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Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2.

Rett syndrome is caused by loss-of-function mutations in the gene encoding the methyl DNA-binding factor MeCP2. As brain mass and neuronal complexity tend to be diminished in Rett patients, we tested whether MeCP2 directly influences the morphological complexity of developing neurons. Our results show that cultured mouse neurons overexpressing MeCP2beta (MECP2A) develop more complex morphologies, having longer axonal and dendritic processes, and an increased number of axonal and dendritic terminal endings. We then tested whether overexpressing a mutant form of MeCP2beta lacking its carboxyl terminus would elicit the same effects. Interestingly, while neurons overexpressing this mutant failed to enhance axonal and dendritic process elongation, the complexity of their axonal and dendritic processes remained significantly elevated. Taken together, these data support the hypothesis that MeCP2 directly regulates neuronal maturation and/or synaptogenesis, and provides evidence that MeCP2 may influence neuritic elongation and process branching through different mechanisms.

Animals↗

PubMed2004

Neuronal morphological change of size-sieved stem cells induced by neurotrophic stimuli.

Size-sieved stem cells (SSCs) derived from human bone marrow have the ability to differentiate into bone, fat and cartilage. SSCs can differentiate into active neural cells after exposure to antioxidant agents. The aim of the present study is to understand if SSCs can be stimulated to differentiate into neurons in response to neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), pituitary adenylate cyclase-activating polypeptide (PACAP) and dibutyryl cAMP (dbcAMP). SSCs in a serum-free medium transform from a fibroblastic-like form to a multipolar morphology. Treatment of SSCs with GDNF, PACAP, and dbcAMP increased the production of neurofilament light protein (NF-L) and a cytoskeleton protein-alpha-tubulin. Examination of a vesicle protein-synapsin-1 or a neuronal progenitor marker-internexin in SSCs indicated that treatment with GDNF, PACAP, and dbcAMP further elongated cell processes and increased process branching. The findings indicate that neurotrophic signaling and cAMP-dependent signaling might promote the neuronal differentiation of SSCs.

Blotting, Western↗

PubMed2006

Prismatic dentine in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi).

The Australian lungfish, Neoceratodus forsteri, has a dentition consisting of enamel, mantle dentine and bone, enclosing circumdenteonal, core and interdenteonal dentines. Branching processes from cells that produce interdenteonal dentine leave the cell surface at different angles, with collagen fibrils aligned parallel to the long axis of each process. In the interdenteonal dentine, crystals of calcium hydroxyapatite form within fibrils of collagen, and grow within a matrix of non-collagenous protein. Crystals are aligned parallel to the cell process, as are the original collagen fibrils. Because the processes are angled to the cell surface, the crystals within the core or interdenteonal dentine are arranged in bundles set at angles to each other. Apatite crystals in circumdenteonal dentine are finer and denser than those of the interdenteonal dentine, and form outside the fibrils of collagen. In mature circumdenteonal dentine the crystals of circumdenteonal dentine form a dense tangled mass, linked to interdenteonal dentine by isolated crystals. The functional lungfish tooth plate contains prisms of large apatite crystals in the interdenteonal dentine and masses of fine tangled crystals around each denteon. This confers mechanical strength on a structure with little enamel that is subjected to heavy wear.

Animals↗

PubMed2003

Immunohistochemical identification of antigen presenting cells in rat salivary glands.

Mucosal dendritic cells affect immune responses through secretion of cytokines and exposure of naïve B- and T-lymphocytes to foreign matter as antigen presenting cells (APCs). APC in oral tissues may play a role in the development of local and secretory immune responses [Crit. Rev. Oral Biol. Med. 7 (1996) 36]. Previous studies have shown that APC are present in the interstitial tissues of rat salivary glands [Arch. Oral Biol. 40 (1995) 1015]. This study sought to further define the distribution of APC in salivary glands. The major glands and ducts of male Sprague-Dawley and Wistar rats were fixed with 4% paraformaldehyde and prepared for immunofluorescence and pre- and post-embedding immunoelectron microscopy. Monoclonal antibodies to the dendritic cell marker Ia antigen (OX-6 antibody), monocyte lineage cytoplasmic antigen (ED-1), and resident tissue macrophage antigen (ED-2) were visualized with FITC-conjugated secondary antibodies for light microscopy and HRP- and gold-labelled secondary antibodies for electron microscopy. Light microscopy revealed numerous OX-6-positive cells with branching processes in the epithelium of striated and excretory ducts of both rat strains, as well as in the connective tissue stroma. ED-1-positive cells had a similar distribution but exhibited a more compact shape with fewer processes. ED-2-positive cells were found only in the connective tissue. Acinar and duct epithelial cells were unreactive. Electron microscopy confirmed that both OX-6-positive and ED-1-positive, non-epithelial cells were present within the duct epithelium. The presence of APC in the duct epithelium suggests that these ducts may be exposed to antigens, possibly by retrograde access from the oral cavity, and that APC located in the salivary gland epithelium may participate in local immune responses.

Animals↗

PubMed2003

Growth velocities of branched actin networks.

The growth of an actin network against an obstacle that stimulates branching locally is studied using several variants of a kinetic rate model based on the orientation-dependent number density of filaments. The model emphasizes the effects of branching and capping on the density of free filament ends. The variants differ in their treatment of side versus end branching and dimensionality, and assume that new branches are generated by existing branches (autocatalytic behavior) or independently of existing branches (nucleation behavior). In autocatalytic models, the network growth velocity is rigorously independent of the opposing force exerted by the obstacle, and the network density is proportional to the force. The dependence of the growth velocity on the branching and capping rates is evaluated by a numerical solution of the rate equations. In side-branching models, the growth velocity drops gradually to zero with decreasing branching rate, while in end-branching models the drop is abrupt. As the capping rate goes to zero, it is found that the behavior of the velocity is sensitive to the thickness of the branching region. Experiments are proposed for using these results to shed light on the nature of the branching process.

Actin Depolymerizing Factors↗

PubMed1996

Theoretical uncertainty of measurements using quantitative polymerase chain reaction.

Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.

Biometry↗

PubMed1999

A computer exploration of some properties of non-linear stochastic partnership models for sexually transmitted diseases with stages.

In this paper, branching process approximations to non-linear stochastic partnership models for sexually transmitted diseases in heterosexual populations were used to find points in the parameter space such that an epidemic would occur. At selected points in the parameter space, samples of Monte Carlo realizations of the process were computed and analyzed statistically to gain insights into the stochastic evolution of epidemics seeded by one infective single female and male. Non-linear difference equations were embedded in the stochastic processes, making it possible to compare trajectories computed according to the deterministic model with those computed from samples of Monte Carlo realizations. From these trajectories it was shown that stochastic fluctuations may have a profound effect on the long-term evolution of an epidemic, and examples demonstrate that an investigator may be misled if a deterministic model alone were used to project an epidemic, particularly when there is a significant probability of extinction.

Computer Simulation↗

PubMed2003

Establishment success and extinction risk in autocorrelated environments.

We consider establishment success (and extinction risk of small populations) in fluctuating environments, by means of an inhomogeneous branching process model. In this model it is assumed that individuals reproduce asexually during discrete reproduction periods. Within each period individuals reproduce independently and have random numbers of offspring. Expected numbers of offspring vary over reproduction periods due to random environmental changes. Previous simulation results indicated that there is a positive autocorrelation between the establishment probabilities of invaders in successive reproduction periods when environmental states are independently distributed. This result was never formally proved. In this paper we prove that this is indeed true, regardless of the form of the distribution of environmental states or the offspring distribution (under a monotonicity condition, which holds for biologically realistic models). Furthermore, we prove that it is also true for positively autocorrelated environmental states. We show by a counterexample that in environments with a strong negative autocorrelation establishment probabilities can be negatively autocorrelated. This was further examined through simulations. Our results imply that in independent, positively autocorrelated and weakly negatively autocorrelated environments the probability of success of invasion in different independently varying sites is the highest, followed by sequential invasion. For environments with a strong negative autocorrelation, sequential invasion has the highest probability of success. Effects of autocorrelation were further examined with simulations. From the results it appears that the expected length of 'runs of bad luck' is the most crucial factor for establishment success.

Algorithms↗

PubMed2003

The distribution of surviving blocks of an ancestral genome.

What is the chance that some part of a stretch of genome will survive? In a population of constant size, and with no selection, the probability of survival of some part of a stretch of map length y < 1 approaches y/log(yt/2) for log(yt) > or = 1. Thus, the whole genome is certain to be lost, but the rate of loss is extremely slow. This solution extends to give the whole distribution of surviving block sizes as a function of time. We show that the expected number of blocks at time t is 1+yt and give expressions for the moments of the number of blocks and the total amount of genome that survives for a given time. The solution is based on a branching process and assumes complete interference between crossovers, so that each descendant carries only a single block of ancestral material. We consider cases where most individuals carry multiple blocks, either because there are multiple crossovers in a long genetic map, or because enough time has passed that most individuals in the population are related to each other. For species such as ours, which have a long genetic map, the genome of any individual which leaves descendants (approximately 80% of the population for a Poisson offspring number with mean two) is likely to persist for an extremely long time, in the form of a few short blocks of genome.

Genome↗

PubMed1998

HLA-DR-positive dendritic cells of the normal human choroid plexus: a potential reservoir of HIV in the central nervous system.

In a previous study of choroid plexus (CPx) from patients with the acquired immunodeficiency syndrome (AIDS), we found a population of stromal cells infected with the human immunodeficiency virus (HIV). To determine whether these represented antigen-presenting dendritic cells, we examined the phenotype of normal human choroid plexus by light and electron microscopy (EM) and established the HIV-infected cell type by immunohistochemistry in AIDS cases with HIV-infected CPx. Monoclonal antibodies were used to detect class II major histocompatibility antigens (MHC), S-100 and S-100beta protein, lymphocytes, monocytes/macrophages, and HIV glycoprotein. A variable number of stromal cells had slightly elongated nuclei and long branching processes that were strongly immunoreactive for class II MHCs, rarely reactive for S-100 and S-100beta and immunonegative for monocyte/macrophage markers. Phagocytic activity was absent by EM and immunomarkers. They were numerous in the subepithelial region, and their processes occasionally extended toward the stromal capillaries or between the CPx epithelial cells. The HIV-infected cells were intensely immunoreactive for class II MHC markers and often displayed a dendritic morphology. These results document the presence of dendritic cells in the normal human CPx whose morphology and immunophenotype closely resemble those of DCs elsewhere in the body. They also show that these immunoreactive MHC class II cells are the cell type infected by HIV. We suggest that the functional activity of the CPx DCs is similar to that of antigen-presenting dendritic cells elsewhere in the body. This includes the potential to harbor HIV during the prolonged period of clinical latency, acting as a central nervous system reservoir of infection before the onset of AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Identification of interstitial cells of Cajal in the rabbit portal vein.

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.

Aniline Compounds↗

PubMed2001

ATP and adenosine induce ramification of microglia in vitro.

Microglial cells in the healthy adult brain possess a characteristic ramified morphology with multiple branched processes, small somata and down-regulated inflammatory properties. In contrast, microglial cells isolated from new-born rat brain inevitably show a non-ramified amoeboid phenotype, which is observed in vivo after pathologic activation or during development. To identify factors that control microglial morphology we investigated the effects of purines alone or in combination with astrocyte-conditioned medium (ACM). Under optimized culture conditions postnatal rat microglial cells developed an amoeboid to ovoid phenotype. Addition of 0.6-1 mM ATP or adenosine induced the outgrowth of numerous processes after 2-3 days that could be observed also in the presence of ACM as previously reported. Culture in ACM plus ATP or adenosine yielded an optimized ramified phenotype. ATP or adenosine, but not ACM alone, also prevented the formation of a flat, amoeboid morphology induced by lipopolysaccharide (LPS); however, at 0.6-1 mM they did not reduce the initial LPS-induced activation of the transcription factor NF-kappaB. By using specific agonists or antagonists the morphological transformations could not be confined to a distinct purinoreceptor subtype, but appeared to be mediated by long-term presence of adenosine in the medium to which phosphorylated purines were rapidly hydrolyzed by microglial cells. Since ACM did not contain sufficient concentrations of ATP or adenosine, purines are not the only ramification-inducing factors present in ACM; however, they are a valuable tool to induce microglial ramification in vitro.

Adenosine↗

PubMed1996

Temporal characterization of microglia, IL-1 beta-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures.

These studies demonstrate that murine hippocampal slice cultures possess neural-immune elements that show responses parallel to comparable in vivo models of neural-immune activation. Using immunocytochemical techniques, this study characterized the phenotypes of specific glial elements and the expression of the cytokine, interleukin-1 (IL-1 beta), in the hippocampal dentate gyrus over a period of 10 days in vitro (DIV). Preparation of organotypic slice cultures of neonatal mouse hippocampus produced cellular damage including axotomy of afferent fibers within the molecular layer of the dentate gyrus. This form of lesion-induced injury caused activation of neural-immune elements in the slice cultures. Staining with the microglial specific biotinylated Griffonia simplicifolia B4-isolectin revealed reactive microglia were most prevalent at 2 DIV and decreased in number from 4 to 10 DIV, whereas the initial population of resting microglia at 2 DIV increased approximately four-fold from 4 to 10 DIV. The presence of a round IL-1 beta-like immunophenotype closely paralleled the temporal and spatial distribution of the reactive form of microglia observed in the dentate gyrus. In addition, between 4 and 10 DIV, some IL-1 beta-like immunoreactive cells exhibited a stellate-like morphology with numerous branching processes, similar to resting microglia. At 2 DIV astrocytes showed minimal labeling with antibodies directed against glial fibrillary acidic protein (GFAP), while from 4 to 10 DIV, a dramatic hypertrophic astrocytic response occurred, resulting in a gliotic scar forming over the entire dentate gyrus. We conclude that neural-immune activation in the hippocampal organotypic slice culture preparation closely parallels similar responses observed in vivo and thus slice cultures represent an excellent model for further studies of neural-immune interactions resulting from lesion-induced injury in the central nervous system.

Animals↗

PubMed1998

Properties of human glial cells associated with epileptic seizure foci.

We studied physiological properties of glial cells from acute slices of biopsies from patients operated for intractable mesio-temporal lobe epilepsy using whole-cell patch-clamp recordings. Cells were filled with Lucifer Yellow (LY) during recordings to allow morphological reconstruction and immunohistochemical cell identification. Seizure-associated astrocytes had complex, arborized, highly branched processes giving them a stellate appearance, and cells stained intensely for the intermediate filament GFAP as previously reported for 'reactive' astrocytes. GFAP-positive astrocytes from epilepsy biopsies consistently expressed voltage-activated, TTX-sensitive Na+ channels that showed fast activation and inactivation kinetics. Unlike comparison astrocytes, derived from tissues that were not associated with seizure foci, these astrocytes expressed Na+ channels at densities sufficient to generate slow action potentials (spikes) in current clamp studies. In these cells, the ratio of Na+ to K+ conductance was consistently 3-4-fold higher than in comparison human or control rat astrocytes. Four of 17 astrocytes from epilepsy patients versus 14/14 from control rat hippocampus and four of five in comparison human tissue showed a lack of inwardly rectifying K+ currents, which in normal astrocytes are implicated in the control of extracellular K+ levels. These results suggest that astrocytes surrounding seizure foci differ in morphological and physiological properties, and that glial K+ buffering could be impaired at the seizure focus, thus contributing to the pathophysiology of seizures.

Adult↗

PubMed1996

Odontogenic myxoma of the jaws: a clinical, radiologic, immunohistochemical, and ultrastructural study.

Ten cases of odontogenic myxoma were evaluated radiologically, histologically, and histochemically. Ultrastructural examination was performed in five cases. Stellate cells with branching processes were seen in an abundant acid mucopolysaccharide background. Occasionally macrophages were found in the tumors. In two cases islands of inactive odontogenic epithelium were identified. The ultrastructural features suggest that many lesional cells are very similar to a myofibroblast. With the avidin-biotin complex immunohistochemical technique, the lesions were tested with antibodies to S- 100 protein, neuron-specific enolase, neurofilaments, glial fibrillary acid protein, keratin, desmin, muscle-specific actin, and vimentin. All the lesions were found to be positive for vimentin and muscle-specific actin, and negative for the rest. The results of our study seem to confirm the muscle-specific actin-positivity of odontogenic myxomas as previously reported.

Actins↗

PubMed2003

Phosphorylation-dependent paxillin-ERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis.

Activation of the hepatocyte growth factor (HGF) receptor c-met results in the regulation of cell-matrix interactions, including the MAPK-dependent stimulation of epithelial cell morphogenesis. In the present study we demonstrate that HGF stimulates the localization of ERK to sites of cell-matrix interactions and that this is mediated by the tyrosine phosphorylation-dependent association of inactive ERK and the focal adhesion complex protein paxillin. In addition, paxillin was found to associate with the upstream MAP kinases Raf and MEK, resulting in a complex that can mediate localized ERK activation. Mutation of the ERK binding site in paxillin prevented HGF-stimulated ERK-paxillin association and eliminated HGF-induced cell spreading and branching process formation. These experiments reveal that paxillin-dependent ERK activation at sites of cell-matrix interaction is critical for HGF-stimulated epithelial morphogenesis.

Animals↗

PubMed1991

The ultrastructure and immunogold labelling of pancreatic polypeptide-immunoreactive cells associated with the egg-forming apparatus of a monogenean parasite, Diclidophora merlangi.

An electron microscopical examination has been made of the fine structure and disposition of pancreatic polypeptide immunoreactive cells associated with the egg-forming apparatus in Diclidophora merlangi. The cell bodies are positioned in the parenchyma surrounding the ootype and taper to axon-like processes that extend to the ootype wall. The terminal regions of these processes branch and anastomose and, in places, the swollen endings or varicosities form synaptic appositions with the muscle fibres in the ootype wall. The cells are characterized by an extensive GER-Golgi system that is involved in the assembly and packaging of dense-cored vesicles. The vesicles accumulate in the axons and terminal varicosities, and their contents were found to be immunoreactive with antisera raised to the C-terminal hexapeptide amide of pancreatic polypeptide. It is concluded that the cells are neurosecretory in appearance and that, functionally, their secretions may serve to regulate ootype motility and thereby help co-ordinate egg production in the worm.

Animals↗

PubMed1998

Within-host transmission strategies of transovarial, feminizing parasites of Gammarus duebeni.

The amphipod Gammarus duebeni harbours several species of vertically transmitted microsporidian parasites. G. Duebeni were collected from 3 localities in the UK. Animals from Budle Bay, Northumberland, were infected with Octosporea effeminans, and those from Millport, Isle of Cumbrae and Fenham Flats, Northumberland were infected with microsporidia of the genus Nosema. We derived expected distributions of parasites per host embryonic cell by modelling parasite transmission as a multitype, Galton-Watson branching process. Parasite prevalence (proportion of females infected) was significantly heterogeneous among localities. Parasite burden in zygotes was much higher for females infected with Nosema than in animals infected with O. effeminans. There was no significant difference between localities in the number of Nosema in the zygotes. Comparison of models and data from 64-cell host embryos showed that the distributions of parasites per cell were consistent with the hypothesis that sorting of parasites into daughter cells is biased for at least 1 cell lineage. Host embryos infected with O. effeminans could expect to contain a growing number of parasites in each cell generation within such biased cell lineages; similar estimates for Nosema predict a decline in the number of parasites per cell within a biased lineage. We discuss the possibility that the 2 species of parasite may be employing different strategies in order to ensure transmission to the next host generation.

Animals↗