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Displaced horizontal cells and biplexiform horizontal cells in the mammalian retina.

We have used the neurofibrillar method of Gros-Schultze to stain the axonless horizontal cells of capybara, agouti, cat, and rabbit retinae. In all of these species, we have found two unusual horizontal cell morphologies: displaced horizontal cells and biplexiform horizontal cells. The displaced horizontal cells have perikarya located in the ganglion cell layer and dendrites branching in the inner plexiform layer. Many dendrites take an ascending trajectory to branch in the outer plexiform layer. The biplexiform horizontal cells are normally placed horizontal cells with descending processes that branch in the inner plexiform layer. Both cell types occur mainly in the retinal periphery, near the ora serrata. They are more numerous in the capybara retina, where they represent as much as 50% of the axonless horizontal cells of the retinal periphery.

Animals↗

The molecular basis of lung morphogenesis.

To form a diffusible interface large enough to conduct respiratory gas exchange with the circulation, the lung endoderm undergoes extensive branching morphogenesis and alveolization, coupled with angiogenesis and vasculogenesis. It is becoming clear that many of the key factors determining the process of branching morphogenesis, particularly of the respiratory organs, are highly conserved through evolution. Synthesis of information from null mutations in Drosophila and mouse indicates that members of the sonic hedgehog/patched/smoothened/Gli/FGF/FGFR/sprouty pathway are functionally conserved and extremely important in determining respiratory organogenesis through mesenchymal-epithelial inductive signaling, which induces epithelial proliferation, chemotaxis and organ-specific gene expression. Transcriptional factors including Nkx2.1, HNF family forkhead homologues, GATA family zinc finger factors, pou and hox, helix-loop-helix (HLH) factors, Id factors, glucocorticoid and retinoic acid receptors mediate and integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Signaling by the IGF, EGF and TGF-beta/BMP pathways, extracellular matrix components and integrin signaling pathways also directs lung morphogenesis as well as proximo-distal lung epithelial cell lineage differentiation. Soluble factors secreted by lung mesenchyme comprise a 'compleat' inducer of lung morphogenesis. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as FGFR, EGFR, IGFR, PDGFR and c-met stimulate lung morphogenesis. On the other hand, cognate receptors with serine/threonine kinase intracellular signaling domains, such as the TGF-beta receptor family are inhibitory, although BMP4 and BMPR also play key inductive roles. Pulmonary neuroendocrine cells differentiate earliest in gestation from among multipotential lung epithelial cells. MASH1 null mutant mice do not develop PNE cells. Proximal and distal airway epithelial phenotypes differentiate under distinct transcriptional control mechanisms. It is becoming clear that angiogenesis and vasculogenesis of the pulmonary circulation and capillary network are closely linked with and may be necessary for lung epithelial morphogenesis. Like epithelial morphogenesis, pulmonary vascularization is subject to a fine balance between positive and negative factors. Angiogenic and vasculogenic factors include VEGF, which signals through cognate receptors flk and flt, while novel anti-angiogenic factors include EMAP II.

Animals↗

Characterization of strand exchange activity of yeast Rad51 protein.

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.

Adenosine Triphosphate↗

[Ultrastructure of synapses in the nucleus of the trapezoid body of the bat].

The subsynaptic structure of the synapses in the medial nucleus of the trapezoid body was studied in the bat Myotis oxygnatus. The synaptic endings in the nucleus are represented by large-cup-shaped and small loop-shaped terminations. The cup-shaped terminations are formed of large branches originating from a thick myelinated fibre after loss of myelin from it. Each branch forms a series of contacts alternating with vast enlargements of extracellular space, on the body of the cell and its processes. Large branches are filled with synaptic vesicles, neurofilaments and neurotubules, mitochondria; all these components are distributed rather regularly along the branch diameter. In fine branches of the cup the synaptic vesicles are the main and often the only component. The pattern of the cup branch changes as the distance from the main fibre increases, namely the amount of neurofilaments and neurotubules diminishes up to their disappearance, while the amount and the density of synaptic vesicles increases. The small loop-shaped treminals are different from the cup-shaped ones by the composition of the synaptic vesicles and the structure of the contact zone. In addition to agranular vesicles there are also granular ones. Both types of terminations--cup-shaped and loop-shaped ones -- are found both on the bodies and dendrites. On distal portions of dendrites the terminations are disposed in nests.

Animals↗

Four types of amacrine in the cat retina that accumulate GABA.

Roughly one-quarter of neurons in the amacrine cell layer accumulate exogenous gamma-aminobutyric acid (GABA). Some of these (8%) are interplexiform cells; the remainder are true amacrine cells. We partially reconstructed, from serial electron microscopy autoradiograms, 25 GABA-accumulating amacrines and distinguished four types based on cytoplasmic appearance, soma size and shape, and the form of primary and secondary processes. Type 1 had a large (609 +/- 60 microns3), dark soma, and multiple, medium-diameter (0.6 microns) processes splayed from the soma margins like the appendages from a crab. Type 2 had a medium (360 +/- 40 microns3), helmet-shaped, pale soma, and medium-diameter (0.8 microns) processes that branched in sublamina alpha. Type 3 had a small (267 +/- 44 microns3), dark, pyriform soma. The latter formed a single stout (3.0 microns) process that bifurcated in the middle of sublamina alpha. Type 4 had a very large, pale soma (860 microns3). This was pyriform, tapering into a stout (2.0 microns) process that descended into the middle of sublamina alpha where it emitted smaller tangential processes. It is to be expected that each of these amacrine cell types will have distinct functions in neurotransmitter retinal circuitry.

Animals↗

Synapses of the inner plexiform layer in the retina of cyprinid fish.

Two types of synapses are known to occur in the inner plexiform layer of the vertebrate retina. The typical ribbon synapse has two postsynaptic elements, whereas the conventional synapse displays only one. Ribbon synapses are characteristic of bipolar cells, and the conventional synapses are seen in amacrine and interplexiform cells. Two varieties of conventional synapses with one postsynaptic element are described in the present study. One has the well-known presynaptic cluster of synaptic vesicles, whereas the other has several clusters surrounding dense globules. Furthermore, there are conventional synapses with two postsynaptic elements, called branched conventional synapses. They are most numerous in the middle of the inner plexiform layer and are characterized by a presynaptic aggregation of synaptic vesicles usually clustering around a dense globule. The neuronal processes with branched conventional synapses also have conventional synapses with one postsynaptic element and are therefore thought to be amacrine cell processes.

Animals↗

Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation.

The lacrimal gland provides an excellent model with which to study the epithelial-mesenchymal interactions that are crucial to the process of branching morphogenesis. In the current study, we show that bone morphogenetic protein 7 (Bmp7) is expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, we find that Bmp7-null mice have distinctive reductions in lacrimal gland branch number, and that inhibition of Bmp activity in gland explant cultures has a very similar consequence. Consistent with this, exposure of whole-gland explants to recombinant Bmp7 results in increased branch number. In determining which cells of the gland respond directly to Bmp7, we have tested isolated mesenchyme and epithelium. We find that, as expected, Bmp4 can suppress bud extension in isolated epithelium stimulated by Fgf10, but interestingly, Bmp7 has no discernible effect. Bmp7 does, however, stimulate a distinct response in mesenchymal cells. This manifests as a promotion of cell division and formation of aggregates, and upregulation of cadherin adhesion molecules, the junctional protein connexin 43 and of alpha-smooth muscle actin. These data suggest that in this branching system, mesenchyme is the primary target of Bmp7 and that formation of mesenchymal condensations characteristic of signaling centers may be enhanced by Bmp7. Based on the activity of Bmp7 in promoting branching, we also propose a model suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland.

Animals↗

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Base Composition↗

Isolation and purification of osteocytes.

An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1-2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone.

Animals↗

Formation of a RuvAB-Holliday junction complex in vitro.

The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration. In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions. As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay. We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex. In the absence of RuvA, the RuvB protein fails to bind Holliday junctions. The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g. complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-[gamma-thio]triphosphate). In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction. These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis. In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.

Adenosine Triphosphatases↗

Alzheimer disease brain extract stimulates branching of laminin-mediated neuronal processes.

Patients with Alzheimer disease (AD) suffer mental deterioration associated with neurofibrillary tangle and senile plaque formation in the brain. Here we have determined the effects of brain extracts from normal and from AD patients on neuronal process formation by a pheochromocytoma (PC-12) and a neuroblastoma x glioma hybrid cell line (NG108-15). PC12 cells show a dose-related stimulation of branching of neuronal processes by AD brain extracts with cells cultured on a laminin substrate. The neurotrophic effects of extracts of AD brains may be related to the abnormal sprouting and neurofibrillary tangle formation observed in the brain in this disorder.

Adrenal Gland Neoplasms↗

[Learning and chronobiological regulation following experimental damage to the coronary-artery wall of albino rats].

Forty male albino rats were used to study the relationship between experimentally (concentrated hydrochloric acid) produced local structural changes of the coronary artery wall and central nervous information processing and chronobiological events, respectively. The alterations of the coronary arteries caused by this treatment did not produce infarction but initiated on the whole length of the arterial branch proliferative processes that are complete within 12 weeks following the intervention. Central nervous information processing and chronoregulatory processes showed, immediately after exposure to hydrochloric acid, pronounced malfunctions followed by a stage of hyperreactivity (4 weeks following the damage). At 8 weeks from the intervention, the parameters of information processing and chronobiological events of the CNS again equalled the control values. Since the central nervous functions were back to normal earlier than the structural alterations, the results are interpreted as reflecting CNS-controlled interactions between the complex of visceral afference and efference, the regulation of which is determined by the CNS after coronary damage in such a way as to restore the adaptational capacity of the organism by a predominance of sanogenetic processes.

Animals↗

The types of neurons in the neostriatum of bison bonasus. Nissl and Golgi study.

The caudate nucleus and the putamen of bison bonasus have been investigated. The Golgi procedure and Nissl stain had been used to reveal their cellular organization. In the Golgi preparation striatal neurons belong to the two fairly divergent main groups in which several types of cells can be distinguished. The first class, comprising the bulk of neuronal population, consists of only one type of efferent cells with the long axons. The spines cover dendrites except its proximal portion. The second one is composed of four types of interneurons which perikarya and dendritic processes are devoid of spines: aspiny neurons with tortuous dendrites, aspiny cells with dendritic swellings, large aspiny nerve cells, and long-axoned neurons with sparsely distributed but regularly branching dendritic processes.

Animals↗

Regulation of GFAP expression in glial-like cells of the rat pituitary intermediate lobe by lactation, salt-loading, and adrenalectomy.

Glial-like cells in rat pituitary intermediate lobe were localized and characterized by immunohistochemistry with antisera against glial fibrillary acidic protein (GFAP) and S-100. Individual GFAP immunoreactive (IR) cells possessed several processes that often branched into secondary and tertiary processes, terminating with end-feet. The GFAP-immunopositive cell population was distributed in specific rostrocaudal and dorsoventral patterns. The distribution and numbers of cells differed between male and female rats. Examination of altered physiological states, e.g., adrenalectomy, lactation, and salt-loading, revealed state-specific changes in the appearance and distribution of GFAP-IR cells. Adrenalectomy and lactation increased GFAP-IR glial-like cell numbers, whereas salt-loading decreased their numbers and the typical pattern of distribution. By contrast, S-100-expressing cells were evenly distributed in male and female rats, and its expression was not affected by the experimental conditions. Double-label immunocytochemistry indicated that GFAP-IR cells are a subpopulation of S-100-IR cells. These results suggest that cells normally expressing only S-100 may be induced to express GFAP under altered physiologic conditions.

Adrenalectomy↗

Selective localization of glycine-accumulating cells in reaggregate culture of rat retina.

Uptake of [3H]glycine into the cells either in monolayer or reaggregate cultures of retina from two-day-old rat pups was studied. The glycine-accumulating cells (glycine cells) had short processes with several branches. Only 5% of process-bearing cells were labelled in the monolayer cultures. The major cell type, previously identified as photoreceptor cells, was unlabelled. In reaggregate cultures, the glycine cells were localized mainly in the outermost layer of the reaggregate. But the proportion of positive cells among all the cells in that layer was not so large. Although the cell type of the glycine cells has not yet been unambiguously identified, these results demonstrate a possible example of selective sorting out of a group of biochemically distinct cells from a cell mixture.

Animals↗

Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension.

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

Animals↗

Structural dynamics of individual Holliday junctions.

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.

Base Sequence↗

Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment.

Neurons from the abdominal ganglion of the mollusc Aplysia californica regenerate neurite processes in dissociated cell culture. Both the nature of neurite outgrowth and the morphology of the cells are influenced by the presence of adult Aplysia hemolymph in the growth medium and the presence of a portion of a cell's original axonal process. Aplysia hemolymph enhances cell survival, the initiation of neurite outgrowth from multiple sites on the cell body surface, the linear growth of the processes, and the amount of branching by those processes. Hemolymph also decreases the diameter of the outgrowing neurite fascicles and the diameter of the individual neurites within the fascicles. The presence of a cell's original axon reduces the time required for the initiation of neurite outgrowth and restricts the formation of multipolar processes. In addition, the presence of an initial axonal segment is essential for neurite regeneration from large adult neurons.

Animals↗