Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi     

Virulence and pathogenicity of human and environmental isolates of Cladosporium carrionii in new born ddY mice

Three strains of Cladosporium carrionii, two human isolates and one from a xerophilous plant, were used to study the effect of culture conditions in 106 newborn ddY mice. Growth in a complex medium (YPG) and a basal synthetic medium (BSM) was compared. Filamentous forms developed during static incubation while conidia were readily formed with shaking. Mice inoculated intraperitoneally were sacrified and autopsied after 4 weeks. Mortality was related only to sporulated exponential phase growing cells. Invasiveness ability was preserved in all experimental conditions. BSM medium that inhibited exopigment formation appeared more suitable than YPG to obtain intact cells for further studies.Biochemical and physiological alteration associated with shape changes during differentiation of vegetative cells into spores could play an important role in virulence of C. carrionii     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Use of pectinases complexed to colloidal gold for the ultrastructural localization of polygalacturonic acids in the cell walls of the fungus Ascocalyx abietina

Summary Three pectinase—gold complexes were used to localize polygalacturonic acids in the fungusAscocalyx abietina (Lagerberg) Schlaepfer-Bernhard. With the pectinesterase and pectin lyase—gold complexes, the labelling was uniformly distributed over the fungus walls and did not seem to be significantly influenced by the tissue preparation. With the polygalacturonase—gold complex, differences in the labelling distribution were noted according to the fixation procedure indicating, therefore, that osmication of the tissues could greatly interfere with the localization of the specific enzyme binding sites. These results demonstrate, for the first time, the possibility of detecting polygalacturonic acids by means of different gold-complexed pectinases.     

3-Hydroxyacyl-CoA epimerases of rat liver peroxisomes and Escherichia coli function as auxiliary enzymes in the beta-oxidation of polyunsaturated fatty acids

The beta-oxidation of 2-trans,4-cis-decadienoyl-CoA, an assumed metabolite of linoleic acid, by purified enzymes from mitochondria, peroxisomes, and Escherichia coli was studied. 2-trans,4-cis-Decadienoyl-CoA is an extremely poor substrate of the beta-oxidation system reconstituted from mitochondrial enzymes. The results of a kinetic evaluation lead to the conclusion that in mitochondria 2-trans,4-cis-decadienoyl-CoA is not directly beta-oxidized, but instead is reduced by NADPH-dependent 2,4-dienoyl-CoA reductase prior to its beta-oxidation. Hence, the mitochondrial beta-oxidation of 2-trans,4-cis-decadienoyl-CoA does not require 3-hydroxyacyl-CoA epimerase, a conclusion which agrees with the finding that 3-hydroxyacyl-CoA epimerase is absent from mitochondria (Chu, C.-H., and Schulz, H. (1985) FEBS Lett. 185, 129-134). However, 2-trans,4-cis-decadienoyl-CoA can be slowly oxidized by the bifunctional beta-oxidation enzyme from rat liver peroxisomes, as well as by the fatty acid oxidation complex from E. coli. The observed rates of 2-trans,4-cis-decadienoyl-CoA degradation by these two multi-functional proteins were significantly higher than the values calculated according to steady-state velocity equations derived for coupled enzyme reactions. This is attributed to the direct transfer of L-3-hydroxy-4-cis-decenoyl-CoA from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase on the same protein molecule. All observations together lead to the suggestion that the chain shortening of 2-trans,4-cis-decadienoyl-CoA in peroxisomes and in E. coli occurs simultaneously by two different pathways. The major pathway involves the NADPH-dependent 2,4-dienoyl-CoA reductase, whereas 3-hydroxyacyl-CoA epimerase functions in the metabolism of D-3-hydroxyoctanoyl-CoA which is formed via the minor pathway.     

Sorption von Cellulase an Weizenstroh und seine Komponenten

By comparing different activity data of the buffered cellulase solution before and after contact with the substrate the interaction between Penicillium janthinellum cellulase and wheat straw, resp. its components (holocellulose and isolated lignin) has been investigated. The loss of activity due to sorption or denaturation has been found to differ widely between the different activity data and between the various substrates. A remarkable loss of enzyme activity was observed after contact with isolated straw lignin. The differences in activity decrease between the cellulose and the lignin moiety were found to be largent with the cellobiase activity.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.