Red light activated "caged" reagents for microRNA research.

"Caged" reagents for miRNA research (siRNA targeting EGFR, involved in miRNA maturation, and mimics of miR-20a, playing a key role in tumor formation and metastasis) were prepared. It was demonstrated that these reagents can be activated by non-toxic to cells red light both in cells and in cell free settings.

Golgi apparatus studied in vitreous sections.

Cryo-electron microscopy of vitrified specimen is the method of choice to explore cellular ultrastructure at high resolution as close as possible to the native state and environment. In this study, we investigated the Golgi apparatus - the main organelle of the secretory pathway. Cultured mammalian cells were fixed by high-pressure freezing, sectioned in vitreous ice and subjected to cryo-electron microscopy and cryo-electron tomography. Although the overall morphology of Golgi stacks was comparable to well prepared and plastic-embedded samples, in detail we reached much higher resolution in terms of distinction between biological structures based on their native density. On cisternal buds and peri-Golgi vesicles--some associated with microtubules--we detected two different subtypes of COPI coats: (1) a homogenous coat and (2) an inhomogeneous spiky coat, providing an 8-9 nm regularity, clearly distinct from clathrin coat. Next, we monitored the secretion of cargo, namely, procollagen I, through the Golgi complex. Temporally correlated with fluorescence microscopy, we performed three-dimensional cryo-electron tomography analysis and detected Golgi cisternae enlarged to saccules, containing cargo and showing inter-cisternal connections. Our work provides a first step towards the high-resolution description of the secretory pathway in native vitrified samples and describes the challenges associated with this attempt.

The potential of high-content high-throughput microscopy in drug discovery.

Fluorescence microscopy is a powerful method to study protein function in its natural habitat, the living cell. With the availability of the green fluorescent protein and its spectral variants, almost any gene of interest can be fluorescently labelled in living cells opening the possibility to study protein localization, dynamics and interactions. The emergence of automated cellular systems allows rapid visualization of large groups of cells and phenotypic analysis in a quantitative manner. Here, we discuss recent advances in high-content high-throughput microscopy and its potential application to several steps of the drug discovery process.

Purification and characterization of the D-hydantoinase from Bacillus circulans.

A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.