Candida pyralidae killer toxin disrupts the cell wall of Brettanomyces bruxellensis in red grape juice.

AIMS: The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B. bruxellensis cells in red grape juice. METHODS AND RESULTS: Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B. bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of ß-glucan, suggesting a compensatory response of the sensitive cells. CONCLUSIONS: The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.

Chromatographic and immunochemical approaches to the analysis of the HIV protease inhibitor saquinavir in plasma.

The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.

5-HT2a receptors in the rabbit retina: potential presynaptic modulators.

Three 5-HT receptors have been implicated in retinal processing but positive identification of the receptors and the localization of receptor subtypes in the retina have not been achieved. In this study, molecular techniques were used to identify one class of 5-HT receptor--5-HT2a--in the retina, and immunohistochemical techniques were used to localize the receptor in the retinal network. Reverse transcription polymerase chain reaction (RT-PCR) techniques were used to identify a segment of the rabbit 5-HT2a gene; a 422 base fragment was identified, cloned, and sequenced. The fragment shows a high degree (ca. 90%) of nucleotide sequence identity with the 5-HT2a receptor gene from other mammals. 5-HT2a immunoreactivity was seen in both the inner and outer plexiform (synaptic) layers of the retina. Using cell-type-specific markers, the 5-HT2a immunoreactivity was shown to be on the terminals of photoreceptor and rod bipolar cells. This association of 5-HT2a receptors with these two synapses suggests that serotonin may be a modulator of synaptic function in the retina.

Metabolism of calcium antagonist Ro 40-5967: a case history of the use of diode-array u.v. spectroscopy and thermospray-mass spectrometry in the elucidation of a complex metabolic pathway.

1. The calcium antagonist, Ro 40-5967, is metabolized to a multitude of products by the rat and drug-related material is excreted predominantly via the bile. 2. Diode-array u.v. spectroscopy, following reverse phase h.p.l.c. separation of the partially purified metabolites, has been used to classify these compounds into six spectral classes which have been correlated with different metabolic reactions. 3. Connection of a mass spectrometer directly to the h.p.l.c. equipment by a thermospray interface, produced useful mass spectra. These, together with the u.v. spectra, enabled the structures of many metabolites to be elucidated. 4. Confirmation of structural assignments was provided by n.m.r. spectra of the major metabolites. 5. Major metabolic pathways included N-demethylation (16% of the biliary metabolites), hydrolysis of the ester side-chain (32%), hydroxylation at 4- (19%) and 5- (29%) positions of the benzimidazole ring, aromatization of the tetrahydronaphthyl system (26%), loss of the benzimidazole (15%) and glucuronidation of hydroxyl groups (81%).

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces.

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.