Stripping voltammetry (SV) is used to quantitatively determine concentrations of the anti-neoplastic drug mitomycin C (MMC) alone and in mixtures with 5-fluorouracil and cisplatin, both of which are used in combined chemotherapy with MMC. If the accumulation is performed at the potentials of MMC reduction (-0.35 V vs. SCE), reduced MMC is strongly adsorbed at the electrode. It is possible to prepare a MMC-modified electrode, which, after a washing step, is transferred to the background electrolyte to determine MMC by voltammetry. This procedure, which is termed transfer stripping voltammetry (TSV), helps to eliminate interferences and can be applied for a direct determination of MMC alone or in mixtures with other drugs in urine.

A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.

In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 μM) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

The intestinal absorption of analgesic peptides (leucine enkephalin and kyotorphin) and modified peptides in rat were studied. Although these peptides were not absorbed, the absorbability (absorption clearance) of these peptides were increased in the presence of peptidase inhibitors. In order to kinetically analyze these phenomena, we proposed the metabolic inhibition model, which incorporated the metabolic clearance (metabolizability) with the absorption clearance. Metabolic activity was determined with intestinal homogenates. The higher the metabolic clearance was, the lower was the absorption clearance. The relationships between the absorption clearance and the metabolic clearance of the experimental data as well as of the theoretical values were hyperbolic. This model predicted the maximum absorption clearances of cellobiose-coupled leucine enkephalin (0.654 μl/min/cm) and kyotorphin (0.247 μl/min/cm). Details of the experimental methods are described.

Four methods (an enzyme activity assay, western blotting, RT-PCR, and northern hybridization) to detect the enzyme D-amino-acid oxidase are described.

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three diffirent methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity.

Several micro-scale chromatography-based procedures for purification of the β-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

Deng and Lynch (1, 2) proposed to characterize deleterious genomic mutations from changes in the mean and genetic variance of fitness traits upon selfing in outcrossing populations. Such observations can be readily acquired in cyclical parthenogens. Selfing and life-table experiments were performed for two such Daphnia populations. A significant inbreeding depression and an increase of genetic variance for all traits analyzed were observed. Deng and Lynch's (2) procedures were employed to estimate the genomic mutation rate (U), mean dominance coefficient ( ), mean selection coefficient ( ), and scaled genomic mutational variance ( ). On average, , , and (^ indicates an estimate) are 0.84, 0.30, 0.14 and 4.6E-4 respectively. For the true values, the and are lower bounds, and and upper bounds.

The chemical modification of synthetic oligonucleotides has recently been investigated to improve their pharmacological utilization. In addition to chemical alterations of the backbone and of the heterocyclic bases, their conjugation with amphiphylic moieties, such as the polyethylene glycol has been proposed. The large scale production of these molecules as demanded for commercial purposes is hampered by the heterogeneity of the solid-phase processes and by the low reactivity of high-molecular weight PEGs in solution. A new synthetic procedure based on the recently developed liquid-phase method (HELP), has been set up to overcome these limitations.

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

Six extracellular yeast glycoproteins were prepared from three yeast species in osmotic equilibrium and unequilibrium environments and used as non-penetrating cryoadditives. Glycoproteins secreted by the strain Dipodascus australiensis into growth medium containing NaCl (8% w/v) were found to be the most effective cryoadditives. It was possible to use these glycoproteins alone (without DMSO as penetrating agent) for the cryoprotection of the studied yeasts.