Automated quantitative analysis of single and double label autoradiographs.

A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H-thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C-thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains.     

Morphometry and cytometry correlated to quantitative autoradiography

Studies performed in various cell systems and designed to establish correlations between morphometric and functional parameters of individual cells are reviewed. Functional parameters were evaluated by utilizing quantitative 14C-autoradiography and measuring DNA, RNA and protein synthesis. Morphologic parameters were derived from the scanning of Feulgen-stained nuclei and the calculation of features related to shape, optical density and texture. A series of correlations between parameters of these two groups of features was established. Results such as the possibility of allocating cells to the G1, S and G2 phases by textural and densitometric features alone, without making use of the total DNA content and nuclear size, point to the power of this approach. The data, however, are not yet comprehensive enough to allow the interpretation of morphologic parameters in terms of cellular function on a large scale. It is emphasized that the measurement of the RNA synthesis rate will further promote the functional understanding of structural details and may help in making this approach useful for diagnostic purposes.     

An elementary approach to catastrophe theory

The recently proved classification theorem of René Thom has led to the development of the new science of Catastrophe Theory. This theory has been widely held to be particularly relevant in biological applications. The present paper presents a simplified proof of Thom’s Theorem and assesses its importance in biology.     

The organism and its double: An approach to the individual

The individual is certainly the most complex entity that we know until now. Current research converges on a triad-like understanding of the biological individual as an interface of a mechanism and an ecosystem. The present article wants to propose a more generic concept of individual by integrating the functional role of its qualitative characteristics. Modules-problems ensuing from this generic model will serve as a matrix in the elaboration of a future integral biomathics, while category theory will be its syntactic base.     

To label or not to label: applications of quantitative proteomics in neuroscience research

Proteomics has provided researchers with a sophisticated toolbox of labeling-based and label-free quantitative methods. These are now being applied in neuroscience research where they have already contributed to the elucidation of fundamental mechanisms and the discovery of candidate biomarkers. In this review, we evaluate and compare labeling-based and label-free quantitative proteomic techniques for applications in neuroscience research. We discuss the considerations required for the analysis of brain and central nervous system specimens, the experimental design of quantitative proteomic workflows as well as the feasibility, advantages, and disadvantages of the available techniques for neuroscience-oriented questions. Furthermore, we assess the use of labeled standards as internal controls for comparative studies in humans and review applications of labeling-based and label-free mass spectrometry approaches in relevant model organisms and human subjects. Providing a comprehensive guide of feasible and meaningful quantitative proteomic methodologies for neuroscience research is crucial not only for overcoming current limitations but also for gaining useful insights into brain function and translating proteomics from bench to bedside.     

An experimental test of quantitative genetic theory

Theoretical and Applied Genetics - An experiment was conducted withDrosophila melanogaster to test a theory of quantitative inheritance. The covariances among half and full sibs did not increase...     

Standardization of tritium-sensitive film for quantitative autoradiography

A method for the calibration of plastic tritium standards for use with LKB Ultrofilm is described and validated. This method uses 12-microns cryostat slices of frozen liver which have been labeled with [3H]-formaldehyde and extracted with chloroform-methanol to remove lipids. Quantitative autoradiographic measurement of 3H radioactivity in the liver slices was underestimated by 35% when lipids were not extracted. Plastic sections impregnated with tritium were calibrated in terms of lipid-extracted, tissue-equivalent radioactivity content. Calibrated standard curves for these plastic standards were closely fit (p = 0.99) by second order polynomial equations for exposures of 1, 4, 7, 13, 28, and 104 days. The equations are generally useful for any plastic tritium standards.     

An integrative approach for the identification of quantitative trait loci

The genetic dissection of complex traits is one of the most difficult and most important challenges facing science today. We discuss here an integrative approach to quantitative trait loci (QTL) mapping in mice. This approach makes use of the wealth of genetic tools available in mice, as well as the recent advances in genome sequence data already available for a number of inbred mouse strains. We have developed mapping strategies that allow a stepwise narrowing of a QTL mapping interval, prioritizing candidate genes for further analysis with the potential of identifying the most probable candidate gene for the given trait. This approach integrates traditional mapping tools, fine mapping tools, sequence-based analysis, bioinformatics and gene expression.     

Methods of quantitative autoradiography using incident light microphotometry.

Several different approaches of automated grain counting of microautoradiographic grain densities have been reported in the literature. Application of grain counters to cell biology is limited, however, primarily due to shortage of methods allowing the interpretation of grain counts on a molecular basis. Two suitable methods of quantitative autoradiography at the cellular level are reviewed, developed for the isotopes 14C and 125 I. They permit evaluation of absolute radioactivity in autoradiographs and, thus, determination of synthesis processes such as deoxyribonucleic acid synthesis, and of antigen densities on cell surfaces. In this approach towards quantitative autoradiography, grain densities are compared photometrically over labeled cells and over a standard source on the same autoradiograph. Allowance has to be made for the specific geometric factors of the isotope used. This can be advantageously done with an integrating type of measurement using incident light bright-field. With this type of recording, there is an exponential dependence of the photometric values on the radioactive dose. As an example of application, results are presented of the deoxyribonucleic acid synthesis rate of human myelocytes in aplastic anemia and of the immunoglobulin G density on lymphocyte membranes in the normal state and in chronic lymphocytic leukemia.     

Calibration of the emulsion layers in quantitative electron microscopic autoradiography]

The quantitative results in electron microscope autoradiography are affected by many factors. The principal problem of this method is a low mechanization of all the operations made. This paper gives a short review of technical improvements. The use of a calibration permits to except those factors which exert their influence on the sensitivity of the method. Tritium sources were used for the determination of the sensitivity of the method.     

An approach to quantitative assessment of hemodynamic differences between unruptured and ruptured ophthalmic artery aneurysms

Background and purpose: Hemodynamic parameters are important in the pathogenesis, evolution and rupture of intracranial aneurysm. Energy loss (EL) has been applied for the rupture risk prediction of artery aneurysms recently. We proposed a new EL and further investigate its effects on the rupture of aneurysms. Materials and methods: Sixty-four patient-specific ophthalmic aneurysm datasets were divided into ruptured and unruptured groups based on their clinical history. Based on patient-specific 3D-DSA data, realistic models were retrospectively reconstructed and then analyzed by using computational fluid dynamic method. Results: The flow field feature EL in ruptured cases was significantly higher than that in unruptured cases. The average wall shear stress (WSS) and the maximum WSS in ruptured cases were higher than those in unruptured cases. Modified pressure loss coefficient (PLC_M) in ruptured cases was slight higher than that in unruptured cases but the difference has no statistical significance. Multivariate logistic regression analysis demonstrated flow field feature EL (p < 0.05) and the maximum WSS (p < 0.05) were the only independently significant variables to predict rupture of ophthalmic aneurysm. There were no differences in PLC_M, the maximum oscillatory shear index (OSI), the average OSI and AR between the two groups. Conclusion: Flow field feature EL may be a reliable factor to predict the rupture risk of aneurysms.     

A quantitative approach to the cranial ontogeny of the puma

The cranial ontogeny of specialized mammals is relevant to the understanding of the connection of form and function in a developmental, ecological, and evolutionary context. As highly specialized carnivores, felids are of especial interest. We studied the postnatal ontogeny of the skull in Puma concolor (Mammalia: Carnivora: Felidae) using a quantitative approach. We interpreted our results in the light of a previous qualitative assessment of ontogenetic changes in the species. This represents one of the few integrative studies of skull development in any extant species of wild felids. We report patterns of multivariate allometry of 19 linear skull dimensions measured in 48 Argentine specimens. We examined the (jackknife resampled) departures from isometry as well as the interplay of isometric and allometric trends in shaping the puma skull. Both the qualitative and quantitative results indicate that the major ontogenetic changes are directly linked to cranial structures that support a developing masticatory apparatus and its associated jaw and neck musculature, which are essential for the action of canines and carnassials during the killing bite and slicing flesh. Sexual differences suggest allometric scaling (hypo- or hyper-morphosis) as key processes in the development of the puma skull.     

Conjugation of lectins with fluorochromes: An approach to histochemical double labeling of carbohydrate components

Summary Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyanate (TRITC) are reported. 20 g of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.     

Localization of aldosterone and corticosterone in the central nervous system,assessed by quantitative autoradiography

Nuclear localization of tritiated aldosterone in the CNS was studied in rats by numerical evaluation of silver grains, deposited over neuronal cell nuclei in thawmounted autoradiograms, and compared with the localization obtained after prior administration of a 100-fold excess of radioinert aldosterone, corticosterone or 18-hydroxy-11-deoxycorticosterone (18-OH-DOC). Corticosterone and 18-OH_DOC completely prevented nuclear localization in most regions examined. However, in contrast to pretreatment with aldosterone, pretreatment with corticosterone and 18-OH-DOC did not completely prevent the concentration of radio-activity in the cell nuclei of the indusium griseum. Traces of radioactivity were, furthermore, retained in areas CA₁ and CA₂ and the dentate gyrus in rats exposed to corticosterone, but not to 18-OH-DOC, prior to [³H]aldosterone. A similar profile of silver grain distribution to that noted with aldosterone was found for corticosterone except that with tritiated corticosterone the most intense concentration of radioactivity occurred in hippocampal areas CA₁ and CA₂ and not in the indusium griseum. Prior administration of excess deoxycorticosterone acetate abolished nuclear accumulation of tritiated corticosterone. Dihydrotestosterone, on the other hand, failed to compete with tritiated corticosterone at a dose 200-fold in excess of the tritiated steroid.We conclude that (1) a receptor readily shared by aldosterone, corticosterone, 18-OH-DOC and DOC, but not by dihydrotestosterone, is widely distributed throughout the CNS, (2) a receptor shared by aldosterone and 18-OH-DOC, but not by corticosterone may be present in hippocampal areas CA₁ and CA₂, (3) that both these as well as the receptor accepting dihydrotestosterone can be located within the same cell.Dedicated to K. A. C. Elliott on his 80th birthday.