Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Aspects of audit. 3: Audit in British general practice.

Compared with hospitals, general practice has many handicaps in developing medical audit. This is especially true with regard to defining objectives and outcomes of care. Many methods have been proposed to overcome these problems, and several audit studies that have been published are presented here. Further work should concentrate on evaluating the effectiveness of audit in bringing about change rather than on generating information.     

Differential phosphorylation of multiple sites in protein 4.1 and protein 4.9 by phorbol ester-activated and cyclic AMP-dependent protein kinases

The phosphorylation of the membrane skeleton components protein 4.1 and protein 4.9 in intact erythrocytes is shown to increase in the presence of either 1 microM 12-O-tetradecanoyl phorbol 13-acetate or 2 mM dibutyryl cAMP. The phosphorylation induced by these protein kinase activators is compared by two-dimensional tryptic peptide mapping. In both proteins, the pattern of peptides phosphorylated in the presence of 12-O-tetradecanoyl phorbol 13-acetate differs from the pattern of peptides phosphorylated in the presence of dibutyryl cAMP. The relative locations of the phosphorylated sites on protein 4.1 have been determined using limited proteolysis by alpha-chymotrypsin.     

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody

A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.     

Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids.

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.     

Oral care of people with disability: a qualitative exploration of the views of nursing staff

This study aimed to identify influences, attitudes and actions of nursing staff in relation to oral care for people with disabilities. Individual in-depth interviews of twenty two nursing staff were conducted. Enabling and inhibiting factors in the reported attitudes, approaches and practices were identified. The main enabling factors were that nursing staff saw mouth care as part of their role and took an empathic and caring approach to its delivery. They aimed to make the residents socially acceptable, improve their self-esteem and make their mouths clean and breath fresher. Inhibiting factors were: a lack of training, time constraints associated with workload, and poor understanding of the processes causing dental disease. The study concludes that improvements in oral care by nursing staff can be encouraged by working with nurses'attitudes, values and beliefs. Once the desired behaviour change has occurred then the lack of knowledge can be addressed.     

Multiple mating and sperm displacement in a natural population of Drosophila melanogaster

Summary Mother-offspring data for alcohol dehydrogenase genotypes of a vineyard cellar population of D. melanogaster are best explained by a model that allows 21% of females in the population to mate twice with an 83% level of sperm displacement. A population model with multiple mating and sperm displacement is examined theoretically. A formula for the effective population size is derived under this model. Multiple mating increases the effective population size relative to single mating.     

Intracellular Ca2+ concentration and the antidiuretic hormone-induced increase in water permeability: effects of ionophore A23187 and quinidine

The hydroosmotic responses induced by oxytocin and 8-bromo-cyclic AMP, in frog and toad urinary bladders, were recorded minute by minute. 3HHO and 45Ca unidirectional fluxes as well as prostaglandin B2 liberation were also measured. It was observed that: (1) Addition of the calcium ionophore A23187 or quinidine to the serosal bath inhibited the response to oxytocin, but not to 8-bromo-cyclic AMP, while increasing prostaglandin E1 liberation into the serosal but not into the mucosal bath. (2) Addition of A23187 to the mucosal bath induced a transient and temperature-dependent inhibition of the response elicited by 8-bromo-cyclic AMP. The time-course of this reduction in water permeability and its sensitivity to medium temperature were similar to those observed after the withdrawal of agonist, but clearly different of those observed after intracellular acidification. (3) The hydroosmotic response was also transitorily inhibited when the Ca2+ concentration was step-changed in the mucosal bath. (4) When added to the mucosal or to the serosal baths, the ionophore increased either the apical or the laterobasal Ca2+ permeabilities. It is concluded that manipulation of intracellular Ca2+ interferes with the hydroosmotic response at two different levels. (1) A first target point located 'pre-cyclic-AMP production'. This effect would be mediated by prostaglandin liberation. (2) A second target point located after cyclic AMP production and before the 'temperature-dependent rate-limiting step'. This effect is probably related to the mechanism controlling the insertion and removal of water channels.     

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.