The influence of contact guidance on the orientation of colonies of subcultured vascular smooth muscle cells

Summary Subcultures of smooth muscle cells derived from rat thoracic aorta were grown on plane plastic substrata and on plastic substrata having ridges molded in them by a heated, ruled template. The cells were found to have a very high degree of contact guidance when distributed sparsely on the ridged substrata. When the cell density increased multilayered, elongated colonies formed. On plane substrata these were irregular, curved, and disposed in all directions. On the ridged substrata, however, the colonies were straight, evenly spaced, and positioned at right angles to the ridges. Supported by Grant MT1011 from the Medical Research Council of Canada.     

Investigations of the performance of a high-performance liquid chromatography system with an electrochemical detector

A procedure for the determination of norepinephrine and dopamine, based on high-performance liquid chromatography, is evaluated using an electrochemical detector system. The use of an inorganic mobile phase to provide resolution of low retention amines and extend column life is discussed. A high degree of correlation between estimations of endogenous catecholamine levels is reported using both electrochemical and fluorometric detector systems. Inter-assay reproducibility of the extraction method, and sensitivity and linearity of response of the electrochemical detector system are shown to be consistent across trials. The system described is determined to be accurate, sensitive, and reliable over time.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.