The hybrid resolution approach to automated autoradiographic analysis.

The use of radioactive isotopes in the investigation of cells and their relations to an organism as a physiologic unit is an important area of research to the biologist. The manual analysis of the autoradiographs which result from the use of radioactive tracers is, however, very time consuming and prone to error. This paper describes the utilization of hybrid resolution image processing techniques for the rapid and automatic analysis of tissue region autoradiographs. Nine regional features can be extracted and six properties characterized using the present system implementation. Priliminary measurements on two tissue types show good agreement between manual and automated readings.     

Incorporation and stabilization of 3H-tetracycline in embryonic chick bone: an autoradiographic study

Tibiae from 11-day-old chick embryos, injected with 3H-tetracycline, were autoradiographically analyzed at different stages to localize and study the dynamics of the initial phases of bone mineralization. 3H-tetracycline was localized within newly formed trabeculae, but only at the surfaces of older trabeculae, indicating that the incorporation of tetracycline into bone occurs at active sites of calcification. It takes between 24 and 36 h for injected tetracycline to become stabilized and incorporated into the mineralized matrix. Absence of 3H-tetracycline grains over the osteoblasts suggests a paracellular pathway for incorporation of tetracycline into the mineralizing bone matrix.     

Somatostatin receptors in the senescent rat brain: a quantitative autoradiographic study.

To examine the effects of aging on the density and distribution of somatostatin receptors (SS-R) in the rat brain, receptor autoradiography for SS-R was carried out in rats aged 3 and 24 months using 125I-labeled Tyr11-SS-14. Autoradiograms were quantitatively assessed by an image analyzer to evaluate changes in the expression of SS-R due to senescence. Statistically significant decreases in SS-R binding were found in specific regions of the brains of senescent rats as compared to young adult rats. The regions affected included the periaqueductal gray matter (73% loss versus young adult rats), the interpeduncular nucleus (73% loss), the pontine nucleus (63% loss), the superior colliculus (46% loss), the ventral tegmental area (46% loss), the temporal cortex (39% loss), the frontal cortex (34% loss), the hippocampus (33% loss), the amygdala (27% loss) and the claustrum (26% loss). There was no significant change in SS-R expression in the spinal cord with aging. Significant reductions in SS-R binding in these brain regions may be involved in the impairment of sensory and cognitive function that can occur with aging.     

Early endosteal bone response to incorporated plutonium-238 in mice

Ten Swiss albino ICR SPF female mice 110 days old (weight about 30 g) were exposed for 48 hours to a solution of plutonium-238 nitrate (spec. act. 5 MBq/1 m1, pH 2.7) injected in amounts of 0.01 ml into the popliteal area of the right femur, each thus receiving about 500 kBq per 30 g body weight. Of the injected activity, 50% was retained in the right femur, 2% in the left femur and approximately 2-3% in the excrements collected separately from each animal during the whole exposure period. Ultrastructurally, electron micrographs revealed a variety of changes, including hypertrophy and destruction of endosteal cell organelles (primary damage), deformation and hypertrophy of osteocytes (secondary damage) and the irregularities in the osteocyte self-burial process leading to an abnormal formation of bone tissue structure (tertiary damage). Qualitatively, these changes in the irradiated bone ultrastructure were analogous to those occurring with age. This was confirmed by comparing two groups of control mice 110 and 330 days old. Assessed quantitatively, changes due to irradiation were more pronounced than those associated with aging.     

Pattern of labelling of the rat brain stem after intraventricular administration of 3H-leucine; low and high resolution autoradiographic study

The pattern of labelling of proteins of the periventricular grey matter was studied two hours after intraventricular administration of 3H-leucine by low- and high-resolution autoradiography. The pattern was investigated by computer-controlled densitometry. The deposition of radioactive, proteins in the periventricular grey surrounding the mesencephalic part of the aquaeductus Sylvii was compared with that surrounding the fourth ventricle. In the former case, the distribution, of grains was in a circular area 500-600 micrometer in diameter; the densitometric tracing revealed a homogeneous distribution of the label; in the latter case, the distribution was nonhomogeneous and was limited by the tissue components forming the wall of the fourth ventricle. A comparison of the intensity of labelling (performed by a combination of low- and high-resolution autoradiography indicated: a) relatively substantial labelling of proteins of ependymal cells, b) very sparce labelling of subependymal layers, c) very high labelling ot neurones, adjacent to the subependymal layers. The significance of these findings for the interpretation of studies using the intraventricular administration of labelled amino acids for investigating brain macromolecular metabolism is discussed.     

Towards construction of a high resolution map of the mouse genome using PCR-analysed microsatellites.

Fifty sequences from the mouse genome database containing simple sequence repeats or microsatellites have been analysed for size variation using the polymerase chain reaction and gel electrophoresis. 88% of the sequences, most of which contain the dinucleotide repeat, CA/GT, showed size variations between different inbred strains of mice and the wild mouse, Mus spretus. 62% of sequences had 3 or more alleles. GA/CT and AT/TA-containing sequences were also variable. About half of these size variants were detectable by agarose gel electrophoresis. This simple approach is extremely useful in linkage and genome mapping studies and will facilitate construction of high resolution maps of both the mouse and human genomes.     

Plutonium,241Am,90sr,and137cs concentrations in some antarctic matrices

A radioecological survey in Antarctica shows that the^239+240Pu,²³⁸Pu,²⁴¹Am,⁹⁰Sr, and¹³⁷Cs activities were detectable in nearly all the samples. The activity level of^239+240Pu,²⁴¹Am, and¹³⁷Cs in antarctic sediments was about 5–20 times lower than in the northern Adriatic Sea sediments, but the²³⁸Pu activities were relatively high. It was interesting to note that the⁹⁰Sr concentrations in all the sediments tended to be low, which could be the result of the easier exchangeable behavior of⁹⁰Sr in water. High concentrations were detected in mosses and lichens and their activity levels were comparable to those in central Italy. The radionuclide ratio analyses show that the major part of^239+240Pu,²⁴¹Am,⁹⁰Sr, and¹³⁷Cs was a result of nuclear weapon tests. The higher²⁴¹Am/^239+240Pu ratio was observed and it could perhaps be the result of fallout of nuclear weapon tests prior to 1962. The²³⁸Pu/^239+240Pu ratio in the antarctic matrices was about seven times higher than in the Northern hemisphere and it could be inferred that the major part of²³⁸Pu was originating from the SNAP-9A satellite accident.     

Hyphal growth ofHypomyces chlorinus Tul.: An autoradiographic study

Summary The incorporation of the chitin precursor N-acetyl-D-(1-³H) glucosamine byH. chlorinus has been studied by light and electron microscopic autoradiography. Light microscopic autoradiography showed that the incorporation occured preferentially at the hyphal apex. Autoradiograms from electron microscopy were quantitatively evaluated to determine the relative radioactivity incorporation between the cell wall and cytoplasm: this showed that (³H) incorporation took place mainly in the plasmalemma-wall complex. However a small amount of N-acetyl glucosamine can enter into the cytoplasmic space and is then transported by endomembranes (Golgi apparatus-vesicles) to the plasmalemma-cell wall interface before polymerization.Abbreviations PATAg Periodic acid-thiocarbohydrazide-silver proteinate - PTA phosphotungstic acid     

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.     

Brain somatostatin receptors in spontaneously hypertensive rats: an autoradiographic study.

The apparent densities of brain somatostatin (SRIF) receptor sites were compared in adult spontaneously hypertensive rats (SH) and their normotensive genetic counterparts (Wistar-Kyoto; WKY) using quantitative receptor autoradiography. Globally, the distribution of brain [125I][Tyr0, D-Trp8]SRIF14 binding sites was very similar in both strains. However, apparent densities of specific labeling were either higher (subfornical organ, 3.2 x; locus coeruleus, 1.9 x; lateroanterior hypothalamic nucleus, 1.3 x) or lower (basolateral amygdaloid nucleus, 0.8 x; spinal trigeminal sensory nucleus, 0.6 x) in SH than WKY rats in areas especially relevant to CNS cardiovascular integration. This provides further evidence for the possible involvement of brain SRIF neurons in cardiovascular regulation.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.     

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.