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Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly.

A cell line has been established in continuous culture of human cerebral cortical neurons obtained from a patient with unilateral megalencephaly, a disorder associated with continued proliferation of immature neuronal cells. When differentiated in the presence of nerve growth factor, 1-isobutyl-3-methylxanthine, and dibutyryl adenosine 3',5'-monophosphate (cAMP), the cells display mature neuronal morphology with numerous long, extensively branched processes with spines and varicosities. The cells stain positively for neurofilament protein and neuron-specific enolase (selective neuronal markers) but are negative for glial markers, such as glial fibrillary acidic protein, S-100, and myelin basic protein. The cells also stain positively for the neurotransmitters gamma-aminobutyric acid (GABA), glutamate, somatostatin, cholecystokinin-8, and vasoactive intestinal polypeptide. These cells may facilitate characterization of neurons in the human central nervous system.

1-Methyl-3-isobutylxanthine↗

Axonal transport and Alzheimer's disease.

In contrast to most eukaryotic cells, neurons possess long, highly branched processes called axons and dendrites. In large mammals, such as humans, some axons reach lengths of over 1 m. These lengths pose a major challenge to the movement of proteins, vesicles, and organelles between presynaptic sites and cell bodies. To overcome this challenge axons and dendrites rely upon specialized transport machinery consisting of cytoskeletal motor proteins generating directed movements along cytoskeletal tracks. Not only are these transport systems crucial to maintain neuronal viability and differentiation, but considerable experimental evidence suggests that failure of axonal transport may play a role in the development or progression of neurological diseases such as Alzheimer's disease.

Aging↗

Interstitial cells in deep muscular plexus of canine small intestine may be specialized smooth muscle cells.

The fine structures and properties of cells between the inner and outer circular muscle layers in the canine small intestine were studied by transmission electron microscopy (TEM), immunocytochemistry, and scanning electron microscopy (SEM). A nerve plexus (deep muscular plexus) supported by enteroglial cells, fibroblasts around blood vessels, macrophages, and thin and branched cells previously identified as interstitial cells of Cajal was observed. The interstitial cells of the deep muscular plexus (IC-DMP) were rich in mitochondria, dense bodies, and caveolae, and they were closely associated with nerve fibers. The IC-DMP had incomplete basal laminae. These cells also had numerous interconnecting gap junctions, and they also formed gap junctions with the surrounding smooth muscle cells of the outer circular muscle layer. IC-DMP were rich in myofilaments, which were primarily actin thin filaments, but myosin thick filaments, identified with anti-myosin light-chain antibodies, were also apparent. IC-DMP and circular smooth muscle cells both expressed immunoreactivity to anti-smooth muscle actin antisera, but these two types of cells differed in their intermediate filament proteins: IC-DMP featured vimentin immunopositive filaments, and circular smooth muscle cells featured desmin immunoreactivity. SEM showed that IC-DMP had thin and flat cell bodies with numerous branching processes. These cells came into close contact with nerve fibers and circular smooth muscle cells. The findings that IC-DMP cells contained myosin thick filaments and were immunopositive for anti-smooth muscle actin suggest that they may be more properly categorized as a type of smooth muscle cell.

Animals↗

An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice.

Regulation of the surfactant protein B gene (SP-B) is developmentally controlled and highly tissue specific. To elucidate the SP-B gene temporal/spatial expression pattern in lung development at the transcriptional level, a transgenic mouse model line carrying the human SP-B (hSP-B) 1.5-kb 5'-flanking regulatory region and the lacZ gene was established. Expression of hSP-B 1.5-kb lacZ gene started at the onset of lung formation [embryonic day 9 (E9)] and was restricted to epithelial cells throughout prenatal and postnatal lung development. In the adult lung, hSP-B 1.5-kb lacZ gene expression was restricted to bronchiolar and alveolar type II epithelial cells. In lung explant culturing studies, the hSP-B 1.5-kb lacZ gene was highly expressed in newly formed epithelial tubules during the respiratory branching process. In a second transgenic mouse line, an enhancer region, which binds to thyroid transcription factor-1, retinoic acid receptor, signal transducers and activators of transcription 3, and nuclear receptor coactivators (SRC-1, ACTR, TIF2, and CBP/p300), was deleted from the hSP-B 1.5-kb lacZ gene. The deletion abolished hSP-B lacZ gene expression in bronchiolar epithelial cells and significantly reduced its expression level in alveolar type II epithelial cells in transgenic mice.

Animals↗

Renal afferent and efferent arterioles of the rabbit.

The present study employed our microdissection-digestion technique to determine the three dimensional organization of the walls of the afferent and efferent arterioles of the adult rabbit kidney. Each smooth muscle cell of an afferent arteriole is characterized by a somal enlargement and two lateral processes. The cells of the distal segment of the afferent arterioles are modified into myoepithelioid cells, which are closely apposed to one another. The cells of the proximal efferent arteriole are irregularly shaped and regions of adjacent cells may be separated from one another so that gaps exist and areas of the underlying endothelium are not covered by smooth muscle cells. The cells of the distal segments of efferent arterioles are multipolar pericytes, of which the processes branch. Thus the afferent arteriole possesses a complete muscular wall while only the proximal efferent possesses smooth muscle cells with a sphincteric orientation. The distal efferent arteriole possesses pericytes that appear to have a limited capability to modulate renal hemodynamics.

Animals↗

HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase.

The association of hepatocyte growth factor (HGF) with its high-affinity receptor, c-met, has been shown to induce mitogenesis, motogenesis, and morphogenesis in renal epithelial cells (L. G. Cantley, E. J. G. Barros, M. Gandhi, M. Rauchman, and S. K. Nigam. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F271-F280, 1994), suggesting that HGF may be critical to the orchestration of both renal development and regeneration following injury. Although signal transduction pathways activated by c-met include the phosphatidylinositol 3-kinase (PI-3-kinase), phospholipase C gamma, ras, and others, the activation of PI-3-kinase has been the most striking in vivo. We therefore investigated whether the pathways that mediate phenotypic changes in inner medullary collecting duct cells are altered by inhibition of PI-3-kinase with the fungal metabolite, wortmannin. In these cells, the mean inhibitory concentration for in vitro wortmannin inhibition of PI-3-kinase was approximately 0.2 nM. At this low concentration, motogenesis (quantified by chemotaxis) and morphogenesis (by branching-process formation within collagen matrix) were inhibited in a striking and parallel fashion, while mitogenesis was inhibited to a lesser degree. These experiments suggest that activation of PI-3-kinase is critical for c-met-mediated chemotaxis and tubulogenesis.

Androstadienes↗

Folliculo-stellate cells of human pituitary adenomas: immunohistochemical study of the monocyte/macrophage phenotype expression.

Folliculo-stellate cells (FS) represent a small percentage of anterior pituitary elements of still undetermined embryological origin. They are sparse among endocrine pituitary cells and are characterized by the lack of secretory granules and by the presence of few branching processes inserted between hormone-secreting cells. Although FS cell role is still under discussion, recent reports showed that they produce monocyte-derived cytokines able to influence the hormone production and modulate the immunoendocrine connections. In this study we applied three monocyte-macrophage markers (HAM56, KP1, HLA-DR) to 15 pituitary adenomas in order to ascertain whether FS cells belong to the macrophage lineage. In this case FS cells could be considered the resident macrophages of the pituitary. FS cells were identified according to the reactivity to S-100, GFAP and vimentin. We confirm that S-100 represents the most useful marker for these cells that were detected scattered between tumor cells in more than half of the adenomas. GFAP stained only a percentage of FS cells, while vimentin recognized in addition to stellate cells endothelia, perivascular and infiltrating macrophages. We were unable to detect the expression of the macrophage markers on S-100 and GFAP reactive cells. Indeed, HAM56, KP1 and HLA-DR-positive cells were mostly round, small size and located in the perivascular and septal positions where FS cells were never detected. Lack of expression of monocyte-macrophage lineage markers by FS cells in pituitary adenomas suggests their preferential neuroectodermal origin. However, further studies on normal human pituitary will be needed before ruling out a possible role for FS cells as resident pituitary macrophages.

Adenoma↗

Langerhans' cells in squamous metaplasia of the human uterine cervix.

The number and shape of Langerhans' cells (LC) were studied by determining cytoplasmic formaldehyde-resistant ATPase activity in whole mounts of normal and metaplastic human cervical epithelium. In normal epithelium the number of LC per square millimeter was 52.75 +/- 2.21. A similar number was found in completely differentiated metaplastic squamous epithelium (49.11 +/- 2.42), but their shape was different with less branching processes. When metaplasia was still incomplete, and numerous mucous cells remained, no LC were present. On the basis of these results it is speculated that mucous cells provide a negative chemotactic stimulus which prevents migration of LC into metaplastic epithelium. When the latter is completely squamous it is repopulated by LC in a fashion similar to normal squamous epithelium.

Adult↗

Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.

Angiotensin II↗

Hsp27 and axonal growth in adult sensory neurons in vitro.

BACKGROUND: Neurite growth can be elicited by growth factors and interactions with extracellular matrix molecules like laminin. Among the targets of the signalling pathways activated by these stimuli are cytoskeletal elements, such as actin, tubulin and neurofilaments. The cytoskeleton can also be modulated by other proteins, such as the small heat shock protein Hsp27. Hsp27 interacts with actin and tubulin in non-neuronal cells and while it has been suggested to play a role in the response of some neurons to injury, there have been no direct studies of its contribution to axonal regeneration. RESULTS: We have investigated neurite initiation and process extension using cultures of adult dorsal root ganglion (DRG) sensory neurons and a laminin stimulation paradigm. Employing confocal microscopy and biochemical analyses we have examined localization of Hsp27 at early and later stages of neurite growth. Our results show that Hsp27 is colocalized with actin and tubulin in lamellopodia, filopodia, focal contacts and mature neurites and growth cones. Disruption of the actin cytoskeleton with cytochalasin D results in aberrant neurite initiation and extension, effects which may be attributable to alterations in actin polymerization states. Inhibition of Hsp27 phosphorylation in our cultures results in an atypical growth pattern that may be attributable to an effect of pHsp27 on the stability of the actin cytoskeleton. CONCLUSION: We observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early stages of neurite growth, and in processes, branch points and growth cones at later stages, suggests that Hsp27 may play a role in neuritogenesis and subsequent neurite extension, and potentially in the patterning of this growth. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells. Our results suggest that this may also be the case in neurons and support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin.

Animals↗

Tiling of the Drosophila epidermis by multidendritic sensory neurons.

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.

Animals↗

Effect of calcium on oxytocin-induced contraction of mammary gland myoepithelium as visualized by NBD-phallacidin.

The effect of calcium on oxytocin-induced contraction of myoepithelial cells was visualized with NBD-phallacidin, a fluorescent stain for filamentous actin. In the absence of oxytocin, the cells appeared relaxed; long, branching processes radiated from the cell bodies. In the presence of 50 nM-oxytocin, myoepithelial cells contracted into smaller spoke-shaped bodies in which the arms were shorter and thicker. Electron microscopy confirmed the morphological differences between oxytocin-treated and untreated myoepithelium. To determine a role for extracellular calcium, tissue was incubated in EGTA, then exposed to oxytocin, with or without added calcium. Contraction occurred in the presence of oxytocin plus additional calcium but not in the absence of calcium. When the tissue was incubated with the calmodulin antagonist trifluoperazine (TFP) in calcium-containing medium, oxytocin did not induce myoepithelial cell contraction. These data support previous results obtained with a myosin light-chain phosphorylation assay implicating calcium and calmodulin in oxytocin-induced contraction. Furthermore, NBD-phallacidin visualization of myoepithelial cells demonstrates that the effect of calcium on contraction is physiologically significant.

Amanitins↗

Long-term growth in vitro of isolated, fully differentiated neurones from the central nervous system of an adult insect.

A method is described for the isolation and growth in vitro of fully differentiated neurones from the thoracic ganglia of adult cockroaches. The presence of insect blood in the culture system is shown to promote growth. The morphology of the growing neurones and the plasticity of the branching processes are described and growth rates are measured. Using a fluorescent Ca2+ indicator dye, changes of intracellular calcium levels in the growing neurones in response to K+ depolarization have been measured. The results, indicating the presence of voltage-dependent Ca2+ channels on neuronal processes in vitro, show that neurones can be maintained in a functional state for several weeks by this technique. Such preparations could prove useful for studying a variety of physiological and pharmacological properties of neurones, including the mechanisms controlling growth, synapse formation and neuronal interactions with other cell types.

Animals↗

The Hill-Robertson effect and the evolution of recombination.

In finite populations, genetic drift generates interference between selected loci, causing advantageous alleles to be found more often on different chromosomes than on the same chromosome, which reduces the rate of adaptation. This "Hill-Robertson effect" generates indirect selection to increase recombination rates. We present a new method to quantify the strength of this selection. Our model represents a new beneficial allele (A) entering a population as a single copy, while another beneficial allele (B) is sweeping at another locus. A third locus affects the recombination rate between selected loci. Using a branching process model, we calculate the probability distribution of the number of copies of A on the different genetic backgrounds, after it is established but while it is still rare. Then, we use a deterministic model to express the change in frequency of the recombination modifier, due to hitchhiking, as A goes to fixation. We show that this method can give good estimates of selection for recombination. Moreover, it shows that recombination is selected through two different effects: it increases the fixation probability of new alleles, and it accelerates selective sweeps. The relative importance of these two effects depends on the relative times of occurrence of the beneficial alleles.

Alleles↗

Histological identification of the interstitial cells of Cajal in the guinea-pig small intestine.

In order to clarify the contraversial structure that Cajal (1889, 1893, 1911) called "interstitial cells" in the intestine, we have compared the descriptions by Cajal with our recent findings. Cajal defined three groups of interstitial cells in the intestine: those in the mucosa, the deep muscular plexus, and the myenteric plexus. In the deep muscular plexus, he described cells with small perikarya and long, branching processes. Neither glial cells nor ZIO positive fibroblast-like cells, the only cell types seen in the numbers and location necessary to support Cajal's observations, conformed with the morphology he described. Cajal might have described a composite cell, or chimera, the cell body being that of a glial (or perhaps a fibroblast-like) cell and the processes being simultaneously stained neurites that ran in close association with the cell. In the myenteric plexus, staining with S-100 protein for glial cells and ZIO for fibroblast-like cells reveal cells between the external muscle layers. Cells stained with S-100 protein resemble the drawings and also his description of the interstitial cells, although these cells appear to be less frequent than Cajal's drawings indicate. There are numerous glial cells in the ganglia and primary strands of the plexus which were not included in Cajal's publications. Conversely, the fibroblast-like cells are incidentally associated with nerve fibers. Although they lie in the same plane, the fibroblast-like cells form a pattern distinct from that of the nerve fibers of the tertiary component of the myenteric plexus. These cells occur in the distribution and numbers of the interstitial cells described by Cajal. They are stained by ZIO and also immunoreactive for gamma-aminobutyric acid. They can be identified by scanning electron microscopy. In the mucosa, Golgi staining reveals a pattern of nerve fiber bundles that is indistinguishable from Cajal's drawings. Glial cell bodies frequently occur at the intersections of these bundles and appear as cell nuclei surrounded by a cytoplasm which is actually the stained nerve fiber bundles. What Cajal depicted as interstitial cells were composite structures consisting of glial cells and contiguous nerve fiber bundles. We conclude that the structures which Cajal called interstitial cells in the intestine do not originate from one cell type. Nevertheless, two groups of fibroblast-like cells, those lying parallel and close to the nerve strands of the deep muscular plexus and those of the myenteric plexus, can be recognized by ZIO staining and scanning electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Three-dimensional visualization of renal cells by NaOH maceration.

The three-dimensional fine structure of cells composing the renal tissue was demonstrated by SEM after the removal of extracellular matrices by NaOH maceration. This paper focuses on glomerular mesangial cells, Goormaghtigh's cells (extraglomerular mesangial cells), and epithelial cells in the thin limbs of Henle's loop in the rat, rabbit and dog. Mesangial cells reveal rough surfaces covered with short microvilli. The cells extend long branching processes in close association with the glomerular capillary, suggesting a role for them of regulating the capillary caliber. The mesangial cells interdigitate with each other by their microvilli, forming an intercellular labyrinth. Goormaghtigh's cells at the glomerular hilus are also covered with microvilli, which form narrow labyrinthine spaces between the cells. The labyrinth among the mesangial cells and that among Goormaghtigh's cells connect with each other at the hilus, giving rise to a channel system leading from the periphery of the glomerulus through the hilus to the interstitial space outside the glomerulus. Renal tubule cells display complicated intra- and intercellular interdigitations on the basal aspect. The pattern of epithelial interdigitation is specific to each tubular segment. The descending and ascending thin limbs of the long loops demonstrate a striking contrast to each other. The former is characterized by moderate intercellular interdigitation and by numerous microvilli on the lateral and basal surfaces; the latter is marked by elaborate, pectineal interdigitations, and by smooth basolateral surfaces.

Animals↗

Colocalization of neuropeptide Y with other neurochemical markers in the guinea-pig small intestine.

The chemical coding and projections of neurons containing neuropeptide Y (NPY) have been investigated in the myenteric plexus of the guinea-pig small intestine. Chemical coding was determined by investigating the colocalization of NPY immunoreactivity with the immunoreactivities for bombesin (BN), 5-HT, nitric oxide synthase (NOS), and somatostatin. Projections were determined by studying the consequences of nerve lesions created by myectomy and myotomy operations. NPY immunoreactivity was localized in four classes of myenteric neuron, anally projecting interneurons, neurons that projected anally and to the circular muscle, neurons projecting to the longitudinal muscle and in a small population of secretomotor neurons that projected to the mucosa. The interneurons and muscle motor neurons both had Dogiel type I morphology, whereas the secretomotor neurons had fine branching processes. Of the NPY-immunoreactive Dogiel type I neurons, 98% were also immunoreactive for NOS; conversely, 82% of NOS-immunoreactive neurons were immunoreactive for NPY. BN was also colocalized with NPY and NOS; 30% of the NPY-immunoreactive neurons were BN/NOS/NPY-immunoreactive. No nerve cells had BN and NPY immunoreactivity without NOS immunoreactivity. The presence of NPY immunoreactivity was investigated in three classes of descending interneurons that are distinguished by their reactivities for somatostatin, 5-HT and NOS. NPY immunoreactivity was never colocalized with 5-HT or somatostatin, but most NPY-immunoreactive descending interneurons whose terminals formed pericellular baskets were also reactive for BN and NOS. The average projection lengths of the NPY interneurons was 2-3 mm, in the anal direction. Evaluation of immunoreactivity for BN, NOS and NPY revealed three major populations of anally directed circular muscle motor neurons, with BN/NOS/NPY, BN/NOS and NOS/NPY immunoreactivities. Examination of simultaneous labeling after nerve lesions showed that NOS/NPY neurons had short anal projections, averaging about 2-3 mm, and neurons with BN immunoreactivity were longer, having average projections of about 5-8 mm.

Animals↗

Evolution of resistance to cancer therapy.

Acquired drug resistance is a major limitation for successful treatment of cancer. Resistance emerges due to drug exclusion, drug metabolism and alteration of the drug target by mutation or overexpression. Depending on therapy, the type of cancer and its stage, one or several genetic or epigenetic alterations are necessary to confer resistance to treatment. The fundamental question is the following: if a genetically diverse population of replicating cancer cells is subjected to chemotherapy that has the potential to eradicate it, what is the probability of emergence of resistance? Here, we review a general mathematical framework based on multi-type branching processes designed to study the dynamics of escape of replicating organisms from selection pressures. We apply the general model to evolution of resistance of cancer cells and discuss examples for diverse mechanisms of resistance. Our theory shows how to estimate the probability of success for any treatment regimen.

Animals↗