Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.     

Intermediate filaments and stress

Before we can explain why so many closely related intermediate filament genes have evolved in vertebrates, while maintaining such dramatically tissue specific expression, we need to understand their function. The best evidence for intermediate filament function comes from observing the consequences of mutation and mis-expression, primarily in human tissues. Mostly these observations suggest that intermediate filaments are important in allowing individual cells, the tissues and whole organs to cope with various types of stress, in health and disease. Exactly how they do this is unclear and many aspects of cell dysfunction have been associated with intermediate filaments to date. In particular, it is still not clear whether the non-mechanical functions now being attributed to intermediate filaments are primary functions of these structural proteins, or secondary consequences of their function to respond to mechanical stress. We discuss selected situations in which responses to stress are clearly influenced by intermediate filaments.     

Change of composition of intermediate filaments in rat telencephalon during early postnatal period of ontogenesis

The goal of the present work was to study composition and spatial-temporal distribution of cells containing various proteins of intermediate filaments (nestin, vimentin, GFAP) in various brain areas at the early postnatal period of rat ontogenesis. By using methods of immunohistochemical determination of proteins of intermediate filaments, it has been found that at the early postnatal period of development, in the course of maturation of the nervous tissue, in the cells of cortex, hippocampus, and subventricular area, there occurred changes of immunohistochemical profile of intermediate filaments (ratio of immunopositive (+) and immunonegative (?) cells): nestin⁺/vimentin⁺/GFAP^? cells become nestin^?/vimentin^?/GFAP⁺.     

Developmental expression of the Glial fibrillary acidic protein (GFAP) gene in the mouse retina

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.     

Differential Silver Enhanced Double Labeling in Immunoelectron Microscopy

A two-sided double labeling method using protein A gold was used to demonstrate the presence of two hormones within the same anterior pituitary cell granule. A single probe size was used for both section faces but one side of the grid was silver enhanced. The use of a single probe size reduced the cost of the study and eliminated the variations in labeling efficiency that result from the use of different probe sizes.     

Abstracts

A simplification of the Schaeffer-Fulton spore stain for bacteria is presented. It is shown that omission of the heating step during staining with malachite green resulted in spore stains as good as when the heat was applied. The simplified procedure involves (1) heat fixation of the smear by 20 passages through the flame, (2) staining with saturated aqueous malachite green for 10 minutes, (3) rinsing, and (4) counterstaining with 0.25% aqueous safranin for 15 seconds. The omission of the heating step in staining has obvious advantages, particularly in the classroom.     

Immunoelectron microscopic study of somatostatin biosynthesis in dog pancreas

The biosynthesis of somatostatin has been studied at the ultrastructural level in pancreatic islets by using rabbit antiserum against synthetic somatostatin. To document that the antiserum specifically bound preprosomatostatin, we have tested the ability of the antiserum to precipitate the product synthesized in vitro. Poly(A) enriched RNA isolated from catfish islets was translated in both the wheat germ extract and nuclease-treated reticulocyte lysate systems. It was found that the in vitro translation product, preprosomatostatin, could be recognized by the antibody against synthetic somatostatin. The morphological study was then performed by immunoelectron microscopy by using the Fab-peroxidase conjugate technique. In dog pancreatic islets, somatostatin immunoreactive reaction product was seen only in the delta cells. In these cells, they were detected on bound ribosomes, in the cisternae of the rough endoplasmic reticulum (ER) and Golgi apparatus, in the Golgi associated vesicles, and in secretory vesicles. These findings suggest that somatostatin precursor molecules are synthesized on bound ribosomes and discharged into the cisternae of the rough ER. They are then transported to the Golgi apparatus and transferred to the secretory vesicles for secretion. The different staining intensities in the secretory vesicles would suggest that the processing of the precursor molecules of somatostatin probably takes place in the secretory vesicles.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Morphological Study of the Role of Calcium in the Assembly of the Rotavirus Outer Capsid Protein VP7

Maturation of rotavirus occurs in the endoplasmic reticulum (ER), a site of intracellular calcium storage. It was demonstrated previously that calcium plays an important role in the maturation of bovine rotavirus. We used protein A colloidal gold conjugated to an antibody to localize VP7, the outer capsid protein of the simian rotavius SA11, in permeabilized infected cells in the presence and absence of calcium in the culture medium. In medium containing calcium, VP7 was associated with nonenveloped double-shelled particles and membranous structures of the ER. In calcium-free medium, gold particles were not associated with the ER or with virus particles. Gold particles were distributed through the cytoplasm and were mainly associated with granular structures, but did not assemble onto virus particles. Our data suggest that in calcium-free medium, VP7 is synthesized, but does not remain incorporated, in the ER.     

The Determination of Comparable Labeling Densities in Quantitative Immunoelectron Microscopic Double Labeling Studies

In quantitative ultrastructural studies using colloidal gold immunocytochemical techniques, labeling intensities vary according to the size of the probe used. Using postembedded indirect two-sided double labeling and single labeling protocols, the labeling characteristics of four antigens were studied using two probe sizes commonly used in double labeling studies. It was determined that the labeling intensity variation resulting from the use of different probe sizes was unpredictable after correcting for the increased probe size alone. It was possible, however, to obtain comparable labeling densities by first determining the labeling intensities for each probe size with its antigen in single label studies on serial sections and using the same procedure as the double labeling studies. A probe size correction factor for each antigen was calculated from these data. This factor was used to obtain comparable measurements of the relative abundance of each label.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.