A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus

Summary Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Glutamate and GABA receptors in vertebrate glial cells

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Molecular and genetic analysis of condensin function in vertebrate cells

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.     

Electron microprobe analysis in periodontal guided tissue regeneration.

Electron microprobe analysis was used to determine the evolution of Ca, P and S in regenerated tissue surrounding incisors roots after periodontal treatment with guided tissue regeneration. Our results, which showed increased Ca and P, and decreased S are discussed in relation to the process of mineralization electron probe microanalysis with potentially provided an accurate means of assessing the degree of mineralization in extremely small tissue samples.     

Genetic analysis of homologous DNA recombination in vertebrate somatic cells

The maintenance of genomic stability and the ability to repair induced DNA damage in vertebrate cells require homologues of the yeast RAD52 epistasis group genes. The homologous recombination carried out by the products of these genes is essential and appears to be closely linked to DNA replication. Defects in recombination and associated activities are implicated in human cancer. This review summarises recent biochemical and genetic findings on the roles played by the vertebrate RAD52 group gene products in recombination. We describe the phenotypic analysis of genetically engineered mammalian and chicken mutants of homologous recombination genes.     

Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L-form cells of streptomycetes

Protoplast type L-form cells of Streptomyces hygroscopicus and S. griseus contain different types of inclusion bodies. Cytoplasmic cores and paracrystalline structures are peculiar inclusions which could not be observed in normal parent bacteria. The cytoplasmic cores are 1-4 micron long and 0.05-0.25 micron broad straight and stiff non-tubular structures consisting of homogeneous mode-rate electron opaque material. Paracrystalline inclusions have side-lengths between 0.2 and 0.5 micron and show a characteristic pattern of 15-20 nm thick straight dark lines and electron lucent intervening spaces of 20-30 nm. Both cytoplasmic cores and paracrystalline inclusions are apparently proteins. Their occurrence in L-form cells indicates an altered synthesis of one or several proteins in these cell types.     

Tip-link integrity and mechanical transduction in vertebrate hair cells.

An attractive hypothesis for hair-cell transduction is that fine, filamentous "tip links" pull directly on mechanically sensitive ion channels located at the tips of the stereocilia. We tested the involvement of tip links in the transduction process by treating bundles with a BAPTA-buffered, low-Ca2+ saline (10(-9) M). BAPTA abolished the transduction current in a few hundred milliseconds. BAPTA treatment for a few seconds eliminated the tip links observed by either scanning or transmission electron microscopy. BAPTA also eliminated the voltage-dependent movement and caused a positive bundle displacement of 133 nm, in quantitative agreement with a model for regulation of tension. We conclude that tip links convey tension to the transduction channels of hair cells.     

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

Response latency of vertebrate hair cells.

An in vitro preparation of hair cells from the bullfrog sacculus produces a transepithelial microphonic potential in response to well-defined mechanical stimuli. If corrected for the electrical time constant of the epithelium, the response follows a fast stimulus with a 40-microsecond delay at 22 degree C. The short latency and its modest temperature dependence limit possible models for transduction by hair cells.     

Ultrastructural analysis of basal neuroepithelial cells in dysraphic mice

Ultrastructural aspects of the cellular pathology in the basal neuroepithelium of the hindbrain and spinal cord were analyzed in dysraphic loop-tail mice at nine days of gestation. Whereas the basal cytoplasm of the neuroepithelium in normal littermates showed a consistent electron density, the neuroepithelium in abnormal embryos was characterized by "light" and "dark" cells scattered randomly along the basal aspect of the hindbrain and spinal cord. In the abnormals, gaps occurred in the neural basal lamina, and the neuroepithelial cells often were in direct contact with cytoplasmic processes from mesenchymal cells and from notochordal cells; in normal littermates, contact was observed only between the intact and continuous neural basal lamina and mesenchymal cells and notochordal cells. Thus, it is possible that the pathological features observed ultrastructurally in the basal neuroepithelium in dysraphic embryos may represent faulty tissue interaction with adjacent notochordal and mesenchymal cells.     

Ultrastructural analysis of membrane-bound polysomes in human myeloma cells

We prepared ultra-thin sections of human myeloma cells, in which the rER was cut tangentially, and studied the make-up and distribution of membrane-bound polysomes electromicroscopically. In IgG myeloma large and small polysomes were detected. The polysome distribution curve showed a high peak at 7 ribosomes and a lower peak at 17-18 ribosomes. IgA-, IgD- and IgE myeloma, as well as macroglobulinemia, showed peaks at 7 and 13 ribosomes. BJP myeloma manifested a sharp peak only at 7 ribosomes. Our results suggest that BJP myeloma has only small polysomes participating in L-chain synthesis, while the other myelomas exhibited large and small polysomes participating in H- and L-chain synthesis, respectively. The quantitative ratio of small and large polysomes was determined on the basis of an analytically corrected direct count.