In vivo airway surface liquid Cl- analysis with solid-state electrodes.

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl-] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl-] in the "thin" ASL (approximately 30 microm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl-] in vivo. In initial experiments, the electrode measured [Cl-] in physiologic salt solutions, small volume (7.6 microl) test solutions, and in in vitro cell culture models, with > or =93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl- measurements were made in the following airway regions and species. First, ASL [Cl-] was measured in normal human nasal cavity and averaged 117.3 +/- 11.2 mM (n = 6). Second, ASL [Cl-] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 +/- 1.8 mM; (n = 6) and 126.9 +/- 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 +/- 4.2 mM (n = 13); and monkey bronchus = 112.3 +/- 10.9 mM (n = 3). Third, Cl- measurements were made in small (1-2 mm) diameter airways of the rabbit (108.3 +/- 7.1 mM, n = 5) and monkey (128.5 +/- 6.8 mM, n = 3). The measured [Cl-], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

A device for the semiautomatic determination of oxygen-radical absorbance capacity

The oxygen-radical absorbance capacity (ORAC) assay has become a standard method to quantify the antioxidative properties of phytonutrients in fruit and vegetable extracts. However, it is usually not possible to determine directly the contribution of specific phytonutrients to the total ORAC value. Separation of the components in the plant extracts by HPLC followed by ORAC analyses of the column fractions might permit the determination of free radical-scavenging profiles. The accurate determination of ORAC values may require 1 to 2 h/sample. Considering the number of samples that would be generated by a single HPLC separation, a device was constructed which permits up to 45 simultaneous ORAC analyses. Varying degrees of automation were included in the design. Furthermore, since the assay has a Q(10) for peroxyl radical-scavenging of about 3, elevation of the assay temperature from the standard 37 to 47 degrees C significantly reduced the assay times. Relatively simple modifications would allow the apparatus to be used in a variety of time-dependent fluorescence and absorbance assays.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.