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.     

Behavioral and Emotional Dynamics of Two People Struggling to Reach Consensus about a Topic on Which They Disagree

We studied the behavioral and emotional dynamics displayed by two people trying to resolve a conflict. 59 groups of two people were asked to talk for 20 minutes to try to reach a consensus about a topic on which they disagreed. The topics were abortion, affirmative action, death penalty, and euthanasia. Behavior data were determined from audio recordings where each second of the conversation was assessed as proself, neutral, or prosocial. We determined the probability density function of the durations of time spent in each behavioral state. These durations were well fit by a stretched exponential distribution, with an exponent, , of approximately 0.3. This indicates that the switching between behavioral states is not a random Markov process, but one where the probability to switch behavioral states decreases with the time already spent in that behavioral state. The degree of this “memory” was stronger in those groups who did not reach a consensus and where the conflict grew more destructive than in those that did. Emotion data were measured by having each person listen to the audio recording and moving a computer mouse to recall their negative or positive emotional valence at each moment in the conversation. We used the Hurst rescaled range analysis and power spectrum to determine the correlations in the fluctuations of the emotional valence. The emotional valence was well described by a random walk whose increments were uncorrelated. Thus, the behavior data demonstrated a “memory” of the duration already spent in a behavioral state while the emotion data fluctuated as a random walk whose steps did not have a “memory” of previous steps. This work demonstrates that statistical analysis, more commonly used to analyze physical phenomena, can also shed interesting light on the dynamics of processes in social psychology and conflict management.     

Effects of cultural conditions on the production of phenylalanine from a plasmid-harboring E. coli strain

Summary Phenylalanine production from E. coli KA 197/pJN6 (plasmid harboring genes for aro F, phe A^FBR, Amp^R and Tc^R) was studied under varying nutritional conditions in batch and continuous cultures. In batch culture experiments where growth was deliberately interrupted by limiting concentrations of sulphate and phosphate the phenylalanine production continued from the non-growing cells. However, the depletion of phosphate resulted in an immediate cessation of phenylalanine production but thereafter a low specific rate of phenylalanine formation resumed, while the decrease in specific rate of product formation was less after sulphate depletion. In the chemostat experiments, however, phosphate limitation was the only case where the specific rate of phenylalanine formation remained constant, while at the corresponding time in sulphate and glucose limited chemostats it was declining respectively had ceased.     

Seed and tuber banks of aquatic macrophytes in the Askö area, northern Baltic proper

Sediment samples from 5 stations in the northern Baltic proper, 6.5 o/oo S, were collected in April 1987 and the emergence of seedlings was recorded over 120 days in a greenhouse at 20°C. Total seedling densities varied from 0 to 3328 m^-2: and of seven species, Zannichellia palustris and Chara spp. were the most abundant among seedlings and sporelings, respectively. Several common macrophytes in the area were rare as seedlings and no seedlings were recorded for the most abundant angiosperms, Potamogeton pectinatus, Potamogeton perfoliatus and Ranunculus baudotii. Except for the few annual species, reproduction by seeds contributed little to the dynamics of the vegetation in the area and no correlation was found between vegetation composition and the seed bank. For perennial species the winter survival of vegetative propagules is the most important factor for vegetation dynamics.     

Enzyme histochemical methods applied in the brain

Catalytic enzyme histochemistry offers the possibility to demonstrate enzymes qualitatively and their activities quantitatively in brain sections at those sites where they are localized. To get an appropriate histochemical demonstration of enzymes, requirements are to be fulfilled with respect to the preparation of brain tissue, the detection methods, and the incubation conditions. For enzyme demonstration at the light microscopic level, brain tissue should be frozen as quickly as possible and for those at the electron microscopic level perfusion fixation using low concentrations of aldehydes seems to be best suited. The detection of enzymes in brain sections is preferentially performed by the so-called precipitation reactions with metallic ions, the tetrazolium and the diaminobenzidine methods. The application of these methods was shown in the example of aspartate aminotransferase, glutamate dehydrogenase, and cytochrome c oxidase. In the detection of enzymes incubation conditions should be chosen so that soluble enzymes cannot diffuse out of the sections into the incubation media and that the activities of enzymes are completely demonstrated. On the whole, all the precipitation reactions result in a water-insoluble reaction product which is precipitated at the enzymatic sites in brain sections. Finally, it is shown that scanning microphotometry is a valuable tool for the quantification of enzyme activities in brain sections. It is concluded that catalytic enzyme histochemistry using improved detection methods could be a source of results complementary to those provided by immunocytochemistry and microchemistry.     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)