Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Unsaturated oligogalacturonic acids are generated by in vitro treatment of pectin with human faecal flora.

Pectins with a degree of esterification (DE) of 95, 66, 34 and 0%, respectively, were incubated in vitro with human faecal flora (pH 7.8). The concentration and composition of oligogalacturonic acids (oligoGalA) generated were determined using high-performance thin-layer chromatography (HPTLC) with UV and colorimetric detection. In the first period of the anaerobic degradation, the pectin macromolecules were fragmented into unsaturated oligoGalA as intermediate products by the action of bacterial pectate lyases. Depending on the incubation time and the DE of pectin, the amount of unsaturated oligoGalA having different degrees of polymerization changed continuously. These oligoGalA were present in the cultures for some hours. Mixtures of unsaturated di-, tri- and tetraGalA were the end products of a pectate lyase action. Later, the oligoGalA disappear as a result of their further fermentation by the gastrointestinal microflora under formation of short-chain fatty acids (SCFA). Low-esterified pectins were depolymerized and fermented faster than the highly esterified by the human faecal flora in vitro. Furthermore, a mixture of unsaturated oligoGalA prepared from pectic acid by the action of pectate lyase from Erwinia carotovora was completely fermented by human faecal flora.     

Dielectric spectroscopy for noninvasive examination of corneal tissue]

Dielectric spectroscopy is a non-invasive contact technique that permits the in vivo measurement of the specific electrical properties of biological tissue induced by an external electrical field. Permittivity, relaxation time and specific conductivity as a function of corneal hydration (wet weight/dry weight) and temperature were measured in 10 porcine corneas. Variation of tissue hydration has a minor influence on the signal, with a significant variation of the signal being detectable only for relatively dry tissue. A much greater influence was found for temperature, in particular on relaxation times. Dielectric spectroscopy provides us with an opportunity to detect structural, in particular temperature-induced, changes in living tissue. In the frequency range investigated, hydration has only a small influence on the dielectric properties of the tissue.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.     

Circuit for the electromanipulation of plant protoplasts

An electric circuit for plant protoplast manipulation is described. The circuit used readily available materials and was designed for use in teaching. This integrated circuit can be placed in a single small box with controls for the aligning voltage, the aligning frequency, the pulse voltage, and the pulse timing. The circuit can be supplied by any suitable source of dc power and can be easily altered for individual requirements. The circuit, as presented here, can be assembled for less than $250.