Analysis of mammalian viable cell biomass based on cellular ATP.

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture.

Mammalian cell culture processes.

Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments.

Effect of substrate organization on the activity and on the mechanism of gentamicin-induced inhibition of rat liver lysosomal phospholipase A1.

Aminoglycoside antibiotics, such as gentamicin, induce a lysosomal phospholipidosis in the kidney cortex of experimental animals and humans. In vitro, gentamicin binds to negatively charged phospholipids, such as phosphatidylinositol, and decreases the activity of lysosomal phospholipases towards a neutral phospholipid (phosphatidylcholine) included in lipid vesicles. The mechanism of such an inhibition was not unequivocally established.

Model of oxygen transport limitations in hollow fiber bioreactors.

Axial and radial oxygen depletion are believed to be critical scale-limiting factors in the design of cell culture hollow fiber bioreactors. A mathematical analysis of oxygen depletion has been performed in order to develop effectiveness factor plots to aid in the scaling of hollow fiber bioreactors with cells immobilized in the shell-side. Considerations of the lumen mass transport resistances and the axial gradients were added to previous analyses of this immobilization geometry.

Mammalian cell and protein distributions in ultrafiltration hollow fiber bioreactors.

The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 x 10(4) D cutoff).

Functional expression of 8-hydroxy-5-deazaflavin-dependent DNA photolyase from Anacystis nidulans in Streptomyces coelicolor.

The gene encoding Anacystis nidulans 5-deazaflavin-dependent photolyase (phr) was inserted into the Streptomyces vector pIJ385 to form a transcriptional fusion with the neomycin resistance (aph) gene. The resulting plasmid, pANPL, was introduced into Streptomyces coelicolor, a host which exhibits no detectable photolyase activity and provides 5-deazaflavins. Transformants expressed functional photolyase and could be cultured at much higher cell densities than A.

Effect of acidic phospholipids on the activity of lysosomal phospholipases and on their inhibition induced by aminoglycoside antibiotics--II. Conformational analysis.

In a companion paper (Mingeot-Leclercq et al. Biochem Pharmacol 40: 489-497, 1990), we showed that the inhibitory potency of gentamicin on the activity of lysosomal phospholipases, measured towards phosphatidylcholine included in negatively-charged liposomes, is markedly influenced by the nature of the acidic phospholipid used (phosphatidylinositol, phosphatidylserine, phosphatidic acid), whereas the binding of the drug to the three types of liposomes is similar. This result challenged previous conclusions pointing to a key role exerted by drug binding to phospholipid membranes and presumably charge neutralization, for phospholipases inhibition (Carlier et al.

Effect of acidic phospholipids on the activity of lysosomal phospholipases and on their inhibition by aminoglycoside antibiotics--I. Biochemical analysis.

Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al.

The Streptomyces coelicolor A3(2) bldB region contains at least two genes involved in morphological development.

Streptomyces coelicolor A3(2) bldB mutants are blocked in the formation of aerial hyphae. A phage library of wild-type S. coelicolor DNA was used to isolate recombinant phages which restore wild-type morphological development to several bldB mutants.

Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585.

We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants.

Immobilized mammalian cell cultivation in hollow fiber bioreactors.

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs.

Heterogeneous genomic amplification in Streptomyces glaucescens: structure, location and junction sequence analysis.

Certain chromosomal markers in Streptomyces glaucescens behave unstably, being lost at high frequency as a result of extensive genomic deletion. Additionally, mutant strains possessing such deletions frequently display intense DNA amplification. With the help of a wild-type cosmid library we investigated the structure of the amplified DNA sequences (ADS) and the corresponding wild-type amplifiable units of DNA (AUD).

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions.

Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens.

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development.

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule.

Sporulation and spore properties of Bacillus brevis and its gramicidin S-negative mutant.

The function(s) of the peptide antibiotic, gramicidin S, in its producer, Bacillus brevis Nagano, was investigated. Particular attention was paid to the possible role of gramicidin S in sporulation and spore properties. Sporulation was similar in both the gramicidin S-producing parental strain and a gramicidin S-negative mutant of this strain.

Germination initiation and outgrowth of spores of Bacillus brevis strain Nagano and its gramicidin S-negative mutant.

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth or L-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments.

Potential causes of spurious programming: report of a case.

The possibility of false or inadvertent reprogramming increases with the availability of different and more complex programming mechanisms. A case of spurious programming during application of the Vitatron MPA1 analyzer to a Medtronic impulse generator (IPG) is described and the nature of the phenomenon is explained. It is concluded that the introduction of a simple and standardized "security-maneuvre" to which every IPG will respond safely, in the event of fortuitous dysprogramming, is required.