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     

The AXB and BXA set of recombinant inbred mouse strains

The recombinant inbred (RI) set of strains, AXB and BXA, derived from C57BL/6J and A/J, originally constructed and maintained at the University of California/San Diego, have been imported into The Jackson Laboratory and are now in the 29th to 59th generation of brother-sister matings. Genetic quality control testing with 45 proviral and 11 biochemical markers previously typed in this RI set indicated that five strains had been genetically contaminated sometime in the past, so these strains have been discarded. The correct and complete strain distribution patterns for 56 genetic markers are reported for the remaining RI strain set, which consists of 31 living strains and 8 extinct strains for which DNA is available. Two additional strains, AXB 12 and BXA 17, are living and may be added to the set pending further tests of genetic purity. The progenitors of this RI set differ in susceptibility to 27 infectious diseases as well as atherosclerosis, obesity, diabetes, cancer, cleft palate, and hydrocephalus. Thus, the AXB and BXA set of RI strains will be useful in the genetic analysis of several complex diseases.     

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.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.