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Putative neuroendocrine cells in the Aplysia cerebral ganglion.

We report on a special population of cells in the Aplysia cerebral ganglion that are characterized by several features compatible with neuroendocrine function. These cells can be recognized in living ganglia by their small size, white color and their typical distribution as a compact cluster in the central medial region of the dorsal ganglion surface. Upon intracellular recording, these cells generate action potentials of relatively long duration (about 25 ms), as compared with the faster action potentials of larger white cells or of non-white cells (about 4 ms). Intracellular injection of the small white cells with Lucifer yellow after recording revealed a dual projection area: single cells have one process which branches extensively into many varicose terminals as it courses through the neuropil, and then sends varicose terminals to the vascular sheath at the periphery of the ganglion. In cryostat sections, these cells were specifically characterized by their content of large granules, the staining characteristics of which distinguish them from lipochondria or lysosomes. Their ability to bind fluorochromes nonspecifically is of particular importance for the interpretation of histochemical localization studies based on immunofluorescence techniques.

Animals↗

Effects of sodium butyrate on the expression of sodium channels by neuronal cell lines derived from the rat CNS.

We have studied the effects of sodium butyrate on cell morphology and the expression of mRNAs encoding voltage-gated sodium channels in five neuronal cell lines, B35, B50, B65, B103 and B104, all derived from the rat CNS. The cells were grown in medium supplemented with 2.5 mM sodium-n-butyrate and examined daily by phase-contrast microscopy. Sodium butyrate caused slowing of cell division and the formation of longer and more highly branched cytoplasmic processes than were present in untreated cells. Expression of sodium channel mRNA was analysed by PCR with primers that allow the transcripts encoding the different types of sodium channel to be distinguished according to the lengths of the PCR products. The identity of the PCR products was confirmed by restriction enzyme digestion. Southern blotting and hybridization with internal radiolabelled probes. Prior to sodium butyrate treatment, expression of sodium channel mRNA was largely restricted to B50 and B104 cells: B50 cells showed expression of rat brain types I and II sodium channel and B104 cells expressed rat brain type III sodium channel. After treatment for 5 days with sodium butyrate, sodium channel mRNA was detected in all five cell lines. In addition to type I and type II sodium channel, B50 cells expressed rat brain type III sodium channel. These three types of sodium channel were also expressed by B35, B65 and B103 cells. Even after butyrate treatment, B104 cells expressed only type III sodium channel. The treatment also induced expression of rat skeletal muscle SkM1 sodium channel in B35 cells but only trace amounts in the other neuronal cell lines.

Animals↗

NADPH-diaphorase histochemistry reveals oligodendrocytes in the rainbow trout (teleosts).

We studied the distribution of NADPH-diaphorase (NADPHd) in the brain of the rainbow trout. Not only neurons but also tanycytes and oligodendrocytes showed NADPHd positivity. Interestingly, staining of oligodendrocytes was delicate and revealed fine characteristics of these cells, comparable to those revealed by classical 'oligodendrocyte methods' in mammals (such as the rich branching of processes and their association with myelin sheaths). NADPHd histochemistry indicated that trout oligodendrocytes are heterogeneous as regards cell size, shape and number of processes, and that there is a positive correlation between the size of axons and that of the associated oligodendrocytes. NADPHd histochemistry thus appears to be a useful method for the study of oligodendrocyte populations in bony fishes. Furthermore, this is the first report of NADPHd activity in oligodendrocytes of any vertebrate.

Animals↗

Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth.

The role calmodulin plays in the growth and differentiation of nerve cells was assessed by altering the levels of calmodulin in the PC12 rat pheochromocytoma cell line and determining the effects of altering these levels on cellular proliferation and differentiation. Calmodulin levels in the PC12 cells were increased or decreased by transfecting the cells with a mammalian expression vector into which the rat calmodulin gene I had been cloned in the sense or antisense orientation, respectively. The cells transfected with the calmodulin sense gene showed increased levels of calmodulin immunoreactivity and increased levels of calmodulin messenger RNA as ascertained by immunocytochemistry and slot-blot analysis, respectively. Cells transfected with the calmodulin antisense construct showed reduced levels of calmodulin immunoreactivity. Reducing the levels of calmodulin by expression of antisense calmodulin messenger RNA resulted in a marked inhibition of cell growth, whereas increasing the levels of calmodulin by overexpressing calmodulin messenger RNA resulted in an acceleration of cell growth. Transfected PC12 cells having reduced levels of calmodulin immunoreactivity exhibited spontaneous outgrowth of long, stable and highly branched neuritic processes. PC12 cells in which calmodulin was overexpressed showed no apparent changes in cell morphology, but did show an altered response to the addition of nerve growth factor. While nerve growth factor slowed cellular proliferation and induced extensive neurite outgrowth, in parental PC12 cells nerve growth factor induced little or no neurite outgrowth and little inhibition of cell proliferation in transfected cells overexpressing calmodulin. These results indicate that calmodulin is essential for the proliferation of nerve cells and for the morphological changes that nerve cells undergo during differentiation. The study also suggests the possibility that a calmodulin antisense approach may be used to inhibit the proliferation of neuronal tumors.

Animals↗

Effects of nerve growth factor and acetyl-L-carnitine arginyl amide on the human neuronal line HCN-1A.

The HCN-1A clonal cell line, derived from the cortical tissue of a patient with unilateral megencephaly, was shown to differentiate into a mature neuronal-like state in the presence of the nerve growth factor, dibutyryl cyclic adenosine, 3',5'-monophosphate and either 1-isobutyl-3-methylxanthine or forskolin. Differentiation was assessed by measuring the percentage of cells that displayed branched, varicose processes that stained for synaptophysin. Treatment of cultures with a cocktail containing forskolin increased immunocytochemical staining for gamma aminobutyric (GABA), neurofilament protein and the nerve growth factor receptor species p75NGFR. Treatment with acetyl-L-carnitine alone had some effects on the cell morphology while acetyl-L-carnitine arginyl amide and nerve growth factor together increased the GABA content. Positive staining levels for the neurotransmitters gamma aminobutyric acid, glutamate, somatostatin, cholecystokinin and vasoactive intestinal polypeptide were measured quantitatively for HCN-1A under basal conditions.

1-Methyl-3-isobutylxanthine↗

High gelsolin content of developing oligodendrocytes.

The actin-binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5-bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5-positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL-1-, GC-, and MBP-positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP-positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Animals↗

Immunocytochemical localization of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase, in the hypothalamus of cattle.

Immunocytochemical staining for the presence of catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, was used to characterize the regional distribution of catecholaminergic neurons in the hypothalamus and adjacent areas of domestic cattle, Bos taurus. In steers, heifers and cows, tyrosine hydroxylase-immunoreactive perikarya was located throughout periventricular regions of the third cerebral ventricle, in both anterior and retrochiasmatic divisions of the supraoptic nucleus, suprachiasmatic nucleus, and ventral and dorsolateral regions of the paraventricular nucleus, dorsal hypothalamus, ventrolateral aspects of the arcuate nucleus, along the ventral hypothalamic surface between the median eminence and optic tract, and in the posterior hypothalamus. Immunostained perikarya ranged from small (10-20 microns, parvicellular) to large (30-50 microns, magnocellular) and were of multiple shapes: round, triangular, fusiform or multipolar, often with 2-5 processes of branched arborization. There were no dopamine-beta-hydroxylase immunoreactive perikarya observed within the hypothalamus and adjacent structures. However, both tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive fibers and punctate varicosities were observed throughout regions of tyrosine hydroxylase immunoreactivity perikarya. Generally, the location and pattern of hypothalamic tyrosine hydroxylase immunoreactivity and dopamine-beta-hydroxylase immunoreactive were similar to those reported for most other large brain mammalian species, however, there were several differences with commonly used small laboratory animals. These included intense tyrosine hydroxylase immunoreactivity of perikarya within the retrochiasmatic division of the supraoptic nucleus (ventral A15 region), the absence of tyrosine hydroxylase immunoreactive perikarya below the anterior commissure or within the bed nucleus of stria terminalis (absence of the dorsal A15 region), an abundance of tyrosine hydroxylase immunoreactive perikarya within the ependymal layer of the median eminence, heavy innervation of the arcuate nucleus with dopamine-beta-hydroxylase immunoreactive fibers and varicosities, and the paucity of dopamine-beta-hydroxylase immunoreactive throughout the median eminence.

Animals↗

Integrated transcriptomic and metabolomic analyses provide new insights into the response of black rockfish (Sebastes schlegelii) larvae to temperature fluctuations.

Sebastes schlegelii usually encounter elevated and fluctuating water temperatures near its upper thermal limit in summer, yet the hepatic responses of larvae to repeated temperature fluctuation regimes remain unclear. To address this question, S. schlegelii larvae were exposed for 8&#xa0;days to four thermal regimes: constant 18&#xa0;&#xb0;C (CT), constant 28&#xa0;&#xb0;C (HT), intermittent cooling from 18 to 8&#xa0;&#xb0;C followed by recovery to 18&#xa0;&#xb0;C (FL), and intermittent warming from 18 to 28&#xa0;&#xb0;C followed by recovery to 18&#xa0;&#xb0;C (FH). Survival rate was evaluated, and integrated liver transcriptomic and metabolomic analyses were performed. Final survival rates were 96.67% in the CT group, 97.78% in the FL group, and 77.78% in the FH group. Survival rate in the HT group (38.89%) was significantly lower than that in the other three groups (P&#xa0;<&#xa0;0.05). HTvsCT, FLvsCT, FHvsCT, and FHvsHT comparisons identified 2598, 1207, 622, and 2404 differentially expressed genes and 627, 606, 690, and 610 differential metabolites, respectively. KEGG enrichment analyses of DEGs and SDMs in HTvsCT highlighted HSP-mediated proteostasis, endoplasmic-reticulum protein processing, branched-chain and sulfur amino acid metabolism, glutathione metabolism, and central carbon metabolism, with upregulated hsp90aa1, bckdha, gclc, and pfkp and reduced levels of branched-chain amino acids and methionine. Compared with HT, FH showed attenuated disturbances in proteostasis, amino acid and redox regulation, and central carbon metabolism, together with recovery-associated glycerophospholipid turnover. FL primarily induced polyunsaturated fatty acid (PUFA)-related membrane lipid remodeling. These findings indicate that hepatic responses differed between continuous high-temperature exposure and temperature fluctuations and between fluctuation regimes.

Animals↗

Selective binding of meiosis-specific yeast Hop1 protein to the holliday junctions distorts the DNA structure and its implications for junction migration and resolution.

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex (SC), plays an important role in both gene conversion and crossing over between homologs, as well as enforces meiotic recombination checkpoint control over the progression of recombination intermediates. In hop1Delta mutants, meiosis-specific double-strand breaks (DSBs) are reduced to 10% of the wild-type level, and at aberrantly late times, these DSBs are processed into inter-sister recombination intermediates. However, the underlying mechanism by which Hop1 protein regulates these nuclear events remains obscure. Here we show that Hop1 protein interacts selectively with the Holliday junction, changes its global conformation and blocks the dissolution of the junction by a RecQ helicase. The Holliday junction-Hop1 protein complexes are significantly more stable at higher ionic strengths and molar excess of unlabeled competitor DNA than complexes containing other recombination intermediates. Structural analysis of the Holliday junction using 2-aminopurine fluorescence emission, DNase I footprinting and KMnO4 probing provide compelling evidence that Hop1 protein binding induces significant distortion at the center of the Holliday junction. We propose that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates with the process of branch migration of Holliday junction.

Crossing Over, Genetic↗

Generation of bone marrow-derived neural cells in serum-free monolayer culture.

Bone marrow-derived cells (BMCs) are reported to trans-differentiate into neural lineages, and are expected to offer a valuable resource for treating severe diseases of the central nervous system. BMCs that show neural differentiation may differ morphologically from typical marrow stromal cells. The present study aimed to obtain efficient generation of cells with neural features from bone marrow. Serum-free culture was applied to BMCs to prevent the growth of differentiated cells. Using basic fibroblast growth factor and extracellular matrix, rodent BMCs capable of proliferation and neural differentiation expanded in monolayer form. Cultured cells were small, round or spindle-shaped, and expressed nestin. Under neural differentiation conditions, cells developed long, thin cellular processes with branches, and expressed neuronal and glial phenotypes. Intracerebrally transplanted BMCs survived and migrated into the subcortex of syngenic animals.

Animals↗

A random walk model of oligodendrocyte generation in vitro and associated estimation problems.

A branching stochastic process proposed earlier to model oligodendrocyte generation by O-2A progenitor cells under in vitro conditions does not allow invoking the maximum likelihood techniques for estimation purposes. To overcome this difficulty, we propose a partial likelihood function based on an embedded random walk model of clonal growth and differentiation of O-2A progenitor cells. Under certain conditions, the partial likelihood function yields consistent estimates of model parameters. The usefulness of this approach is illustrated with computer simulations and data analyses.

Algorithms↗

Secretory granule-cytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role.

A survey covering the serous (granular) cutaneous glands in several anuran families from the Old and New Worlds (Bombinatoridae, Discoglossidae, Ranidae, Hylidae, Pseudidae and Leptodactylidae) has revealed consistent patterns of complex interactions between the syncytial secretory unit and serous deposits (granules). These relationships involve outgrowths from the syncytial cytoplasm encircling the granules and complex invaginations of the perigranular compartment (halo) into the syncytium. The outgrowths are branched, cytoplasm processes resembling ramified microvilli, or can be larger, dome-like to cylindrical structures. Despite their different features and origins, all these structures are efficient devices for amplifying the cytoplasmic surfaces round the granules, so improving exchange between the secretory syncytium and serous product. These complex secretory granule-cytoplasm interactions affect the product released from the Golgi apparatus and are consistent with the hypothesis of prolonged serous maturation following the initial phase of biosynthesis. Post-Golgian maturation modifies the secretory material on a centripetal gradient, causing condensation and, possibly, the transfer of component molecules from and/or to the cytoplasm.

Animals↗

Arborization of dewlap motoneurons in the green anole lizard (Anolis carolinensis) is not sexually dimorphic.

Male anoles extend a bright red throat fan, called a dewlap, during both courtship and aggressive encounters. Female anoles perform this behavior less often than males and only in aggression towards both sexes. The cartilage, muscle fibers, and motoneuron somata controlling the display are larger in males than females. In the present study, we used the Golgi technique in an effort to characterize more completely the morphology of these dewlap motoneurons, and to investigate whether the dendritic arborization is different between the sexes. In addition to describing the morphology, we report that the length of processes, and numbers of primary processes and branch points are comparable in males and females. This similarity in arborization represents an intriguing contrast to other sexually dimorphic neuromuscular systems.

Animals↗

Digital fluorescence imaging of elementary steps of neurosteroid synthesis in rat brain glial cells.

With fluorescence microscopic imaging, we have examined Ca2+ signaling, LDL uptake and distribution of cytochrome P450 scc on individual rat brain glial cells in order to investigate the molecular mechanisms of neurosteroid synthesis. Astrocytes and oligodendrocytes were cultured from newborn rat brain. Ca2+ signaling was observed in Calcium Green-1 loaded astrocytes upon neurotransmitter stimulations using video-enhanced microscopy. Upon stimulation of serotonin and glutamate, we observed typically three types of Ca2+ signaling which were Ca2+ oscillations, a transient increase in Ca2+ concentration and Ca2+ oscillations superimposed on a transient Ca2+ increase. On the other hand, histamine and ATP induced only a transient increase in Ca2+ without oscillatory response. Uptake of octadecyl rhodamine (R18) labeled LDL by astrocytes and oligodendrocytes was observed in the time scale of 30 min with confocal laser scanning microscopy. Some localization of LDL in the cytoplasm was observed for astrocytes. For oligodendrocytes, incorporated LDL was distributed over the entire cytoplasmic region of both cell body and multiple branched cell processes. The presence of a significant amount of cytochrome P450 scc was demonstrated with immunofluorescence staining in both astrocytes and oligodendrocytes. The density of P450 scc in both glial cells was suggested to be around 1% of that in bovine adrenocortical fasciculata cells. The results lead to an improved quantitative picture of neurosteroid synthesis in glial cells.

Adrenal Cortex↗

Aprotinin reduces injury of the spinal cord in transient ischemia.

OBJECTIVE: The protective effect of aprotinin, which is a protease inhibitor, was assessed in a rabbit spinal cord ischemia model. DESIGN: Randomized, controlled, prospective study. SETTING: University research laboratory. SUBJECTS: New Zealand white rabbits (36) of both sexes. METHODS: In 24 animals, ischemia was induced with midline laparotomy and clamping the aorta just distal to left renal artery and proximal to aortic bifurcation for 20 min. Aprotinin was given 30000 KIU as a short intravenous injection after anesthesia, and was followed by 10000 KIU/h by continuous infusion in group 1 (n = 12). Similar volume of saline solution was used in control group of animals (group 2, n = 12). Group 3 of animals (sham group, n = 12) were anesthetized and subjected to laparotomy without aortic occlusion. Physiological parameters and somatosensory evoked-potentials (SEP) were monitored in animals before ischemia, during ischemia and in the first 60 min of reperfusion. Their neurological outcome was clinically evaluated up to 48 h postischemia. Their motor function was scored, and the intergroup differences were compared. The animals were sacrificed after two days of postischemia. Their spinal cord, abdominal aorta, and its branches were processed for histopathological examination. RESULTS: In group 3, SEP amplitudes did not change during the procedures, and all animals recovered without neurologic deficits. At the end of ischemic period, the average amplitude was reduced to 53+/-7% of the baseline in all ischemic animals. This was followed by a gradual return to 89+/-8 and 81+/-13% of the initial amplitude after 60 min of reperfusion in group 1 and group 2 correspondingly (P > 0.05). The average motor function score was significantly higher in group 1 than group 2 at 24 and 48 h after the ischemic insult (P < 0.05). Histological observations were clearly correlated with the neurological findings. CONCLUSION: The results suggest that aprotinin reduces spinal cord injury and preserves neurologic function in transient spinal cord ischemia in rabbits.

Animals↗

DNA hybrids stabilized by heterologies.

The double D-loop DNA hybrid contains four DNA strands following hybridization of two RecA protein coated complementary single-stranded DNA probes with a homologous region of a double-stranded DNA target. A remarkable feature of the double D-loop DNA hybrids is their kinetic stabilities at internal sites within linear DNA targets after removal of RecA protein from hybrids. We report here that heterologous DNA inserts in one or both probe strands affect the kinetic stability of protein-free double D-loop hybrids. DNA heterologies normally distort DNA-DNA hybrids and consequently accelerate hybrid dissociation. In contrast, heterologous DNA inserts impede dissociation of double D-loops, especially when the insert sequences interact with each other by DNA base pairing. We propose a mechanism for this kinetic stabilization by heterologous DNA inserts based on the hypothesis that the main pathway of dissociation of double D-loop DNA hybrids is a DNA branch migration process involving the rotation of both probe-target duplexes in the hybrids. Heterologous DNA inserts constrain rotation of probe-target duplexes and consequently impede hybrid dissociation. Potential applications of the stabilized double D-loops for gene targeting are discussed.

Base Sequence↗

Mechanistic insights in charge-transfer-induced luminescence of 1,2-dioxetanones with a substituent of low oxidation potential.

We have investigated the decomposition pathway of dioxetanones 1c with a phenoxide anion group by the B3LYP/6-31+G(d) method together with the second-order multireference Møller-Plesset perturbation (MRMP) theory and propose charge-transfer-induced luminescence (CTIL) with polarization-induced branching excitation processes. In the gas phase, the thermal decomposition of 1c occurs by an asynchronous two-stage pathway without a discrete intermediate; that is, the initial O-O bond breaking to generate a charge-transfer (CT) diradical species is immediately followed by the subsequent C-C bond breaking with simultaneous back CT, which is responsible for the surface crossing at the avoided crossing. The activation energy is dramatically reduced from 19.4 to 3.8 kcal mol(-)(1) by the deprotonation of phenol meta-1d to its anion meta-1c, showing an important role of the endothermic CT. The odd/even selection rule for the chemiluminescence efficiency can be explained by the orbital interaction for the back CT between the carbonyl pi orbital and either a HOMO or a LUMO of the generated light emitters. To examine the accessibility of the chemically initiated electron exchange luminescence (CIEEL) route, we considered the solvent effects on the free-energy change of meta-1c by using continuum solvent models. The bending vibration mode of the CO(2) fragment is specifically considered. Borderline features emerges from the solution-phase CT reaction of meta-1c, which depends on the solvent polarity: one is a nonadiabatic or adiabatic back CT process (polarization-induced concerted CTIL), and the other is a radical dissociation, i.e., complete one-electron-transfer process (CIEEL).

Journal Article↗