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Cellular heterogeneity in cerebral cortex: a study of the morphology of pyramidal neurones in visual areas of the marmoset monkey.

The morphological characteristics of the basal dendritic fields of layer III pyramidal neurones were determined in visual areas in the occipital, parietal, and temporal lobes of adult marmoset monkeys by means of intracellular iontophoretic injection of Lucifer yellow. Neurones in the primary visual area (V1) had the least extensive and least complex (as determined by Sholl analysis) dendritic trees, followed by those in the second visual area (V2). There was a progressive increase in size and complexity of dendritic trees with rostral progression from V1 and V2, through the "ventral stream," including the dorsolateral area (DL) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively). Neurones in areas of the dorsal stream, including the dorsomedial (DM), dorsoanterior (DA), middle temporal (MT), and posterior parietal (PP) areas, were similar in size and complexity but were larger and more complex than those in V1 and V2. Neurones in V1 had the lowest spine density, whereas neurones in V2, DM, DA, and PP had similar spine densities. Neurones in MT and inferotemporal cortex had relatively high spine densities, with those in ITr having the highest spine density of all neurones studied. Calculations based on the size, number of branches, and spine densities revealed that layer III pyramidal neurones in ITr have 7.4 times more spines on their basal dendritic fields than those in V1. The differences in the extent of, and the number of spines in, the basal dendritic fields of layer III pyramidal neurones in the different visual areas suggest differences in the ability of neurones to integrate excitatory and inhibitory inputs. The differences in neuronal morphology between visual areas, and the consistency in these differences across New World and Old World monkey species, suggest that they reflect fundamental organisational principles in primate visual cortical structure.

Animals↗

Cellular heterogeneity and drug resistance in two ovarian adenocarcinoma cell lines derived from a single patient.

Two ovarian cell lines were derived from the ascites of a patient before and after the onset of resistance to chemotherapy involving cis-platinum, chlorambucil and 5-fluorouracil. Characterization of these lines shows them to have various features in common and some significant differences. Cytologically the lines cannot be distinguished and they both contain high concentrations of oestrogen receptor. However, they do differ with respect to their growth characteristics, karyotype, glutathione content and sensitivity to cis-platinum. The karyotypes of the 2 lines show several marker chromosomes in common but the resistant line contained a chromosome 8 and a 17 which were absent from the earlier sensitive line. This suggests a clonal origin with subsequent divergence to a heterogeneous population.

Adenocarcinoma↗

Molecular and cellular heterogeneity of Wilms' tumor.

We developed a Wilms' tumor-cell culture system to investigate the molecular basis of nephrogenesis and oncogenesis. Several distinct fractions of cells were isolated and characterized from the same tumor specimen. The cells exhibited striking differences in morphology, immunocytochemical staining profiles and cytogenetics. One fraction contained cells with features of epithelium; other cell fractions resembled partially differentiated mesenchyme (blastema or stroma). While the Wilms' tumor-suppressor gene WT1 was not altered, loss of heterozygosity (LOH) and an insertion in intron I of the p53 tumor-suppressor gene occurred in the tumor and the cultured cell types. LOH for RB was detected only in the cultured cells. These findings are consistent with a model of tumor initiation in a pluripotent cell that is able to undergo subsequent differentiation along multiple different lines and which mimics normal nephrogenesis.

Cell Separation↗

Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors.

Thrombin binding to formaldehyde-fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot-like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of 125I-thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin-binding to high-affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4 degrees C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of 125I-thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.

Amyloid beta-Protein Precursor↗

Transforming growth factor beta 1-induced cellular heterogeneity in the periosteum of rat parietal bones.

We examined the osteogenesis process in transforming growth factor beta 1 (TGF-beta 1)-treated neonatal and adult rats, aiming to investigate the age difference in the effect of TGF-beta 1 on mesenchymal cell differentiation. Recombinant human (rh) TGF-beta 1 (20 and 200 ng) was injected onto the outer periostea of the right side of the parietal bone of each rat once a day for 1-12 days starting at the age of either 1 day or 12 weeks. On the day after the final injection, the calvaria was excised and evaluated histologically. In the neonates, the 12-day treatment with rhTGF-beta 1 increased the number of osteoprogenitor cells, resulting in intramembranous ossification. In the adult rats, rhTGF-beta 1 induced differentiation of chondrocytes. Cartilage masses were surrounded by mesenchymal cells, which would differentiate into chondrocytes. The cartilage matrix was partially calcified, with chondrocytes buried therein. In the calcified matrix, marrow cavities containing some multinuclear osteoclasts were formed. These findings indicate that rhTGF-beta 1 stimulated the differentiation of mesenchymal cells into chondrocytes and produced the cartilaginous matrix. rhTGF-beta 1 induced intramembranous ossification of the parietal bone in neonatal rats, and it induced enchondral ossification in adults. This result suggests that the different responses of mesenchymal cells in the periosteum to rhTGF-beta 1 may depend on the age of the animals used: namely, they may reflect the respective osteogenic stages of modeling and remodeling.

Aging↗

Characterization of human mammary cell types in primary culture: immunofluorescent and immunocytochemical indicators of cellular heterogeneity.

Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal cells and large, flat cells are related to mammary epithelial cells. whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types.

Adult↗

Analysis of cellular heterogeneity in mouse thymus cultures.

Analysis of 5 to 6 d primary cultures of cells derived from murine thymus glands revealed a heterogeneous population of cells rather than "pure" reticuloepithelial cell cultures as was assumed previously by other investigators. The monolayer cultures consisted of at least three cell types: thymus epithelial cells, macrophagelike epithelioid cells, and fibroblasts. Surprisingly, about 50% of the cells had positive cytochemical staining reactions for acid phosphatase and nonspecific esterase. The sme cells phagocytized carbon particles, latex beads, and yeast. Furthermore, these cells could be removed from the initial cell suspension by phagocytosis of carbonyl iron, followed by magnetic separation, but once they had adhered to the substratum they were resistant to trypsin removal. All of these findings supported the conclusion that about 50% of the cells in the monolayers were macrophages. The other cells present were thymus epithelial cells and a small number of fibroblasts. Both of the latter types of cell were cytochemically negative, did not phagocytize particulate material, and were not removed by carbonyl iron treatment, but were removed by treating the monolayer with trypsin. The findings in this report indicated that epithelioid morphology alone was inadequate to identify correctly the cell types found in thymus cultures and that the use of such cultures as a model to study in vitro the maturation of certain immunological functions has been based on assumptions here shown to be incorrect.

Acid Phosphatase↗

Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells.

Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice.

Animals↗

Cellular heterogeneity in the expression of the delta-crystallin gene in non-lens tissue.

RNA transcripts of the delta-crystallin genes, which code for the major chicken lens protein, have been detected at low levels in many non-lens tissues. Here it is demonstrated by in situ hybridisation that these transcripts are concentrated at a high level in small, infrequent clusters of cells in many non-lens tissues. While the nuclei of these cells are very heavily labelled, there is only light labelling of the cytoplasm. The unlabelled cells surrounding the labelled clusters are of similar morphology and staining properties as the labelled cells, and all have the characteristic morphology of cells of the embryonic tissue used. With the exception of neural retina, it is not yet known whether the labelled clusters are found in specific locations in the tissues, or whether they arise at random.

Animals↗

Cellular heterogeneity in the ependymal layer of the chicken's lumbosacral spinal cord.

Cytological development in the ventricular zone of the chicken's lumbosacral spinal cord was studied by light and transmission electron microscopy before and after hatching. Two morphologically distinct types of cell were first noted by embryonic stage 41 and were present in the 2-day-old chick. From cell bodies located at the border of the ependymal layer, a lightly stained process extended among the more numerous and typical ependymal cells to reach the central canal. Synapses were observed only on the cell bodies of the lightly stained cells.

Animals↗

The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.

The nucleocapsid (N) protein of SARS coronavirus (SARS_CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is related to the pre-mRNA splicing in the nucleus and translation regulation in the cytoplasm. In this report, based on the relevant biophysical and biochemical assays, the nucleocapsid protein of SARS_CoV (SARS_N) was discovered to exhibit high binding affinity to human hnRNP A1. GST pull-down results clearly demonstrated that SARS_N protein could directly and specifically bind to human hnRNP A1 in vitro. Yeast two-hybrid assays further indicated in vivo that such binding relates to the fragment (aa 161-210) of SARS_N and the Gly-rich domain (aa 203-320) of hnRNP A1. Moreover, kinetic analyses by surface plasmon resonance (SPR) technology revealed that SARS_N protein has a specific binding affinity against human hnRNP A1 with K(D) at 0.35 +/- 0.02 microM (k(on) = 5.83 +/- 0.42 x 10(3) M(-1)s(-1) and k(off) = 2.06 +/- 0.12 x 10(-3)s(-1)). It is suggested that both SARS_N and hnRNP A1 proteins are possibly within the SARS_CoV replication/transcription complex and SARS_N/human hnRNP A1 interaction might function in the regulation of SARS_CoV RNA synthesis. In addition, the determined results showed that SARS_N protein has only one binding domain for interacting with human hnRNP A1, which is different from the mouse hepatitis virus (MHV) binding case where the nucleocapsid protein of MHV (MHV_N) was found to have two binding domains involved in the MHV_N/hnRNP A1 interaction, thereby suggesting that SARS_N protein might carry out a different binding mode to bind to human hnRNP A1 for its further function performance in comparison with MHV_N.

Clone Cells↗