Tetrin polypeptides are colocalized in the cortex of Tetrahymena.

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Members of the RCK potassium channel family are differentially expressed in the rat nervous system.

mRNAs encoding four members of the RCK potassium channel family, named RCK1, RCK3, RCK4 and RCK5 have been analyzed by RNA blot hybridization experiments using specific RNA probes. Each probe recognizes a single mRNA species, their sizes ranging from approximately 4600 nucleotides up to approximately 11,000 nucleotides. The expression of RCK mRNAs as well as their developmental appearance in different regions of the central and peripheral rat nervous system has been investigated. The two most abundant RCK potassium channel mRNAs (RCK1 and RCK5) are predominantly expressed in the adult nervous system. RCK3 and RCK4 mRNAs are present throughout all developmental stages studied. The temporal and regional patterns observed are specific for each RCK potassium channel mRNA indicating that specific regulation of expression occurs. Differential mRNA expression might provide one mechanism for the generation of potassium channel diversity in vivo.     

Regulation of the rat brain endothelin system by endogenous beta-endorphin

Several lines of evidence indicate that the central endogenous opioid and endothelin (ET) system regulate each other. To explore this idea further, we determined the effect of intracerebroventricular (i.c.v.) administration of anti-beta-endorphin IgG (rabbit) on the expression level of the opioid, corticotropin-releasing hormone and endothelin receptors, and tissue concentration of ET-1. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-beta-endorphin IgG (2.5 microg/microl) on days 1, 3 and 5. On day 6, animals were euthanized and caudate, cortex and hippocampus collected for Western blot analysis. Anti-beta-endorphin IgG down-regulated ET-A receptor protein expression in the caudate (51%), but had no effect on the expression of mu, delta, kappa opioid, ET-B, CRH-1 and CRH-2 receptors in any brain region. Anti-beta-endorphin IgG increased tissue ET-1 levels in the caudate by 30.3%. [35S]GTP-gamma-S binding assays demonstrated that anti-beta-endorphin IgG increased the efficacy of [D-Ala2-MePhe4, Gly-ol5]enkephalin without altering its potency in caudate. Control experiments showed that there was no detectable rabbit IgG in caudate, cortex and hippocampus samples. These results suggest that beta-endorphin in the CSF coordinately regulates ET-1 levels and the ET-A receptor in rat caudate. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between opioid and ET system.     

Morphometric analysis of the state of the intramural nervous apparatus of rat duodenum]

Structural organization of the intramural apparatus of the duodenum has been studied in normal white rats. By means of a planimeter, the area of the nerve cells and their nuclei has been estimated in serial sections of the cranial and caudal parts of the duodenum; a definite regularity on the nerve cell types distribution has been stated in different parts of the duodenum. Concentration of afferent nerve elements has been noted in the cranial part of the duodenum.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Distribution of prosaposin in the rat nervous system

Prosaposin is the precursor of four sphingolipid activator proteins (saposins A, B, C, and D) for lysosomal hydrolases and is abundant in the nervous system and muscle. In addition to its role as a precursor of saposins in lysosomes, intact prosaposin has neurotrophic effects in vivo or in vitro when supplied exogenously. We examined the distribution of prosaposin in the central and peripheral nervous systems and its intracellular distribution. Using a monospecific antisaposin D antibody that crossreacts with prosaposin but not with saposins A, B, or C, immunoblot experiments showed that both the central and peripheral nervous systems express unprocessed prosaposin and little saposin D. Using the antisaposin D antibodies, we demonstrated that prosaposin is abundant in almost all neurons of both the central and peripheral nervous systems, including autonomic nerves, as well as motor and sensory nerves. Immunoelectron microscopy using double staining with antisaposin D and anticathepsin D antibodies showed strong prosaposin immunoreactivity mainly in the lysosomal granules in the neurons in both the central and peripheral nervous systems. The expression of prosaposin mRNA, examined using in situ hybridization, was observed in these same neurons. Our results suggest that prosaposin is synthesized ubiquitously in neurons of both the central and peripheral nervous systems. Funding: This study was supported by the Ehime University INCS and in part by grants-in-aid for Scientific Research to S.M. (Exploratory Res. 19659380) from the Japan Society for the Promotion of Science and to AS (Priority Areas 18023029) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Co-existence of the enkephalinergic system and the melanotropinergic system in the rat duodenum shown by immunohistochemistry

The immunohistochemical distribution of alpha-melanotropin (alpha-MSH) and Met-enkephalin (Met-ENK) immunoreactivities in the rat duodenum was examined by using immunofluorescence microscopy. Alternately staining of adjacent frozen serial sections with specific antisera directed to alpha-MSH or Met-ENK revealed that within a subpopulation of myenteric plexus perikarya alpha-MSH immunostaining co-exists with that of Met-ENK. Some myenteric plexus nerve fibres also contain both Met-ENK and alpha-MSH immunoreactivity. These findings might indicate that the genes encoding for the precursors of melanotropins (the pro-opiomelanocortin precursor) and enkephalin (the pro-enkephalin precursor) are generated from a common, large and single ancestor gene which remained conserved during evolution in the rat enteric nervous system.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Neurocalcin-like immunoreactivity in the rat esophageal nervous system

Neurocalcin is a newly identified neuronal calcium-binding protein. We tried here to investigate the immunohistochemical distribution of neurocalcin in the rat esophagus. Nerve cell bodies having neurocalcin immunoreactivity were found throughout the myenteric plexus. In the myenteric ganglia, two types of nerve terminals showed neurocalcin immunoreactivity. One was varicose terminals containing numerous small clear vesicles and forming a synapse with nerve cells. The other terminals were characterized by laminar or pleomorphic structure and many mitochondria. These laminar terminals were supposed to be sensory receptors of the esophageal wall. In the motor endplates of the striated muscles, nerve terminals containing many small clear vesicles and mitochondria also had neurocalcin immunoreactivity. After left vagus nerve cutting under the nodose ganglia, the number of immunopositive thick nerve fibers, laminar endings and nerve terminals on the striated muscles decreased markedly. Retrograde tracing experiments using Fast Blue showed extrinsic innervation of esophagus from ambiguus nucleus, dorsal motor nucleus of vagus, superior cervical ganglia, celiac ganglia, nodose ganglia and dorsal root ganglia. In the celiac ganglia, nodose ganglia and dorsal root ganglia, retrogradely labeled nerve cells were neurocalcin-immunoreactive. Neurons in the celiac ganglia may project varicose terminals, while nodose and dorsal root neurons project laminar terminals. Although cell bodies of motoneurons in the ambiguus nucleus lacked neurocalcin immunoreactivity, these neurons may contain neurocalcin only in the nerve terminals in the motor endplates. Neurocalcin immunoreactivity is distributed in many extrinsic and intrinsic neurons in the esophagus and this protein may play important roles in regulating calcium signaling in the neurons.     

Acetaminophen distribution in the rat central nervous system

Based on the evidence that the antinociceptive effects of acetaminophen could be mediated centrally, tissue distribution of the drug after systemic administration was determined in rat anterior and posterior cortex, striatum, hippocampus, hypothalamus, brain stem, ventral and dorsal spinal cord. In a first study, rats were treated with acetaminophen at 100, 200 or 400 mg/kg per os (p.o.), and drug levels were determined at 15, 45, 120, 240 min by high performance liquid chromatography (HPLC) coupled with electrochemical detection (ED). In a second study, 45 min after i.v. administration of [3H]acetaminophen (43 microCi/rat; 0.65 microg/kg), radioactivity was counted in the same structures, plus the septum, the anterior raphe area and the cerebellum. Both methods showed a homogeneous distribution of acetaminophen in all structures studied. Using the HPLC-ED method, maximal distribution appeared at 45 min. Tissue concentrations of acetaminophen then decreased rapidly except at the dose of 400 mg/kg where levels were still high 240 min after administration, probably because of the saturation of clearance mechanisms. Tissue levels increased with the dose up to 200 mg/kg and then leveled off up to 400 mg/kg. Using the radioactive method, it was found that the tissue/blood ratio was remarkably constant throughout the CNS, ranking from 0.39 in the dorsal spinal cord to 0.46 in the cerebellum. These results, indicative of a massive impregnation of all brain regions, are consistent with a central antinociceptive action of acetaminophen.     

Effect of acute ethanol in vivo and in vitro on the beta-endorphin system in the rat

Acute ethanol treatment in vivo (i.p. injection of 3.5 g ethanol/Kg B. wt.) stimulated the release of beta-endorphin like peptides by the pituitary gland as was indicated by the increased content of beta-endorphin like immunoreactivity (beta-EPLIR) in the plasma. Furthermore, a significant decrease in the anterior lobe content of beta-EPLIR was observed, while the decrease in the neurointermediate lobe beta-EPLIR content at 45 min after the i.p. ethanol injection was not statistically significant. In vitro incubation of neurointermediate lobes, from animals injected with either ethanol or saline, in the presence of 3H phenylalanine indicated that the content of beta-EPLIR in the incubation medium was increased, the content of the newly biosynthesized 3H-phenylalanine labelled proteins in the neurointermediate lobe extract was decreased, while the content of 3H-phenylalanine labelled pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobes extract were not significantly changed by the ethanol treatment, though a small increase was observed. When neurointermediate lobes from untreated control animals were incubated for 3 hrs with 3H-phenylalanine in the presence or absence of 300 mg ethanol per 100 ml incubation medium, there was no significant difference in the beta-EPLIR content in the incubation medium, or in the content of 3H-phenylalanine labelled proteins, pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobe extract. These results suggest that ethanol has little or no direct effect on the beta-endorphin peptides in the pars intermedia cells.     

Adrenocorticotropin production by the intermediate lobe of the rat pituitary

Summary Tissue from the four chambers of the heart of the plaice (Pleuronectes platessa, L.) has been examined in the electron microscope in order to describe the morphology of the heart at a fine structural level.The sinus venosus is a thin walled chamber between 60–90 thick consisting of a connective tissue matrix in which are situated the plexus of the parasympathetic cardiac ganglion and localised bundles of myocardial cells. The myocardial cells do not form a continuous layer but are associated in particular with the region of the cardiac ganglion and are innervated by it.The sino-auricular junction has hitherto been described as a pacemaker region but the myocardial cells in this region are identical in morphology to myocardial cells in other parts of the heart. There is a large complex of nerves, derived from the cardiac plexus, that runs around the junction before branching to innervate the auricle.The myocardial tissues consist of an outer layer of myocardium forming the wall of the heart and a profusion of trabeculae. The endocardium invaginates into the endocardium to divide up the cells into populations of approximately 25 cells in profile. There is no well-defined coronary blood supply although capillaries are occasionally seen. The myocardial cells themselves are small in diameter (3.5–5.5 ) and show some primitive features which are: a short sarcomere (1.4–2.0 ), the absence of any sarcoplasmic reticulum, and very scarce fasciae occludentes. In the atrium in particular, there are many groups of 1500 Å membrane-bound, dense-cored vesicles in the myocardial cells. Ventricular cells contain more myofilaments and mitochondria than do atrial cells and have many vesicles of 0.1–0.3 diameter whose function and contents are unknown.Connective tissue is very evident in the plaice heart, being an integral part of the sinus venosus and the auriculo-ventricular junction and being the sole constituent of the auriculoventricular valve and the bulbus arteriosus.This investigation was carried out during the tenure of an S. R. C. studentshyp awarded to R. M. S.     

Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Morphofunctional stateof the cholinergic nervous apparatus of the normal duodenum and the duodenum after subdiaphragmatic vagotomy

Histochemical study of the intramural nervous apparatus of the duodenum was carried out under normal conditions and following bilateral subphrenic vagotomy. Morphometric and microspectrofluorimetric methods gave informations on the reduction of the number of the cholinergic nervous fibers and of the acetylcholinesterase activity in them after a brief increase of these indices during the first 24 hours after vagotomy, with their subsequent return to the initial values.     

Hyaluronan and chondroitin sulfate proteoglycans are colocalized to the ciliary zonule of the rat eye: a histochemical and immunocytochemical study

 In previous studies, chondroitin sulfate proteoglycans have been localized to the periphery of the zonular fibers and the individual zonular fibrils (or microfibrils) after Cuprolinic blue staining in conjunction with chondroitinase digestions and immunogold labelling with 2-B-6 antibody. In the present study, we wished to determine if these proteoglycans are linked to hyaluronan to form a large multimolecular aggregate. To accomplish this, we localized the hyaluronan using a biotinylated hyaluronan-binding protein fragment of chondroitin sulfate proteoglycan, containing also the link protein, purified from bovine nasal cartilage. The results showed that the ciliary zonule of the rat eye was reactive with the biotinylated hyaluronan-binding probe as demonstrated by streptavidin-peroxidase-diaminobenzidine staining and streptavidin-gold labelling. Hyaluronan-gold labelling showed that the gold particles were mostly localized on the periphery of the zonular fibers, which was similar to the localization pattern of the zonule associated-proteoglycans. This hyaluronan-binding probe also strongly labelled the sites of zonule insertion over the basement membrane of the inner ciliary epithelium at the pars plana and the lens capsule at the equatorial region, which suggests its probable role in the attachment of ciliary zonule to the basement membranes. To demonstrate whether these two molecules are linked to one another, ultrastructural colocalization of both hyaluronan and chondroitin sulfate proteoglycans was performed on the same sections by double-gold labelling, and combined Cuprolinic blue staining and hyaluronan-gold labelling. Gold particles of 15 and 10 nm in sizes labelling both hyaluronan and chondroitin 4-sulfate, were colocalized to the surface of the zonular fibers. The combined Cuprolinic blue staining and hyaluronan-gold labelling showed that the gold particles were localized towards the ends of the Cuprolinic blue-stained rodlets, which strongly suggests that these chondroitin sulfate proteoglycans are linked to the hyaluronan chain to form a large aggregate surrounding the periphery of the zonular fibers. These ciliary zonule-associated proteoglycan-hyaluronan aggregates may play a role in organizing the individual zonular fibrils (microfibrils) into bundles of zonular fibers. Accepted: 5 November 1996