Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures.

Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses. They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems.     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.     

Tubulin structure and biochemistry.

In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands--both microtubule-associated proteins and anti-mitotic drugs--are becoming clearer at the molecular level.     

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.     

Mechanism of action of levonorgestrel: in vitro metabolism and specific interactions with steroid receptors in target organs.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestional and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [³H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [³H]5-dihydro LNG (5-LNG) and [³H]3-5-tetrahydro LNG (3,5-LNG) were identified as the major metabolic conversion products, while [³H]3ß, 5-LNG formation occured to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5-LNG exhibited a diminished though significant interactions with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.     

Reactive synaptogenesis following degeneration of photoreceptor terminals in the fly's optic lobe: a quantitative electron microscopic study.

Following photo-ablation of receptor cells in the retina of the housefly's compound eye, their synaptic terminals degenerate with a timecourse which we have followed over 8 d. Degeneration deprives the monopolar interneurons in the first optic neuropile, the lamina, of their main synaptic input. Simultaneously it deprives one monopolar interneuron (L2) of one of its synaptic targets, as L2 makes numerous feedback synaptic contacts at which it is pre-synaptic upon receptor terminals. Because the feedback synapses are dyadic, input still remains available to the second element post-synaptic at the dyad, which does not degenerate. This element is T1, a higher-order interneuron from the next most proximal neuropile (the medulla). Some of the original feedback synaptic sites soon disappear as a consequence of the photo-ablation, but their loss is partly offset by the production of new synaptic contacts. The new pre-synaptic ribbons resemble those at the original sites except for being smaller. The sites are, moreover, monadic, with T1 now the sole post-synaptic partner. These results show that interneurons in the fly's lamina retain a dynamic capacity for synaptogenesis throughout much of adult life, normally a few weeks in Musca, and that during this synaptogenesis they re-enact the same cell preferences expressed earlier in development.