Studies on catalase, glutathione peroxidase and superoxidismutase activities in aging cells of Paramecium tetraurelia

The nature of the aging process has been the subject of considerable speculation. Now, some data indicate that free radical reactions going on continuously in the cells contribute to aging. Considering these data, we have investigated the activity of enzymes (catalase, glutathione peroxidase, superoxidismutase) present physiologically in the cell to limit to tolerable levels, the rate of free radicals or H2O2. These enzymes activities were assayed in Paramecium tetraurelia as clonal age increased. Catalase activity increases slightly during aging of paramecia, i.e. during maturity and senescence phases (20-150 fissions). No significant changes in glutathione peroxidase and superoxidismutase is found. Catalase activity was also assayed as a function of culture conditions. As the cells begin starving and the percentage of autogamous cells increases, catalase activity decreases. After autogamy, a large increase of catalase activity occurs during the sexual immaturity phase, i.e. during the first 20 fissions. By another way, H2O2 added in the culture medium (from 0 to 15 X 10(-5)M) causes an important increase of catalase activity (from 100 U.I. to 250 U.I.). The possible role of O-.2, OH. and H2O2 in aging is discussed.     

A method to reduce glutathione peroxidase levels in primary endothelial cell cultures

The purpose of this study was to develop culture conditions that would reduce glutathione peroxidase activity in bovine mammary endothelial cells. Conditions of reduce glutathione peroxidase activity were produced in vitro by culturing cells in selenium-deficient media. Low selenium levels were achieved by reducing serum concentrations; however, levels of essential growth factors also were reduced by this method. Therefore, cell proliferation was promoted by supplementation with combinations of defined serum components including insulin, transferrin, linoleic acid, bovine brain extract, and human epidermal growth factor. Out of seven different formulas tested, F12K medium containing 2% fetal bovine serum, insulin, transferrin, and linoleic acid was found to be conducive for cell proliferation. Upon confluence, endothelial cells cultured under these conditions consistently displayed short passage rates, consistent cell numbers, and classic cobblestone morphology when grown in the presence or absence of supplemental selenium. Additionally, these cells retained typical endothelial cell characteristics such as uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carb ocyanine perchlorate acetylated low-density lipoprotein and the expression of cell adhesion molecules and von Willebrand Factor.     

Ultrastructural myeloperoxidase activity and expression of myeloid-associated antigens in acute lymphoblastic leukemia

Ultrastructural myeloperoxidase (MPO) activity and myeloid-associated antigen (MyAg) expression were investigated in 12 adult patients with acute lymphoblastic leukemia (ALL). Ultrastructural MPO was detected by 3 different methods. Immunophenotyping was performed by flow cytometry, using a series of monoclonal antibodies. Ultrastructural MPO-positive blast cells were detected in 6 patients (50%). In 5 of these 6 patients, the methods detecting both MPO and platelet peroxidase (PPO) activities found MPO-positive blast cells more frequently than those detecting MPO activity alone. In 2 patients (17%), at least one kind of MyAg was positive. Ultrastructural MPO activity was detected more frequently than MyAg expression in ALL patients. This method of detecting PPO and MPO is recommended for detection of ultrastructural MPO-positive ALL.     

Localization of motoneurons innervating the stylopharyngeus muscle in the cat

Recent investigations of the nucleus ambiguus (NA) have attempted to identify motoneurons associated with muscles of the larynx and pharynx. However, relatively little attention has been directed to the stylopharyngeus muscle, which is important in elevation of the pharynx in swallowing and speech. The present study was designed to identify the specific location of stylopharyngeus motoneurons within the brainstem. Horseradish peroxidase or fluorescent dye was injected into the stylopharyngeus muscle of 12 cats. Retrogradely labeled cells were located ipsilateral in the rostral NA and retrofacial nucleus. This is the first report to definitively localize stylopharyngeus motoneurons.     

The distribution of the projection from the hippocampal formation to the nucleus accumbens in the rat: an anterograde- and retrograde-horseradish peroxidase study

We have investigated the distribution and organization of the projection from the hippocampal formation to the nucleus accumbens in the rat. In the first experiments, horseradish peroxidase was injected into the fimbria fornicis. This procedure resulted in massive anterograde labelling of fornix fibers, and enabled the hippocampo--accumbens projection to be charted in detail. Labelled fornix fibers are distributed to the entire length of the nucleus accumbens and do not spread lateral to the anterior limb of the anterior commissure, that is, the projection is limited to the medial nucleus accumbens. Terminal labelling is particularly dense in a caudal dorsomedial sector of accumbens immediately adjacent to the septum. In the second part of the study, horseradish peroxidase was microelectrophoretically injected into the nucleus accumbens, in order to confirm the findings from the anterograde experiments. When the deposit was localized within the fornix distribution field, as indicated by the first experiment, retrogradely-labelled cells were observed in the subiculum, prosubiculum, and to a lesser extent, in hippocampal field CAI. When the deposit was located outside of the fornix distribution field, no hippocampal labelling was noted. The topography of the projection, suggested by the retrograde labelling experiments, is discussed. The findings confirm earlier reports of such a projection from the hippocampal formation to the nucleus accumbens, and with the use of a relatively novel anterograde-horseradish peroxidase technique, provide a picture of fiber labelling representing the entire field of origin of the fornix system. The striatal projection field of this pathway is discussed in relation to other striatal afferents, with particular emphasis on limbic afferentation of the striatum. Finally, the so-called 'hippocampal district' of striatum is discussed with respect to its neurotransmitter composition, as well as its functional importance regarding limbic system influence on central motor mechanisms.     

The amygdalostriatal projection in the rat—an anatomical study by anterograde and retrograde tracing methods

Tritiated leucine and proline injected into the amygdaloid complex was found to label a voluminous amygdalostriatal fiber system which is distributed to all parts of the striatum except an antero-dorsolateral striatal sector. The connection is established by way of the longitudinal association bundle as well as the stria terminalis, and includes a modest (10–15%), symmetrically distributed contralateral component conveyed by the anterior commissure. Both autoradiographic findings and subsequent observations in retrograde cell-labelling (horseradish peroxidase) material indicate that the amygdalostriatal projection originates mainly from the nucleus basalis lateralis amygdalae, in much lesser volume from the nucleus basalis medialis, and minimally from the nucleus lateralis amygdalae; no other contributing amygdaloid cell group could be identified.A comparison of the present findings with earlier reports indicates that the amygdalostriatal projection widely overlaps the striatal projections from the ventral tegmental area, the mesencephalic raphe nuclei and the prefrontal cortex. Like the amygdalostriatal projection, these striatal afferents largely or entirely avoid the antero-dorsolateral striatal quadrant, which thus appears to be the striatal region most sparsely innervated by afferents originating from structures within the circuitry of the limbic system. Findings in additional autoradiographic material identify this relatively non-limbic striatal quadrant as the main region of distribution of the corticostriatal projection from the sensorimotor cortex.     

Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin

The distribution of fluoride-resistant acid phosphatase, substance P and somatostatin were investigated in the dorsal horn of the spinal cord and in dorsal root ganglia. In the dorsal horn, the distribution of fluoride-resistant acid phosphatase closely paralleled that of somatostatin and only partly overlapped with that of substance P. In sensory ganglia, none of the fluoride-resistant acid phosphatase-containing neurones contained either substance P or somatostatin. The results suggest the existence of a population of fluoride-resistant phosphatase-positive sensory neurones which is distinct from neurones containing either of these peptides.     

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.     

大鼠丘脑腹后核小细胞部向岛皮质的投射─内脏性传入通路研究之一

本文用ＨＲＰ示踪技术发现丘脑腹后核小细胞部向岛皮质有明确的定位投射，此发现为探索内脏性传入通路提供新的形态学资料．本文还用Ｎｉｓｓｌ法研究了丘脑腹后核小细胞部的细胞构筑，为丘脑腹后核小细胞部的核团划分、形态特征及细胞构筑学积累了资料．     

Various types of corticotectal neurons of cats as demonstrated by means of retrograde axonal transport of horseradish peroxidase

Summary The retrograde labeling of cortical neurons with horseradish peroxidase (HRP) was used to investigate the morphological features of neurons in various cortical areas projecting to the superior colliculus in the cat.Corticotectal cells were found to be labeled in layer V of the entire cerebral cortex. The number of labeled cells and their locations varied according to the sites of injections of HRP in the colliculus. Most of the Corticotectal cells identified in the present study were small (9–20 m in diameter, 66%) and medium (20–40 urn, 30%) pyramidal neurons and only 4% of them were large (more than 40 m). The labeled cells, 261 in total number, had somal diameters of 20.8±8.0 m (mean and SD). The range of sizes of the labeled neurons was different in different cortical areas. For example, the labeled neurons in the Clare-Bishop area had a greater proportion of large diameter cells than in other areas.The present findings are largely in agreement with the previous data of anterograde degeneration methods with respect to the topographical correlation of the Corticotectal projections. However, in some cortical areas, e.g., the sensorimotor and the first visual (area 17) cortex of the lateral surface of the hemisphere, relatively small numbers of Corticotectal neurons appear to have been labeled by retrogradely transported HRP. The sparsity of the labeled neurons in certain cortical areas may reflect the existence of Corticotectal neurons with axon collaterals supplying brain structures other than the superior colliculus.Abbreviations A.c. Aqueductus cerebri - AEct Gyrus ectosylvius anterior - AEs Sulcus ectosylvius anterior - AI Stratum album intermediale - AL Gyrus lateralis anterior - AP Stratum album profundum - AS Gyrus sylvius anterior - Cd Nucleus caudatus - F.l.m. Fasciculus longitudinalis medialis - GI Stratum griseum intermediale - GP Stratum griseum profundum - GS Stratum griseum superficiale - Ic Inferior colliculus - L Left - MEct Gyrus ectosylvius medius - MS Gyrus sylvius medius - MSup Gyrus suprasylvius medius - N.r. Nucleus ruber - O Stratum opticum - P Pontine nuclei - P.c. Pedunculus cerebri - PEct Gyrus ectosylvius posterior - P.g. Periaqueductal gray matter - PSigm Gyrus sigmoideus posterior - PSup Gyrus suprasylvius posterior - R Right - Sc Superior colliculus - S.n. Substantia nigra - Z Statum zonale - II Optic nerve - III and IV Motor nuclei of cranial nerves