Reflectance in situ hybridization (RISH): detection,by confocal reflectance laser microscopy,of gold-labelled riboprobes in breast cancer cell lines and histological specimens

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.     

Extending Prolog to Provide Better Support for Design Code Expert Systems

The CSIR0³ Division of Building Research (DBR) has built a number of text-based and graphics-based design code expert systems in PROLOG. Each of these systems involves several thousand lines of PROLOG and stretches to the limit and the capacity of the IBM ATs on which they run. We have found PROLOG to be a very powerful language for expressing the complex rules and relationships found in building design codes and related applications. Nevertheless, it has a number of weaknesses, particularly in expressing procedural knowledge and arithmetic relationships, and in implementing complex tables and user interfaces. We have designed and implemented extensions to PROLOG to overcome these difficulties. These include new built-in functions to perform functions that are difficult to code in PROLOG, an easy way to add further built-in functions, a meta-interpreter to keep track of intermediate results, sets of routines to support user input and explanation, and a definite clause grammar to implement a more syntactically pleasant language for writing rules. These extensions are being used to develop a larger and much more complete version of the WINDLOADER expert system, and have greatly facilitated this task.     

A pixel‐based likelihood framework for analysis of fluorescence recovery after photobleaching data

A new framework for the estimation of diffusion coefficients from data on fluorescence recovery after photobleaching (FRAP) with confocal laser scanning microscopy (CLSM) is presented. It is a pixel‐based statistical methodology that efficiently utilizes all information about the diffusion process in the available set of images. The likelihood function for a series of images is maximized which gives both an estimate of the diffusion coefficient and a corresponding error. This framework opens up possibilities (1) to obtain localized diffusion coefficient estimates in both homogeneous and heterogeneous materials, (2) to account for time differences between the registrations at the pixels within each image, and (3) to plan experiments optimized with respect to the number of replications, the number of bleached regions for each replicate, pixel size, the number of pixels, the number of images in each series etc. To demonstrate the use of the new framework, we have applied it to a simple system with polyethylene glycol (PEG) and water where we find good agreement with diffusion coefficient estimates from NMR diffusometry. In this experiment, it is also shown that the effect of the point spread function is negligible, and we find fluorochrome‐concentration levels that give a linear response function for the fluorescence intensity.     

Lipid Hydrolysis in Frozen Baltic Herring

SUMMARY— The lipid composition of Baltic herring ( Clupea harengus var . membranus) muscle and the hydrolysis during freeze storage was studied with thin-layer chromatography. The concentrations of the various lipid classes were initially 2–5 times as high in dark muscle as in white muscle. Storage for up to 12 weeks at –15°C resulted in an increase of the free fatty acid content from 50 to 1000 mg/100 g in the dark muscle, and from 17 to 280 mg/100 g in white muscle. The increase was due to hydrolysis of lecithin, cephalin and to a varying extent also of triglycerides. According to indirect estimations, roughly 45 and 75% of the free fatty acid formed in dark and white muscle respectively was a result of phospholipid hydrolysis; the remainder of triglyceride hydrolysis. The hydrolysis of lecithin was faster than that of cephalin in both types of muscles. The enzymatic attack showed no preference for anyone of the different fatty acids in the phospholipids.     

Si-C-O-N High-Pressure Equilibria and ΔGfo for Si2ON2

Phase relations in the Si-C-O-N system were studied by subjecting mixtures of carbon and silica (C/SiO₂= 2 to 3) to high temperatures (up to 2000 K) and elevated nitrogen pressures (0.5 to 2.0 MPa) in a gas autoclave. The condensed-phase assemblages obtained at different temperatures, pressures, and N/Si ratios are reported and compared with thermodynamic calculations. Values of Δ G _f ^o(Si₂ON₂) consistent with the experimental data are -397 kJ/mol at 1780 K and -369 kJ/mol at 1890 K. The influence on the synthesis of seeding the starting material is discussed.