Synapse-like contacts between axons of the pineal tract and the subcommissural organ in Rana perezi (Anra) and their absence in Carassius auratus (Teleostei): ultrastructural tracer studies

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.     

Immunocytochemical and electron-microscopic investigations of the pineal organ in adult agamid lizards,Uromastix hardwicki

Summary Lacertilian species display a remarkable diversity in the organization of the neural apparatus of their pineal organ (epiphysis cerebri). The occurrence of immunoreactive S-antigen and opsin was investigated in the retina and pineal organ of adult lizards, Uromastix hardwicki. In this species, numerous retinal photoreceptors displayed S-antigen-like immunoreactivity, whereas only very few pinealocytes were labeled. Immunoreactive opsin was found neither in retinal photoreceptors nor in pinealocytes. Electron microscopy showed that all pinealocytes of Uromastix hardwicki resemble modified pineal photoreceptors. A peculiar observation is the existence of a previously undescribed membrane system in the inner segments of these cells. It is evidently derived from the rough endoplasmic reticulum but consists of smooth membranes. The modified pineal photoreceptor cells of Uromastix hardwicki were never seen to establish synaptic contacts with somata or dendrites of intrapineal neurons, which are extremely rare. Vesiclecrowned ribbons are prominent in the basal processes of the receptor cells, facing the basal lamina or establishing receptor-receptor and receptor-interstitial type synaptoid contacts. Dense-core granules (60–250 nm in diameter) speak in favor of a secretory activity of the pinealocytes. Attention is drawn to the existence of receptor-receptor and receptor-interstitial cell contacts indicating intramural cellular relationships that deserve further study.Supported by the Deutsche Forschungsgemeinschaft (Ko 758/31) and the Deutscher Akademischer Austauschdienst (Senior DAAD Research Fellowship to M.A.H.)     

Proteins of the Drosophila melanogaster male reproductive system: Two-dimensional gel patterns of proteins synthesized in the XO,XY, and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins

Testes and paragonial glands of Drosophila melanogaster wild-type males were labeled in vitro using [³⁵S]methionine, and the proteins synthesized were analyzed by 2-dimensional gel electrophoresis. Testes and paragonial glands were also labeled in vivo by feeding male larvae ³⁵S-labeled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. [³H]Proline was also used to label testes, and no significant differences from the ³⁵S pattern were noted. Testes and paragonial glands from XO and XYY males were labeled in vitro with [³⁵S]methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Pure sperm were dissected manually from in vivo labeled males and the proteins analyzed. Ninety-two proteins were detected, which were all synthesized in comparable amounts by XO, XY, and XYY males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational or regulatory proteins present only in very small amounts in the adult testes. ³⁵S-labeled males were also mated to unlabeled females and the transferred proteins analyzed on two-dimensional PAGE. The contributions of the testis and paragonial gland to the ejaculate were determined.This work was supported by a grant from the Medical Research Council of Canada and by the U.S. National Institutes of Health (Grant No. GM-22753). J. I. B. is the recipient of a Medical Research Council of Canada studentship.     

Central projections of the pineal complex in the silver lamprey Ichthyomyzon unicuspis

Summary The central projections of the pineal complex of the silver lamprey Ichthyomyzon unicuspis were studied by injection of horseradish peroxidase. The pineal tract courses caudally along the left side of the habenular commissure, and a few fibers penetrate the brain through the caudalmost portion of this commissure. Most of the fibers, however, continue caudally and enter the brain through the posterior commissure. The pineal tract projects bilaterally to the subcomissural organ, the superficial and periventricular pretectum, the posterior tubercular nucleus, the dorsal and ventral thalamus, the dorsal hypothalamus, the optic tectum, the torus semicircularis, the midbrain tegmentum, and the oculomotor nucleus. A few fibers decussate in the tubercular commissure, but the course of these decussate fibers could not be followed owing to the bilateral nature of the projections. No retrogradely labeled cells were found in the brain. With the exception of the projections to the optic tectum and torus semicircularis, the pineal projections in the silver lamprey are similar to those reported in other anamniote vertebrates.     

Efferent connections from the brain to the frontal organ in Rana temporaria demonstrated by labeling with horseradish peroxidase

After injection of horseradish peroxidase into the frontal organ of Rana temporaria, labeled perikarya were found in the medial part of the amygdala, the preoptic area, the nucleus rotundus, the pretectal area, and the lateral parts of the midbrain central griseum.     

The distribution of Concanavalin A binding sites on the intestinal epithelium of the nematodes Ascans suum and Parascaris equorum

Summary Receptors for Concanavalin A (Con A), were localized on the intestinal epithelium of the nematodes Ascaris suum and Parascaris equorum. Fixed tissue incubated in ³H-Con A showed labeling of the microvilli surface and basal membrane. Using Con A coupled with peroxidase, the tips of the microvilli of Ascaris suum and the tips and lateral surfaces of Parascaris equorum were stained. The basal membrane of both species was also labeled. No labeling was observed on control tissue incubated without Con A or on tissue incubated with Con A to which -methyl-D-mannoside was added.     

Ultrastructural localization of lectin-binding sites and laminin-like immunoreactivity in glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts

Summary Lectins with different sugar specificities and labeled with horseradish peroxidase or gold were used to study, at the electron-microscopic level, surface glycoconjugates of glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts. Differential binding to differentiating glial cells and to neurites was demonstrated. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) bound to glial and neurite surfaces with different degrees of labeling. The formation of glial processes and junctional complexes was invariably accompanied by a corresponding increase of Con A- and WGA-receptors. Peanut agglutinin (PNA) failed to bind to glial cells but strongly stained the plasma membrane of neurite junctions. Lotus tetragonolobus a. (LTA) did not bind either to glial cells or to neurites. In addition, staining with an antibody against laminin showed labeling in areas of neurite outgrowth and neurite interactions; this resembled the localization of PNA receptors. These findings provide evidence for the presence of different carbohydrates at the surface of neurites and glial cells of locust. Their predominant localization in glial processes and neurite junctions suggests that these carbohydrates constitute part of a group adhesion glycoproteins that also includes laminin.     

Afferent connections of the optic tectum in channel catfish Ictalurus punctatus

Summary Horseradish peroxidase was injected unilaterally into the optic tectum of the channel catfish, Ictalurus punctatus. The sources of tectal afferents were thereby revealed by retrogradely labeled neurons in various brain centers. Retrogradely labeled cells were seen in both the ipsilateral and contralateral telencephalon. The superficial pretectal area was labeled on both sides of the brain. Ipsilateral projections were also observed coming from the entopeduncular nucleus. Both the anterior thalamic nucleus and the ventro-medial thalamic nucleus projected to the ipsilateral optic tectum. Cells in the ipsilateral nucleus of the posterior commissure were seen to project to the tectum. Labeled fibers were visualized in the lateral geniculate nucleus ipsilateral to the injected tectum, however, no labeled cell bodies were observed. Therefore, tectal cells project to the lateral geniculate nucleus, but this projection is not reciprocal. No labeled cells were found in the cerebellum. Labeled cells occurred in both the ipsilateral and contralateral medial reticular formation; they were also observed in the ipsilateral nucleus isthmi. A projection was seen coming from the dorsal funicular nucleus. Furthermore, labeled cells were shown in the inferior raphe nucleus.Abbreviations AP Area pretectalis - C Cerebellum - DPTN Dorsal posterior tegmental nucleus - H Habenula - IRF Inferior reticular formation - LI Inferior lobe - LGN Lateral geniculate nucleus - LR Lateral recess - MB Mammillary body - MRF Medial reticular formation - MZ Medial zone of the telencephalon - NC Nucleus corticalis - NDL-M Nucleus opticus dorsolateralis/pars medialis - NI Nucleus isthmi - NPC Nucleus of the posterior commissure - OPT Optic tectum - OT Optic tract - PC Posterior commissure - PN Pineal organ - PrOP Preoptic nucleus - PT Pretectum - TBt Tectobulbar tract - TEL Telencephalon - TL Torus longitudinalis - TS Torus semicircularis - VC Valvula cerebelli - VLTN Ventrolateral thalamic nucleus - VMTN Ventromedial thalamic nucleus     

Re-examination of Mg-dechelation reaction in the degradation of chlorophylls using chlorophyllin a as a substrate

The Mg-dechelating activity of extracts of Chenopodium album (goosefoot) was investigated using an artificial substrate, chlorophyllin a. The activity was measured spectrophotometrically by the formation of a reaction product, pheophorbin a (Mg-free chlorin), after release of the central Mg. The Mg-releasing protein was highly purified by successive DEAE, Butyl and HW-55 chromatographies. The molecular weight of the purified protein was 20 k by gel filtration. The protein showed a broad, but single, pH optimum at 7.5. The K _m value for chlorophyllin a was 95.1 nM at pH 7.5. The Mg-releasing protein was not active with chlorophyllide a, a native substrate, although it was active with Zn-chlorophyllin a. Similar results were obtained from horseradish peroxidase. Only a small molecular weight, metal-chelating substance (MCS) had Mg-dechelating activity for the native substrate. An inhibitor study showed involvement of radicals in the Mg-dechelation of the Mg-releasing protein. The purified Mg-releasing protein showed neither peroxidase activity nor absorption bands in the visible region, and this indicates that the Mg-releasing protein is clearly distinct from horseradish peroxidase, which is a heme-containing protein. A likely conclusion is that the Mg-releasing protein and horseradish peroxidase are not involved in the Mg-dechelation in the degradation pathway of chlorophylls. The relevance of the participation of MCS in Mg-dechelation in the breakdown of chlorophylls (Chls) is also discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nervous connections of the parietal eye in adult Lacerta s. sicula Rafinesque as demonstrated by anterograde and retrograde transport of horseradish peroxidase

Summary Subsequent to the injection of horseradish peroxidase into the parietal eye of adult Lacerta sicula, the course of the parietal nerve and its projections were determined.The parietal nerve enters the left habenular ganglion where it branches into a medial and a lateral route. Some nerve fibers decussate within the habenular commissure. Whereas this pathway exhibits a striking asymmetry at the level of the habenular ganglia, its projections to the dorsolateral nucleus of the thalamus, the periventricular hypothalamic area, the preoptic hypothalamic and telencephalic regions, and the pretectal area are arranged in a strictly symmetric manner. A possible innervation of tegmental areas could not be proven due to the presence of endogenous peroxidase within these regions. No parietal nerve fibers were observed in the optic tectum.In a few animals investigated, scattered labeled perikarya were located in the periventricular hypothalamic gray indicating a parietopetal innervation in Lacerta sicula. The injection of horseradish peroxidase into one of the lateral eyes revealed terminal areas of the optic nerve within the preoptic region, and the thalamic and pretectal nuclei, displaying partial overlapping with the projections of the parietal nerve to these areas.From the present investigation further evidence is obtained that the pineal complex of lower vertebrates is a component of the photoneuroendocrine system. Particular emphasis is placed upon the nervous connections between the parietal eye and the hypothalamus, described for the first time in the present study.Supported by the Deutsche Forschungsgemeinschaft (Grant Ko 758/1)In partial fulfillment of the requirements of the degree of Dr. med., Faculty of Medicine, Justus Liebig University of Giessen     

Peroxidase isoenzymes as markers for the rooting ability of easy-to-root and difficult-to-root <Emphasis Type="Italic">Grevillea</Emphasis> species and cultivars of <Emphasis Type="Italic">Protea obstusifolia</Emphasis> (Proteaceae)

Summary One easy-to-root and one difficult-to-root species of the ornamental plant Grevillea were investigated for rooting potential in relation to peroxidase activity. In vitro-grown shoot segments of both species started to root 30 d after transplanting to rooting medium containing indole-3-butyric acid (IBA), however, fewer roots were found on fewer segments of the difficult-to-root species G. petrophioides compared to the easy-to-root species G. rondeau. Total peroxidase (POX) activity was measured during the rooting process. G. petrophioides showed higher total POX activity at the time point of adventitious root formation than G. rondeau. Isoelectric focusing electrophoresis showed that G. rondeau contained more acidic isoforms than G. petrophioides, but the basic isoforms were more prominent in the difficult-to-root species, especially at the time point of lateral root emergence. In addition, the ability of different hormones to induced POX activity in upper and lower stem segments of both species was tested. Indole-3-acetic acid (IAA), IBA and α-naphthaleneacetic acid induced POX activity in the upper stem segments of G. rondeau, whereas the same hormones led to the induction of POX activity in the lower stem segments of G. petrophioides. Similar to the results obtained with Grevillea, the difficult-to-root variety of Protea showed higher POX activity, especially in the middle stem part and the leaves. Feeding of radiolabeled IAA to the Grevillea stem segments resulted in the synthesis of three different compounds in both species. After 1h incubation no differences were found in the uptake of IAA and the appearance of other labeled compounds. However, after 2 and 4 h incubation IAA uptake was faster in the easy-to-root species and IAA was also metabolized to a higher extent in G. rondeau. Three metabolites were found, tentatively identified as IAA-aspartate, IBA, and an IBA conjugate.     

Development of the cartilage canals and the secondary center of ossification in the distal chondroepiphysis of the prenatal human femur.

The cartilaginous epiphysis of the distal femur is vascularized by a network of cartilage canals during prenatal development. The vascular invasion of the epiphysis begins at approximately eight to ten weeks of gestation with the initiation of cartilage canal formation. A complex vascular system develops within the canals and is well defined by fourteen weeks of gestation. The vascular system is fully developed several months prior to the development of the secondary center of ossification. The formation of the secondary center of ossification within the distal femoral epiphysis is preceded by changes that occur simultaneously within both the chondrocytes in the central portion of the epiphysis and the vascular and perivascular elements contained within the cartilage canals in the central portion of the epiphysis. These concurrent changes in the cellular morphology of the central chondrocytes and in the cellular structure of the central cartilage canals appear to be linked with the initiation of the process of osteogenesis.     

Effect of nitrate supply on the in-vivo synthesis and distribution of trifollin A,a Rhizobium trifolii-binding lectin,in Trifolium repens seedlings

In-vivo synthesis of the white-clover lectin, trifoliin A, was examined by the incorporation of labeled amino acids into protein during heterotrophic growth of intact Trifolium repens L. seedlings. Lectin synthesis was quantified by measuring the level of labeled protein immunoprecipitated from root exudate, from the hapten (2-deoxyglucose) eluate of the roots, and from root and shoot homogenates. The presence of labeled trifoliin A was confirmed by non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography and comparison with trifoliin A standards. In-vivo-labeled trifoliin A was detected in seedling root homogenate 2 h after the addition of labeled amino acids and on the root surface by 8 h. Incorporation of labeled amino acids into protein and trifoliin A was greatest with 2-d-old seedlings and was greater when the plants were grown continuously in the dark than when they were exposed to 14 h light daily. Significantly more labeled lectin accumulated on the root surface of seedlings grown with 1.5 mM KNO₃ than of seedlings grown either without N or with 15.0 mM KNO₃. The labeled lectin from the root surface in all nitrate treatments and from the rootexudate samples of seedlings grown N-free and with 1.5 mM KNO₃ was fully able to bind to Rhizobium trifolii. In contrast, only 2% of the immunoprecipitable protein found in the root exudate of seedlings grown with 15.0 mM KNO₃ was able to bind to the bacteria. Thus, excess nitrate does not repress the synthesis of trifoliin A in the root, but does affect the distribution and activity of this newly synthesized lectin in a way which reduces its ability to interact with R. trifolii. By using Western blot analysis, much more total trifoliin A is detected in the homogenates of shoots than roots. However, greater than 80% of the total labeled protein and 85–90% of the total labeled lectin were found in the root homogenates of 2-d-old dark-grown seedlings incubated for 5 h with labeled amino acids. In addition, Western blot analysis indicated that the shoot homogenate contained smaller-molecular-weight peptides which reacted with the specific anti-trifoliin A antibody. These studies indicate that stored trifoliin A in the seed is degraded in the shoots during seedling development, while newly synthesized trifoliin A in the roots is excreted to the root surface and external environment.Abbreviations IgG immunoglobulin G - LPS lipopolysaccharide - PBS 10 mM potassium-phosphate buffer, pH 7.0, containing 0.8% NaCl - PBS-T 20 mM phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al, 1993, FEBS Lett 315: 313–317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.Abbreviations ATG methionine translation initiation codon - bp base pair - cAPX cytosolic ascorbate peroxidase - pAPX plastidial ascorbate peroxidase - RFLP restriction fragment length polymorphism Accession numbers: The APX1b sequence is in the EMBL database under accession number X80036M.S. gratefully acknowledges the support from the Junta Nacional de Investigaçâo Cientifica e Tecnológia, Portugal (grant number BD/394/90-IE). This work was supported by the Biotechnological and Biological Sciences Research Council through a grant-in-aid to the John Innes Centre.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Newly established cell lines fromDrosophila larval CNS express neural specific characteristics

Summary From the central nervous system ofDrosophila melanogaster 3rd instar larvae, eight continuous cell lines have been established (named ML-DmBG1 to 8). Using ML-DmBG2, single colony isolation was carried out and six colonial clones were obtained. All reacted to the antibody to horseradish peroxidase, which is a neuronal marker in insects. Acetylcholine, a known neurotransmitter inDrosophila, was detected in three of the colonial clones by high performance liquid chromatography. Therefore, it is concluded that the established colonial clones are neural cells originating in the larval central nervous system. Among them, some variation was observed with respect to morphology, acetylcholine content, and reactivity to anti-HRP. The variation may reflect the heterogeneity of cells composing the central nervous system.     

Fish cell line (ULF-23HU) derived from the fin of the central mudminnow (Umbra limi): Suitable characteristics for clastogenicity assay

Summary A cell line (ULF-23HU) from the fin of the central mudminnow (Umbra limi) was characterized and tested for its suitability to assess cytogenetic damages induced by chemicals in fish. Cells of this line exhibit a fibroblastlike appearance and grew optimal at 25°C in, TC-199 medium containing 10% fetal bovine serum, but slower growth continued down to 4°C, where they could be stored for prolonged periods. Seeding efficiency of ULF-23HU cells on the plastic substratum was approximately 85% in the above culture medium at 25°C. They had a 32-h cell cycle time taken up by a 20-h S period as determined by the autoradiographic analysis of the fraction of labeled mitosis. Cultures showed relatively high mitotic index (0.84 to 2.35%) during exponential growth phase lasting about 7 d. Karyological analysis of the cells at the different subculture passages revealed constant chromosome modal number of 23 consisting of metacentric or submetacentric chromosomes, which were primarily similar to those of in vivo cells, with one additional chromosome. The spontaneous sister chromatid exchange rate was 5.3 per metaphse. When ULF-23HU cells were exposed toN-nitroso-N-methylurea, a clastogen in the mammalian cells, dose-dependent increases both in sister chromatid exchanges and chromosome aberrations were clearly detected. These results on the growth kinetics and cytogenetic characteristics offered the high possibility of the use of this cell line as a suitable in vitro model for clastogenicity studies in fish. This work was supported by grants from Korea Sciences and Engineering Foundation and Japanese Government Research Awards for Foreign Specialists to E.-H. Park.     

Chlorophyll and cutin in early embryogenesis in Capsella,Arabidopsis, and Stellaria investigated by fluorescence microscopy

Advanced globular embryos of Capsella and early heart-shaped embryos of Arabidopsis begin to show chlorophyll fluorescence. It is not present in the suspensor, epiphysis, radicle and embryo of Stellaria. Cutin fluorescence appears on the protoderm of all advanced globular embryos. Fluorescence disappears during the early torpedo stage. It is not present on suspensors.     

Localization and Central Projections of Primary Afferent Neurons That Innervate the Temporomandibular Joint in Cats

Primary afferent neurons that innervate the temporomandibular joint (TMJ) in cats were labeled by injecting a 2-5% solution of wheatgerm agglutinin bound to horseradish peroxidase into the joint capsule and capsular tissues in 14 cats and processing the brain stem and trigeminal ganglia using the tetramethylbenzidine method described by Mesulam (1978). The perikarya of ganglion cells that innervate the TMJ ranged in diameter from 15 to 109 μm and were primarily located in the posterolateral portion of the trigeminal ganglion. The central processes of these neurons entered the brain stem in middle pons and were distributed to all portions of the sensory trigeminal nuclei. However, the majority of labeled fibers and greatest density of terminal labeling were observed in the dorsal part of the main sensory nucleus and the subnucleus oralis of the spinal trigeminal nucleus. Very few labeled fibers were observed in the spinal tract of the trigeminal nerve below the obex. However, evidence for axon terminals was consistently observed in laminae I, II, and III of the medullary dorsal horn. These findings concur with physiological evidence showing that information from the TMJ influences neurons in rostral (Kawamura et al, 1967) and in caudal (Broton et al, 1985) portions of the trigeminal sensory nuclei.