic bat faunas originated from the establishment of incomers from other areas. Nevertheless, extinction appears in both vicariant and dispersal processes, as an important factor in modelling the current bat communities of the Gulf of Guinea islands.     

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Vertical distribution of planktonic rotifers in a karstic meromictic lake

Vertical distribution of planktonic rotifers is described in relation to temperature and oxygen in Lake La Cruz, a single-doline, closed karstic lake (121 m diameter and 25 m maximum depth) which shows iron meromixis. Samples were taken by peristaltic pumping at 10 cm depth intervals in the oxycline zone from June 1987 to September 1988. A model of rotifer vertical structure in stratified lakes is proposed. Rotifers concentrate their populations at the depths with intense gradients. As stratification develops some rotifer populations show a downward migration following the thermocline and some others show an upward migration following the oxycline. The production-respiration balance in the lake, and so the position of the oxycline with respect to the thermocline and the layer of maximum production, depends on meteorological conditions. A shift in the dominance of congeneric or related species can occur in consecutive years. In Lake La Cruz, mixing conditions and subterranean inflow in spring were much more intense in 1988 than 1987, and the distance between production and decomposition depths was smaller in 1988. Anuraeopsis miraclei, an oxycline-bound species with high abundance in 1987, was displaced by A. fissa in 1988. A. fissa, which was a metalimnetic species during early summer, reached peak densities (3 × 10⁴ ind l^–1) at the oxycline, equaling the abundance of A. miraclei the preceeding year.     

Kinetic study of the cytotoxic effect of α-sarcin,a ribosome inactivating protein fromAspergillus giganteus,on tumour cell lines: protein biosynthesis inhibition and cell binding

-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. -Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by -sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with -sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by -sarcin reveals an -sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external -sarcin concentration. A saturable component for the action of -sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of -sarcin.     

Environmental,geographical and time-related impacts on avian malaria infections in native and introduced populations of house sparrows (Passer domesticus), a globally invasive species

Aim

The increasing spread of vector-borne diseases has resulted in severe health concerns for humans, domestic animals and wildlife, with changes in land use and the introduction of invasive species being among the main possible causes for this increase. We explored several ecological drivers potentially affecting the local prevalence and richness of avian malaria parasite lineages in native and introduced house sparrows (Passer domesticus) populations.

Location

Global.

Time period

2002–2019.

Major taxa studied

Avian Plasmodium parasites in house sparrows.

Methods

We analysed data from 2,220 samples from 69 localities across all continents, except Antarctica. The influence of environment (urbanization index and human density), geography (altitude, latitude, hemisphere) and time (bird breeding season and years since introduction) were analysed using generalized additive mixed models (GAMMs) and random forests.

Results

Overall, 670 sparrows (30.2%) were infected with 22 Plasmodium lineages. In native populations, parasite prevalence was positively related to urbanization index, with the highest prevalence values in areas with intermediate urbanization levels. Likewise, in introduced populations, prevalence was positively associated with urbanization index; however, higher infection occurred in areas with either extreme high or low levels of urbanization. In introduced populations, the number of parasite lineages increased with altitude and with the years elapsed since the establishment of sparrows in a new locality. Here, after a decline in the number of parasite lineages in the first 30 years, an increase from 40 years onwards was detected.

Main conclusions

Urbanization was related to parasite prevalence in both native and introduced bird populations. In invaded areas, altitude and time since bird introduction were related to the number of Plasmodium lineages found to be infecting sparrows.