In situ localization of the sites of paraquat action

Abstract. Effects of paraquat were monitored by electron microscopic, cytochemical, and in vivo spectrophotometric procedures. Electron microscopy of paraquat-treated pea leaf discs indicates that the chloroplasts are affected primarily with both thylakoid dilation and apparent lamellar fusions, resulting in a 'honeycomb' of lamellae. No ultra-structural effects were noted when leaf discs were incubated in both DCMU and paraquat. Paraquat-induced peroxide was located cytochemically along the stroma lamellae, on the ends of the grana stack and in the dilated thylakoids. Less destruction was noted in those samples where peroxide was precipitated by cerium chloride, indicating that peroxide or one of its products is one of the major causes of paraquat toxicily. In C₄ plants mesophyll plastids exhibited much more peroxide deposition than was detected in bundle sheath plastids. No plastid peroxide depositions were noted in a photobleaching mutant that is resistant to paraquat. In situ cytochrome f oxidation-reduction was followed during paraquat treatments and resulted in a hastening of the dark re-reduction of cytochrome f after only 1 h of treatment. This effect was prevented by DCMU or CeCI₃. Electron transport, as measured by cytochrome f oxidation-reduction, was completely obliterated by a 24 h paraquat treatment. These in situ studies on paraquat confirm previous studies that the primary effect in the light is on the plastid and involves peroxide or further products generated by peroxide.     

Distribution and coexistence of urotensin I and urotensin II peptides in the cerebral ganglia of Aplysia californica.

Urotensin I (UI) and urotensin II (UII) were demonstrated in the cerebral ganglia of Aplysia californica by applying immunocytochemical and radioimmunoassay procedures. Sequential analysis of adjacent sections of the cerebral ganglia of Aplysia demonstrated that the UI-immunoreactive (UI-IR) neurons of the F cluster of the cerebral ganglia also contained UII immunoreactivity (UII-IR). Both UI-IR and UII-IR were also observed in a cuff-like arrangement of fibers surrounding the proximal portion of the supralabial nerve, as well as in a few fibers in the anterior tentacular nerves. The UI-IR perikarya of the cerebral ganglia appeared to project to the entire CNS of Aplysia, but the UII-IR fibers appeared only in the neuropile and commissure of the cerebral ganglia. The UI-IR staining was abolished by previous immunoabsorption of the UI antiserum with sucker (Catastomus commersoni) UI, but not with ovine corticotropin-releasing factor (CRF), rat/human CRF, or goby (Gillichthys mirabilis) UII. Immunostaining with UII antiserum was quenched by goby UII, but not by sucker UII-A, UII-B, UII-A(6-12), or carp (Cyprinus carpio) UII-alpha and UII-gamma. The UII staining was not abolished by UI or somatostatin. The F cluster was not stained when a somatostatin antiserum was applied. Radioimmunoassay of dilutions of cerebral ganglia extract, using UII antiserum, revealed a parallel displacement curve to synthetic goby UII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential localization of lectin binding sites and neuropeptides in human dorsal root ganglia

The subpopulations were compared of neurons in human dorsal root ganglia (DRG), as substance P, identified by somatostatin, Glycine max lectin (SBA) specific to terminal N-acetylgalactosamine, and Ulex europaeus I agglutinin (UEA-I) specific to l-fucose. The lectins and neuropeptides all bound to neurons of small diameter. Furthermore, the majority of the SBA binding neurons or somatostatin positive neurons were also UEA-I binding neurons. However, SBA binding neurons were not colocalized with somatostatin or substance P. Less than 20% of substance P positive neurons showed colocalization with l-fucosyl residues, and approximately 10% of l-fucosyl residues showed colocalization with substance P. Our results suggest that both l-fucose and terminal N-acetylgalactosamine containing neurons in the human DRG are subjected to different subpopulations from substance P or somatostatin positive neurons.     

Synthetic egg-laying hormone of Aplysia: quantitative studies of induction of egg laying in Stylocheilus and of the activation of Aplysia buccal neuron B16

1. Synthetic Aplysia egg-laying hormone (ELH-lysine-amide) elicited egg laying in Stylocheilus at a threshold dose of 0.5 microgram per recipient, estimated to be a concentration in the circulation of Stylocheilus of approximately 70 nM. 2. Threshold level and size of egg mass produced by ELH-lysine-amide and bag cell extracts (containing biological ELH) were not significantly different. Latency to lay of recipients of 0.5-4.0 micrograms ELH-lysine-amide (30 +/- 1 min) was significantly longer (P less than 0.05) than for bag cell extract recipients (21 +/- 1 min). 3. ELH-lysine-amide depolarized and activated action potentials in Aplysia buccal neuron B16 in high magnesium, low calcium medium. 4. The lowest concentration of ELH-lysine-amide to activate a supra-threshold response of left and right B16 neurons ranged from 250 nM to 1 microM. 5. Threshold levels for responses to synthetic ELH-lysine-amide and to biological ELH were approximately the same in both egg-laying assay and electrophysiological assay, indicating the likely identity of synthetic and biological ELH. However, the shorter egg-laying latency with bag cell extract suggests that there may be additional factors in the extract that facilitate egg laying.     

Aurora B activity is promoted by cooperation between discrete localization sites in budding yeast

Chromosome biorientation is promoted by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore–microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore, and spindle microtubules. Here we show that a small domain of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This domain, the single alpha helix (SAH), is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of its three locations through targeted mutations and fusion constructs, we determined their individual contributions to chromosome biorientation. We find that only the inner centromere localization is sufficient for cell viability on its own. However, when combined, the inner kinetochore and microtubule binding activities are also sufficient to promote accurate chromosome segregation. Furthermore, we find that the two pathways target the CPC to different kinetochore attachment states, as the inner centromere-targeting pathway is primarily responsible for bringing the complex to unattached kinetochores. We have therefore discovered that two parallel localization pathways are each sufficient to promote CPC activity in chromosome biorientation, both depending on the SAH domain of INCENP/Sli15.     

Structure, bioactivity, and cellular localization of myomodulin B: a novel Aplysia peptide.

Important insights into mechanisms by which neuromuscular activity can be modulated have been gained by the study of experimentally advantageous preparations such as the ARC neuromuscular system of Aplysia. Previous studies have indicated that one source of modulatory input to the ARC muscle is its own two motor neurons, B15 and B16. Both of these neurons synthesize multiple peptide cotransmitters in addition to their primary neurotransmitter acetylcholine (ACh). Peptides present in the ARC motor neurons include SCPA, SCPB, buccalin A and B, and myomodulin A. We have now purified a novel neuropeptide, myomodulin B, which is structurally similar to myomodulin A. Myomodulin B is present in two identified Aplysia neurons that contain myomodulin A; the ARC motor neuron B16 and the abdominal neuron L10. Ratios of myomodulin A to myomodulin B are approximately 6:1 in both cells. Like myomodulin A, myomodulin B potentiates ARC neuromuscular activity; it acts postsynaptically, and increases the size and relaxation rate of muscle contractions elicited either by motor neuron stimulation or by direct application of ACh to the ARC. When myomodulin A is applied to the ARC in high doses (e.g., at about 10(-7) M), it decreases the size of motor neuron-elicited muscle contractions. This inhibitory effect is never seen with myomodulin B. Thus, despite the structural similarity between the two myomodulins, there exists what may be an important difference in their bioactivity.     

Histochemical localization of acetylcholinesterase in the cerebral ganglia of Fasciola hepatica, a parasitic flatworm

Acetylcholinesterase activity was found in the cell bodies and extracellularly in the neuropile of the cerebral ganglia of the adult trematode parasite, Fasciola hepatica. Within neuronal cell bodies of the cerebral ganglion, acetylcholinesterase reaction product was found in the endoplasmic reticulum, in the cisternae of the Golgi apparatus, and in secretory vesicles near the inner (releasing face) cisternae. Acetylcholinesterase reaction product was not seen intracellularly within any nerve processes. The reaction product was found around the somatic cell membranes and in the extracellular space between closely apposed nerve processes in the neuropile. Acetylcholinesterase reaction product was associated with synaptic endings that contained clear spheroidal synaptic vesicles, and the reaction product was localized at the site of synaptic contact between the zone of apposition of the pre- and postsynaptic terminals. This intracellular and extracellular distribution of the enzyme is consistent with its function as the degrading enzyme in cholinergic transmission.     

Activity and localization of cerebral cysteinic cathepsins after the action of ionizing radiation

The effect of total X-ray irradiation (0.155 C/kg) on the activity of cathepsin L (KF 3.4.22.15) from the light mitochondrial fraction and microsomal soluble fraction of the gray substance of the neocortex was studied in rats. The estimations were performed 1, 24, 48, and 120 h after irradiation. The changes in the cathepsin L activity observed in acute radiation pathology were complex and multiphasic.     

Characterization of phosphonoglycosphingolipids containing pyruvate: localization in Aplysia nerve bundles

A phosphonoglycosphingolipid, designated as FGL-IIb, was first identified in nerve fibers of Aplysia kurodai by two-dimensional TLC (Abe, S. et al. (1986) Biomed. Res. 7, 47-51), and its chemical structure has been determined to be 3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNac alpha 1----3(Fuc alpha 1----2)(2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide (Araki, S. et al., submitted). Cryostat and paraffin sections of the nervous tissue and skin of Aplysia were examined immunohistochemically with antiserum against FGL-IIb. With this antiserum, only nerve bundles were stained distinctly: nerve cells in ganglia and in subcutaneous and muscular tissues and other cell elements were not stained. From histochemical findings in cryostat sections pretreated with chloroform-methanol (2 : 1, v/v) and from the results of Western blot analysis of the nervous tissue, the staining was concluded to be due to glycolipid antigens. The antiserum reacted with FGL-IIb and other phosphonoglycosphingolipids named FGL-I, FGL-IIa, FGL-V, and F-9 on TLC plates. This reactivity of FGL-IIb was abolished by mild acid-methanol treatment, and the lost reactivity was recovered by alkaline hydrolysis. These findings suggest that the free carboxyl group of the pyruvic acid of FGL-IIb is essential for the immunological reaction and that all the glycolipids listed above have the same epitope as that of FGL-IIb. Immunohistochemical findings indicated that these glycolipids including FGL-IIb are localized specifically in nerve bundles of Aplysia.     

Studies on extrahypophyseal sites of estrogen action in the induction of ovulation in rats.

To discover possible extrahypophyseal sites of estrogen action in the induction of ovulation, the influence of a s.c. injection of estradiol benzoate (EB) on cell nuclear sizes in the limbic-medial preoptic continuum of progesterone-pretreated cyclic rats was evaluated. The ovulatory dose of 5 mug EB caused a significant increase of nuclear volumes in the medial preoptic nucleus and the anterior and posterior parts of the medial amygdaloid nucleus. Precocious ovulation was induced in prepuberal female rats by unilateral implantation of a molten EB: cholesterol mixture into the posterior part of the mediocortical amygdala (PMCA), but not by implantation into the anterior part of this region (AMCA) or the medial preoptic area (MPA). In adult females injected s.c. with 2.0 mg progesterone on the day post estrus, bilateral implantation of 0.1 or 0.2 mug crystalline EB on the following day did not abolish the delaying effect of progesterone on the preovulatory LH increase and ovulation, when the implants were located in the MPA, lateral septum (LS), bed nucleus of the stria terminalis (BST), AMCA, PMCA or dorsal hippocampus (DHPC), whereas intrapituitary implants were highly effective. However, the bilateral introduction of large tallow pellets containing 0.1 mug EB each, into the LS, BST, AMCA or PMCA advanced ovulation in rats with progesterone-induced 5-day cycles. Equal pellets did neither induced ovulation nor an LH increase after implantation into the MPA or the DHPC. The results suggest that the anterior pituitary, mediocortical amygdala, BST and LS, but not the MPA or DHPC, are sites of the stimulatory feedback of estrogen on gonadotropin secretion in female rats, and that the amygdaloid response to estrogen differs between prepuberal and cyclic females.     

MRI-based localization of electrophysiological recording sites within the cerebral cortex at single-voxel accuracy

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 mum). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 mum within the cortex of 2-3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.     

Ultrastructural localization of concanavalin A-binding sites in the Golgi apparatus of various types of neurons in rat dorsal root ganglia: functional implications

The localization of concanavalin A (con A) binding sites has been determined at the electron-microscopic level in the six types of neurons (A1, A2, A3, B1, B2, C) of rat dorsal root ganglia. In all ganglion cells, con A stained the plasma membrane, the nuclear envelope, the cisternae of the rough endoplasmic reticulum, and the matrix of some multivesicular bodies. In contrast, the con A reactivity of the Golgi apparatus varied according to cell type. In type B1 and B2 cells and possibly in type A3 cells, the lectin was exclusively located in three or four saccules on the cis side of the Golgi stacks, whereas the TPPase-positive saccules and the trans sacculotubular elements were unstained with con A. In type A1, A2, and C neurons, all Golgi saccules as well as the trans sacculotubular elements were stained with the lectin. These results suggest that different types of glycoproteins were produced in these two groups of neurons. In the type A1, A2, and C cells, the persistence of the lectin reactivity in the TTPase-positive saccules or sacculotubular elements on the trans side of the Golgi stacks suggests the presence of glycoproteins with oligosaccharide side chains rich in alpha-D-mannosyl residues in terminal positions. In contrast, the disappearance of the con A reactivity in equivalent elements of the Golgi stacks in type B1, B2, and A3 cells suggests the addition at this level of other sugar residues characteristic of complex oligosaccharide side chains. The majority of the vesicular elements associated with the Golgi apparatus, as well as lysosomes, were unstained with con A.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

Serotonergic stimulation of pituitary-adrenocortical activity in rats: evidence for multiple sites of action

5-Hydroxytryptophan (5-HTP) elevated serum corticosterone concentrations when administered either intraperitoneally (i.p.) or intraventricularly. Inhibition of aromatic L-amino acid decarboxylase outside of the blood-brain barrier antagonized the corticosterone response, but only when the 5-HTP was given i.p. Stimulation of the pituitary-adrenocortical system by fenfluramine was not affected by 5,7-dihydroxytryptamine pretreatment, whereas the stimulation produced by quipazine administration was blocked by lesions of the basomedial hypothalamus. These results suggest that serotonergic drugs can act at multiple sites (i.e., both central and peripheral) to evoke a pituitary-adrenocortical response.