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L G CARO

Publications and source records attributed to L G CARO.

13 recordsLinked to original sources
PubMed1964

PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL. AN AUTORADIOGRAPHIC STUDY.

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H(3) as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H(3); (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H(3) can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at approximately 5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at approximately 20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.

Animals↗
PubMed1962

High-resolution autoradiography. I. Methods.

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.

Autoradiography↗
PubMed1962

High-resolution autoradiogaphy. II. The problem of resolution.

The resolution obtainable in electron microscopic autoradiographs, using a photographic emulsion consisting of a monolayer of silver bromide crystals, was investigated theoretically and experimentally. The expected distribution of exposed crystals around a point source was calculated from the geometry of the preparation and from the range distribution of the beta particles emitted by tritium. From such a distribution an autoradiographic resolution of the order of 1000 A can be predicted. From the point source distribution, the expected distribution of grains around bacteriophages labeled with tritium was calculated. This distribution was also measured experimentally in electron microscopic autoradiographs of bacteriophages T-2 labeled with thymidine-H(3). The two distributions agreed closely. It was also verified, using the nuclear region in thin cross-sections of Bacillus subtilis labeled with thymidine-H(3), that resolutions of the same order were obtained for extended sources. It was concluded that an autoradiographic resolution of 1000 A could be achieved with a presently available commercial emulsion, although emulsions with finer grains might be desirable in some circumstances.

Autoradiography↗
PubMed1961

Electron microscopic radioautography of thin sections: the Golgi zone as a site of protein concentration in pancreatic acinar cells.

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections ( approximately 600 A) of OsO(4)-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4 degrees C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H(3) injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.

Acinar Cells↗
PubMed1961

Localization of macromolecules in Escherichia coli. I. DNA and proteins.

If thin sections of Escherichia coli, labeled uniformly with tritium, are radioautographed calculations, based on the distribution of section sizes show that the number of H(3) decays per section should be very close to a Poisson distribution. We might, therefore, expect that the distribution of radioautographic grain counts among random cross-sections should follow a Poisson distribution. It can then be inferred that a deviation from a Poisson indicates a high concentration of label in a preferred region. This region can then be identified by analysis of serial section and comparison with electron micrographs. Sections of cells labeled with leucine-H3 gave a Poisson distribution of grain counts, and it was concluded that proteins were distributed fairly uniformly throughout the cell. The situation was not changed if labeled cells were placed in chloramphenicol or if very short pulses of label were used. When Escherichia coli is grown in presence of chloramphenicol a major morphological change concerns the nuclear region: it becomes more regular in outline, nearly spherical, and occupies a smaller proportion of the cell length. The previously described association between DNA labeled with thymidine-H3 and the nuclear region was confirmed by showing that the distribution of the label in the cell followed exactly the morphological changes of the nuclear region. It was also shown that the concentration of DNA in the nuclear region was at least 45 times higher than that of the cytoplasm. Several morphological features of cells grown in chloramphenicol and examined in the electron microscope are discussed.

Autoradiography↗
PubMed1961

Localization of macromolecules in Escherichia coli. II. RNA and its site of synthesis.

The distribution of RNA in cells of E. coli 15 T(-)U(-) labeled with uridine-H(3) was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H(3) in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H(3), then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.

Cell Nucleus↗