Biomedical subjects

B Zipser

Publications and source records attributed to B Zipser.

At least 37 records · Page 2Linked to original sources

PubMed1991

A mannose-specific recognition mediates the defasciculation of axons in the leech CNS.

We are studying a mannose-specific recognition mediating the projection of axons in the synaptic neuropil of the embryonic leech CNS. A functional class of neurons, the sensory afferents, can be distinguished by a mannose-containing epitope that is asparagine-linked to a 130 kDa surface protein and is reactive with the monoclonal antibody Lan3-2. Sensory afferents project as a tightly fasciculated bundle through peripheral nerves but, upon arriving in the CNS, defasciculate into the synaptic neuropil. This defasciculation allows the previously bundled sensory afferents to form an arborization in the synaptic neuropil. Three lines of experimental evidence indicate that the defasciculation is mediated by the sensory afferent's mannose-containing Lan3-2 epitope. The defasciculation is inhibited (1) by blocking the Lan3-2 epitope with Lan3-2 Fab fragments, (2) by cleaving the asparagine-linked carbohydrate moieties from surface proteins with the glycosidase N-glycanase, and (3) by competing for a putative mannose-binding protein with the neoglycoprotein mannose-BSA [albumin, p-aminophenyl alpha-D-mannopyranoside (26 mol monosaccharide/mol albumin)]. In addition to inhibiting the defasciculation, the three perturbation reagents also elicited the refasciculation of axons that had defasciculated prior to their application. These three different experimental approaches provide strong evidence that carbohydrate recognition regulates the projections of sensory afferents in the leech synaptic neuropil. Carbohydrate interactions therefore can play a major role in regulating the neuronal architecture in the CNS.

Amidohydrolases↗

PubMed1991

The segmentation of the leech nervous system is prefigured by myogenic cells at the embryonic midline expressing a muscle-specific matrix protein.

In the leech, adult muscle cells and embryonic mesodermal/myogenic cells express the cell-type-specific Laz 10-1 epitope on extracellular matrix-associated proteins. Using this muscle-specific epitope as a marker, we found the following correlations between the development of identifiable myogenic cells at the embryonic midline and the segmentation of the leech CNS into 32 reiterative ganglia. (1) During the production of mesodermal and ectodermal stem cells, cell bodies of midline myogenic cells create 32 anterior-posterior intervals along the midline of the embryonic germinal plate. The mesoblasts then rearrange themselves into 32 somites whose spacing follows the intervals between the midline myogenic cell bodies. (2) Bilateral segmental zones of myogenic differentiation originate in juxtaposition to the midline myogenic cells. (3) The first two types of muscle precursors develop from midline and adjacent bilateral myogenic cells; they are the precursors of the CNS muscles and of three groups of ventral blood sinus muscles. These two types of muscle precursors demarcate the boundaries of the territory within which neuroblasts proliferate and coalesce into segmental hemiganglionic primordia: Cell bodies of muscle precursors, like cornerstones, demarcate the anterior and posterior borders of the hemiganglionic primordia; their longitudinal processes surround the expanding hemiganglionic primordia on the medial, lateral, dorsal, and ventral aspect. The contours of muscle precursors and midline myogenic cells are sharply delineated by immunohistochemical staining of the Laz 10-1 epitopes. In contrast, undifferentiated mesoblasts are surrounded by diffuse layers of stained epitopes, which are expressed at fluctuating levels. Because elevated levels of this matrix epitope are associated with mesodermal/myogenic cells undergoing morphogenetic rearrangements, it may participate in the molecular mechanisms underlying the segmentation of the nervous system.

Animals↗

PubMed1990

Segregation of afferent projections in the central nervous system of the leech Hirudo medicinalis.

Sensory axons originating in peripheral tissues converge onto each segmental ganglion in the central nervous system (CNS) of the leech, where they segregate into well-defined regions of the synaptic neuropil. Here we report on several aspects of the molecular and anatomical organizations of these afferent projections that bear upon the hypothesis that surface markers are involved in organizing these axons as they grow into the CNS. First, we show that the distribution of some surface markers in the adult is restricted to axons of peripheral origin and is not present on the neighboring axons of central neurons. Second, we demonstrate that the number of afferents increases postembryonically as the leech increases in size, suggesting that at least some of the cues employed by afferent axons to grow to appropriate central targets must be present throughout the life of the animal. We then show, using anterograde axonal tracing and immunohistochemistry, that there is both convergence and divergence of afferent axons into highly specific regions of the neuropil. Lastly, we examine the distribution of surface markers present on different subsets of afferents and show that axons having one type of marker segregate from those having the second type. Our results, considered together with previous observations in this system, provide new clues about the organization of afferent projections in the nervous system of the leech. They also suggest how a relatively small number of molecular markers might mediate fiber-fiber interactions to organize afferent axons as they grow into the CNS.

Afferent Pathways↗

PubMed1990

Expression of surface glycoproteins early in leech neural development.

Cell migration and axon growth during neural development rely upon cell-cell and cell-matrix interactions mediated by surface glycoproteins. The surface glycoprotein recognized on leech neurons by monoclonal antibody Lan3-2 has previously been implicated in the process of axon fasciculation during regeneration in adults. In adult leeches, Lan3-2 binds to a carbohydrate epitope of a 130 kD protein. The present study demonstrates that in embryos the antibody binds to the same carbohydrate epitope of glycoproteins with molecular weights of 130 kD and higher. As a first step in evaluating a possible role of the Lan3-2 glycoprotein or the cells that express it during neural development, we determined its distribution in the developing nervous system of the leech Hirudo medicinalis. In embryos, Lan3-2 epitope is expressed on fasciculated sensory afferents and it appears on the cell bodies before neurite outgrowth. The sensory fibers appear rostrally by embryonic day 10, less than halfway through development. Earlier, by 7 days of development at 20 degrees C, Lan3-2 binds to previously undocumented cell types: (1) cells appearing along the embryonic midline and (2) a cluster of cells located at the rostral edge of the germinal plate. These cells only transiently express this antigen and are present at critical left-right and rostrocaudal boundaries during a period of cell proliferation, movement, and migration that produces the nervous system. Thus the Lan3-2 surface glycoprotein or the cells expressing it are candidates for involvement in axon fasciculation, cell migration, and directed axonal growth.

Animals↗

PubMed1990

Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech.

We are reporting on the evolutionary conservation of carbohydrate epitope families from vertebrate to leech. 1) The sulfated L2/HNK-1 carbohydrate epitope (Abo T, Balch CM (1981): J Immunol 127:1024-1029; Kruse J, Mailhammer R, Wernecke H, Faissner A, Timpl R, Schachner M (1984): Nature 311:153-155) is detected on glycoproteins of leech neurons using monoclonal antibodies (mAbs) L2 (336) and HNK-1. 2) Three rat mAbs, L3, L4, and L5, bind to leech nerve and muscle. The L3, L4, and L5 epitopes are localized to a group of mannosidic leech glycoproteins originally identified through mAbs Lan3-2 (Hogg N, Flaster M, Zipser B (1983): J Neurosci Res 9:445-457 and Laz6-189 (McRorie JW III, Zipser B (1988): "Cell Culture Approaches to Invertebrate Neuroscience." London: Academie Press, pp 33-52. MAb Lan3-2, which binds to a mannosidic epitope of the 130 kD sensory protein, has recently been shown to perturb the penetration of sensory afferents into the synaptic area of the central neuropile (Zipser B, Morell R, Bajt ML (1989): Neuron 3:621-630). The L3, L4, and L5 mAbs have been described to recognize different mannosidic epitopes on glycoproteins, some of which have been identified as neural cell adhesion molecules, and on astrocyte-specific proteoglycan from mouse brain (Kücherer A, Faissner A, Schachner M (1987): J Cell Biol 104:1597-1602; Fahrig T, Schmitz B, Weber D, Kücherer-Ehret A, Faissner A, Schachner M (1990): Eur J Neurosci 2:153-161; Streit A, Faissner A, Gehrig B, Schachner M (1990): J Neurochem In Press). The superposition of five different mannosidic epitopes on the axons of sensory afferents suggests complex, concerted participation of mannosidic epitopes in neuronal pathfinding and target recognition.

Animals↗

PubMed1990

The specificity of 130-kDa leech sensory afferent proteins is encoded by their carbohydrate epitopes.

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.

Animals↗

PubMed1989

Glial processes, identified through their glial-specific 130 kD surface glycoprotein, are juxtaposed to sites of neurogenesis in the leech germinal plate.

Glial processes, bearing a unique 130 kD surface protein, are located at key sites of morphogenic movement and neuronal differentiation in the leech germinal plate. A midline glial fascicle resides at the primary axis of embryonic symmetry, alongside which teloblasts move as they generate their bandlets of stem cells. The n-bandlets straddle the midline glia and are known to produce most of the central neuroblasts. The midline glia then defasciculates as neuroblasts begin to aggregate into neuromeres. The defasciculated processes expand into these neuromeres, molding the future central neuropile. Neuroblasts will initiate primary axons toward the midline glia. As the neuromeres mature, midline glial process thin out to demarcate the orientation of the future connectives, which are the major longitudinal axon tracts along the midline. Next, segmental but still primordial glia appear in the neuromeres. Initially, they also project longitudinally, then transversely, demarcating the other two major axonal pathways--the central commissures and peripheral roots. Finally, macroglial processes proliferate as massive axon growth invades the central and peripheral nervous system. Thus, glial processes with different developmental histories accompany different aspects of leech neurogenesis. In other systems, glia have been shown to promote the differentiation and the guidance of neurons. It remains to be seen whether the glial-specific 130 kD protein is a receptor mediating these typical glial functions in the leech germinal plate.

Animals↗

PubMed1989

Defasciculation as a neuronal pathfinding strategy: involvement of a specific glycoprotein.

Leech sensory afferents change their growth behavior as they enter the CNS. Arriving from the periphery in fasciculated tracts, they abruptly defasciculate and expand into diffuse trees before reassembling into four distinct central tracts. In the organ-cultured germinal plate, growing sensory afferents were incubated with monovalent Fab fragments of the Lan3-2 antibody, which recognizes a 130 kd sensory neuron protein by its mannose epitope. Very low concentrations of Lan3-2 (6 and 12 nM) specifically inhibited the central defasciculation of sensory afferents, which then continued growing as a single tract. In contrast, monoclonal antibody Lan3-6, which binds to an internal sensory antigen, failed to yield the same effect. These observations suggest that this sensory neuron 130 kd surface glycoprotein participates in a developmentally significant heterophilic interaction specific for the CNS.

Afferent Pathways↗

PubMed1988

The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech, and rat.

We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [125I]beta-endorphin [( 125I]beta E), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for mu, delta, and kappa opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [125I]beta-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their mu, delta, or kappa specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive sequence homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Identification and characterization of the opiate receptor in the ciliated protozoan, Tetrahymena.

Tetrahymena, a ciliated protozoan, is a highly specialized, differentiated eukaryotic organism. It is known to possess many informational substances, including beta-endorphin (beta E). We wished to investigate the possibility that this organism possesses a functional opiate receptor which might be similar to the well-characterized opiate receptor in the rat brain. Binding assays using both living cells and membrane preparations, verified stereospecific, saturable, reversible 125I-beta E binding. This binding was displaceable by various opiates chosen to represent each of the putative opiate subtypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a disuccinimidyl suberate cross-linked 125I-beta E-receptor complex revealed a pattern of bands which consistently included bands at 110, 58-55, and 29 kDa. These bands, which were all displaceable by the classical antagonist, naloxone, as well as by other opiates, are thought to be prototypic for various opiate receptor subtypes. Limited proteolysis in SDS-PAGE showed that the 110 kDa band could be fragmented into 58-55 and 29 kDa bands and that the 58 kDa band could generate a 29 kDa fragment. The limited digest fragments of the 110, 58-55 doublet and 29 kDa bands were remarkably similar to those generated from the rat brain receptor. Analytical isoelectric focusing of digitonin solubilized 125I-beta E-receptor complexes showed the isoelectric points (pI) from both the rat and Tetrahymena were identical (pI 4.6). Chemotactic experiments with the intact Tetrahymena, demonstrated that these unicellular animals migrated toward a 10(-9) M beta E gradient. Chemotaxis was blocked by (-)-naloxone but not (+)-naloxone, suggesting a stereospecific opiate receptor-mediated response. We conclude that Tetrahymena possesses a functional opiate receptor (recognition molecule) very similar to the opiate receptor of the rat brain.

Animals↗

PubMed1988

Vasoactive intestinal peptide receptors in rat spleen and brain: a shared communication network.

The binding sites for [125I]-vasoactive intestinal polypeptide (125I-VIP) in rat spleen and brain were localized using autoradiography. High affinity VIP receptors are present in rat spleen, and competition studies reflect structure-activity relationship typical of VIP receptors elsewhere. In spleen, specific binding of 125I-VIP occurs on red pulp and, most abundantly, on the periarteriolar lymphoid sheath (PALS) of white pulp. Unlabeled VIP competes for binding to both red pulp and white pulp, whereas secretin displaces binding to PALS more potently than to red pulp. This indicates that expression of VIP and/or secretin type receptors is limited to T lymphocytes of white pulp. In red pulp, VIP receptor bearing cells probably are monocytes/macrophages since this is the most abundant red pulp cell type. In the brain, VIP receptors are widely distributed with the highest densities occurring in "sensory" areas. Receptors are abundant in the olfactory bulb, thalamic nuclei, several cranial nuclei and the area postrema. High levels of 125I-VIP binding occurred on inner walls of blood vessels of the brain and spleen. The distribution patterns of receptors for "VIP-ergic signals" in brain and lymphoid tissue indicate interrelatedness of the two organ systems. This may serve as one biochemical rationale for a bio-psycho-social view of health and disease.

Animals↗

PubMed1987

Regeneration of afferent axons into discrete tracts within peripheral nerves in the leech.

We have analyzed the pathway followed by regenerating afferent axons in peripheral nerves of the leech Hirudo medicinalis by anterograde labeling with horseradish peroxidase. We show that axons are able to reestablish appropriate pathways following a lesion (crush) which greatly disrupts the organization of the nerves. Our results are consistent with a pathway selection mechanism involving axon surface markers which are retained on the distal stumps of crushed axons.

Animals↗

PubMed1987

A group of related surface glycoproteins distinguish sets and subsets of sensory afferents in the leech nervous system.

The distribution of 4 surface glycoproteins on axons of peripheral neurons was studied in the leech Hirudo medicinalis through monoclonal antibodies. All 4 glycoproteins have a similar molecular weight of 130 kDa. Immunohistochemical localization of these glycoproteins on tissue sections of nerves and neuropil reveals tracts of afferent axons organized as nested sets. Their distribution suggests a possible role for these molecules in mediating axon fasciculation.

Animals↗

PubMed1987

The macroglial cells of the leech are molecularly heterogeneous.

Monoclonal antibodies derived from fusions employing either whole leech nerve cords or fractionated proteins (gel bands) bind to the macroglial cells of the nerve cord. Three different antibodies bind to either one, two, or three of the four macroglial cell types in the leech CNS, serving as markers and showing that these four cell types, which differ primarily by anatomical position, all differ molecularly as well. Conventional microscopy confirms the existence of a novel macroglial cell type first noted because it binds antibody. Western (immunoblot) blot analyses demonstrate a polypeptide antigen of 77 kD in the macroglial cells of the connectives and a 130-kD polypeptide antigen associated with the macroglial cells of the connectives, of the root nerves, and of the ganglionic neuropil. The extensive molecular heterogeneity of leech neurons demonstrated by monoclonal antibody techniques is shared by macroglia.

Animals↗

PubMed1987

Cell death during gangliogenesis in the leech: bipolar cells appear and then degenerate in all ganglia.

The bipolar cells can be recognized very early during gangliogenesis in the leech central nervous system by their expression of antigens that are recognized by the monoclonal antibody Laz1-1. They are the first cells to express these antigens, which are later shared with a distinct set of other cells in the leech nervous system. Their processes extend several segments rostrally and caudally along the forming interganglionic connective nerves; they are first found in anterior segments and gradually appear in more posterior ones over the course of 2-3 d. At about the time bipolar cells appear in the neuromeres of the tail ganglion, those in the most anterior segmental ganglia begin to degenerate. Degeneration proceeds caudally over the next 3 d, until all bipolar cells have disappeared. Bipolar cells are, thus, members of that class of cells that exists for only a short period in neurogenesis and then presumably disappears once its functions are no longer required. These cells' morphology and appearance at the earliest stages in the formation of the interganglionic connective nerves are suggestive of a role in the establishment of these longitudinal pathways in the leech CNS.

Animals↗

PubMed1986

Development of segmental differences in the pressure mechanosensory neurons of the leech Haementeria ghilianii.

Using a monoclonal antibody specific for the pressure mechanosensory neurons (P cells) of the leech Haementeria ghilianii, we have examined the segmental differences between P cells in the adult nerve cord, as well as the development of these differences during embryogenesis. The standard segmental ganglion contains two pairs of P cells of about the same size and staining intensity. The sex ganglia appear to be missing the P cells that normally innervate ventral skin, and ganglia 20 and 21 have much smaller ventral P cells than most segments. The pattern of P cells in the head and tail ganglia also differs slightly from that of the standard ganglia. During embryogenesis, when the neurons are first stained by the antibody, there are two pairs of P cells of equal size in each segmental ganglion. Obvious segmental differences arise subsequently, modifying an initially identical set of cells.

Age Factors↗

PubMed1986

Probing structural homologies in cell-specific glycoproteins in the leech CNS.

Three monoclonal antibodies (mAbs) raised against the leech CNS recognize surface antigens on small sets and subsets of neurons or on glial cells. On immunoblots, they all recognize proteins of 130 kDa molecular weight. In addition, they each bind up to several different lower molecular weight forms. The 130 kDa polypeptides recognized by these mAbs are not major proteins on Coomassie blue-stained gels. They behave as glycoproteins on lentil lectin columns but are not major Concanavalin A-binding molecules. These molecules therefore represent a group of lower abundance, cell-type-specific antigens. Structural relationships between these antigens were explored using immunoprecipitation. The glial cell antigen was immunopurified, however, a fraction of the neuronal antigens co-precipitate. The co-precipitation of neuronal antigens raises the possibility that different neuronal antigenic determinants are carried on the same protein molecule. Such a protein may be modified to carry either one or both neuronal determinants, and could serve as a tag to physiologically delineate subsets of neurons nested within larger neuronal sets.

Animals↗

PubMed1985

Antibody staining reveals novel aspects of segmentation within the leech central nervous system.

The leech is a segmented annelid with a well characterized central nervous system. In this report, we use antibodies to map the distribution of neurons confined to selected segmental ganglia in the mud leech Haemopis marmorata. The distribution of these neurons suggest 3 novel aspects of segmentation in the leech nervous system: (1) neurons are assigned to even-numbered ganglia through a mechanism which effectively counts through the leech segmental body plan by units of 2, (2) neurons are assigned to ganglia 7 and 14 through a mechanism which effectively counts in units of 7 and (3) neurons are assigned to the 2nd and 4 fused head ganglia and to the 2nd of 21 unfused midbody ganglia through a mechanism which effectively counts units from the origin of these 2 ganglionic series. These 3 hypothetical counting mechanisms divide the central nervous system (CNS) into supersegmental units. Neurons used to define these supersegmental units have been injected with tracer and identified as interganglionic interneurons. Competitive interactions among embryonic precursors of these neurons may directly eliminate their homologs from intervening ganglia, and thus sculpture supersegmental patterns into the mature nervous system.

Animals↗