The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human liver cytosolic epoxide hydrolases

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.     

Synthesis of dinucleoside tetraphosphates by RNA polymerase B (II) from calf thymus

Highly purified RNA polymerase B (II) from calf thymus catalyses the synthesis of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires a DNA template and bivalent cations such as Mn2+ or Mg2+. It is strongly inhibited by heparin and high concentrations of alpha-amanitin but not by rifampicin. On a given template various dinucleoside tetraphosphates of different sequence are formed although the yield depends on the nature of the template.     

Chromatographic studies on the radiolysis of DNA in aqueous solution

Radiation-induced degradation of double-stranded DNA from calf thymus in aqueous solution with sodium phosphate was studied by conventional gel chromatography and by high-performance liquid-gel permeation chromatography. Comparison of the data after radiolysis of aqueous solutions of DNA under anaerobic and aerobic conditions indicates that double-strand breakage is not enhanced by oxygen. An increase of ionic strength impedes the break-down of the DNA molecules, so that loss of DNA can only be observed at doses above 100 Gy. Only reactions of OH-radicals contribute to the fragmentation of DNA, while the presence of hydrated electrons, H.-or formate radicals does not lead to a loss of highly polymerized DNA up to doses of 1500 Gy. High-performance liquid-chromatography proved to be an excellent method of studying the degradation of macromolecules as a function of dose.     

Role of serum and urinary Tamm-Horsfall protein in the surveillance of kidney transplants

The study of 24-h urinary excretion proves to be quite interesting from a theoretical point of view on account of its topographic origin in the nephron and from a practical point of view to control renal transplant patients with favourable or unfavourable course. In both cases, the results we obtain are in accordance with that of blood creatinine assay and ratio N-acetyl-beta-D-glucosaminidase (NAG)/creatinine in urine. The prolonged study of serum THG concentration confirms the previous data regarding the outcome of renal grafts. Moreover, particularly low concentration rates probably imply the interference of factors such as: renal toxins or anti-THG autoantibodies.     

Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.