Biomedical subjects

B Zipser

Publications and source records attributed to B Zipser.

At least 19 recordsLinked to original sources

PubMed2002

Effect of APOE genotype on microvascular basement membrane in Alzheimer's disease.

APOE4 homozygosity has been associated with an increased risk of sporadic Alzheimer's disease through a mechanism, which has yet to be defined. Recent evidence has suggested that microvascular basement membrane injury may be a critical factor in the pathogenesis of AD-related dementia. In previous studies, we have shown that the synaptic organizing protein agrin can be found in neurons, and is a major component of the brain microvascular basement membrane. Here, we compare the basement membrane surface area of cortical microvasculature in AD brains by staining with an anti-agrin antibody. Quantitative morphometric analysis was used to determine the mean basement area (micro(2)) of prefrontal cortical microvessels. An average of 10 capillaries was measured in each of 35 cases of AD genotyped for APOE status. APOE4,4 homozygotes had smaller capillary basement membrane areas (17.4 micro(2))+/-6.2) than APOE3,3 homozygotes (26.9 micro(2)+/-6.5), p<0.001. The capillary basement membrane areas (CBMA) of heterozygotes APOE3,4 did not differ significantly from APOE3,3 or APOE4,4. Braak stage did not contribute significantly to CBMA. However, a preliminary analysis suggests an interaction between APOE4,4 and Braak V-VI producing smaller CBMA, a finding which needs to be confirmed with a larger sample. These data support the hypothesis that APOE4,4 is associated with thinning of the microvascular basement membrane in Alzheimer's disease.

Aged↗

PubMed2001

Molecular identification and sequence analysis of Hillarin, a novel protein localized at the axon hillock.

The monoclonal antibody Lan3-15 identifies a novel protein, Hillarin, that is localized to the axon hillock of leech neurons. Using this antibody we have identified a full length cDNA coding for leech Hillarin and determined its sequence. The gene encodes a 1274 residue protein with a predicted molecular mass of 144013 Da. Data base searches revealed that leech Hillarin has potential orthologues in fly and nematode and that these proteins share two novel protein domains. The W180 domain is characterized by five conserved tryptophans whereas the H domains share 21 invariant residues. In contrast to the arrangement in fly and nematode the cassette containing the W180 and H domains is repeated twice in leech Hillarin. This suggests that the leech Hillarin sequence originated from a duplication event of an ancestral protein with single cassette structure.

Amino Acid Sequence↗

PubMed2000

Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth.

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif. By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments. The most NH(2)-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain. The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.

Animals↗

PubMed1999

Differential glycosylation and proteolytical processing of LeechCAM in central and peripheral leech neurons.

LeechCAM is a recently described member of the Ig-superfamily which has five Ig-domains, two FNIII-domains, a transmembrane domain, and a cytoplasmic domain. Phylogenetic analysis indicated that LeechCAM is the leech homolog of apCAM, FasII, and vertebrate NCAM. Using a leechCAM-specific monoclonal antibody we show by immunoblot analysis and by Triton X-114 phase separation experiments that in addition to existing in a transmembrane version LeechCAM is likely to be proteolytically cleaved into a secreted form without the transmembrane domain and the intracellular tail. Furthermore, by immunoprecipitation we demonstrate that LeechCAM is glycosylated with the Laz2-369 glycoepitope, an epitope that has been specifically implicated in regulation of axonal outgrowth and synapse formation.

Animals↗

PubMed1999

Sequential steps in synaptic targeting of sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs.

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.

Age Factors↗

PubMed1999

Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice.

Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic beta cells is upregulated by cytotoxic cytokines like IL-1beta. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent beta-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1beta, TNF-alpha, and IFN-gamma for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-gamma + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin-induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide.

Animals↗

PubMed1999

Gliarin and macrolin, two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system.

Using monoclonal antibodies, we have identified two novel intermediate filament (IF) proteins, Gliarin and Macrolin, which are specifically expressed in the central nervous system of an invertebrate. The two proteins both contain the coiled-coil rod domain typical of the superfamily of IF proteins flanked by unique N- and C-terminal domains. Gliarin was found in all glial cells including macro- and microglial cells, whereas Macrolin was expressed in only a single pair of giant connective glial cells. The identification of Macrolin and Gliarin together with the characterization of the strictly neuronal IF protein Filarin in leech central nervous system demonstrate that multiple neuron- and glial-specific IFs are not unique to the vertebrate nervous system but are also found in invertebrates. Interestingly, phylogenetic analysis based on maximum parsimony indicated that the presence of neuron- and glial cell-specific IFs in coelomate protostomes as well as in vertebrates is not of monophyletic origin, but rather represents convergent evolution and appears to have arisen independently.

Amino Acid Sequence↗

PubMed1998

Mannose-specific recognition mediates two aspects of synaptic growth of leech sensory afferents: collateral branching and proliferation of synaptic vesicle clusters.

The developmental role of carbohydrate markers in the genesis of neuronal networks was studied using leech sensory afferents as a model. Leech sensory afferents express a mannose-containing epitope on their cell surface that is recognized by monoclonal antibody Lan3-2. Previously, the elaboration of sensory arbors in the synaptic neuropil of CNS ganglia was experimentally shown to depend on this mannose marker. Sensory arbors were abolished by perturbing sensory afferents in the intact nervous system with Lan3-2 Fab fragments, a glycosidase, or mannose-BSA. To understand the cytological mechanisms underlying mannose-specific recognition for synaptogenesis, we have now studied the effects of antibody perturbation at the ultrastructural level in the sensory afferent target region. A characteristic signature of a normal sensory afferent is its profuse collateral branching, which, with ongoing development, is replaced by a single widened process, the sensory trunk, which possesses numerous synaptic vesicle clusters. The inhibition of mannose-specific recognition leads to a rapid, major reorganization of different stages of sensory afferent growth. Collateral branches at the distal growing region are reduced three- to fourfold. The pruned axons grow at an accelerated rate. Developmentally older sensory trunks experience a threefold reduction in synaptic vesicle clusters. These responses suggest that depriving sensory afferents of mannose-specific recognition aborts their synaptogenesis and causes them to resume behavior typical of tracking through axonal tracts. The current findings also suggest that the mannose marker, by promoting both collateral branching andthe proliferation of synaptic vesicle clusters, plays a critical role in two stages of sensory afferent synaptogenesis.

Animals↗

PubMed1998

Structural analysis of leech galactocerebrosides using 1D and 2D NMR spectroscopy, gas chromatography-mass spectrometry, and FAB mass spectrometry.

Cerebrosides were isolated from the leech species, Hirudo medicinalis, and purified to homogeneity by silicic acid chromatography, followed by preparative thin-layer chromatography. Their structure was determined by spectroscopic and chemical methods. 1D and 2D 1H NMR spectroscopy, DQF-COSY and HMQC indicated that the head group consists of a single galactose residue in the beta configuration. The galacto configuration was determined by the characteristic chemical shift, the spin-spin splitting and the multiplicity of the characteristic resonance of its equatorial H-4 proton, as well as by the splittings of the other ring protons. GC, GC-MS and fast-atom-bombardment mass spectrometry studies indicated that C24:0 and C22:0 are the major saturated fatty acid species. Unsaturated fatty acids present were C25:2, C27:2, C27:3, C28:3, C29:3, C30:3, C33:3. GC-MS indicated the presence of hydroxylated C27:2 and one other unidentified hydroxylated fatty acid. The cerebroside contained an unusual polyunsaturated sphingosine analogue, namely 2-amino-1,3-dihydroxydocsatriene.

Animals↗

PubMed1998

Cholesterol and its derivatives, are the principal steroids isolated from the leech species Hirudo medicinalis.

Steroids were isolated from the blood-sucking leech species Hirudo medicinalis and their structure was studied with one- and two-dimensional NMR spectroscopy (DQF-COSY and HMQC), GC-MS and ESI-MS spectrometry. Fractionating leech lipid using silicic acid chromatography led to the isolation of cholesterol in an early chloroform-eluted peak. Only minor traces of cholest-4-en-3-one, 4 beta-methylcholesterol, and sitosterol were present. The subsequent acetone-eluted fraction contained steroidtriols that were further purified by preparative TLC; these included cholest-7-ene-3,5,6 triol, cholest-4,7-diene-3,6,15 triol and to a lesser amount, cholestane-3,5,6 triol. A developmental study on cholesterol content in the leech showed that it is also the principal steroid in embryonic and freshly hatched leeches prior to feeding. The abundance of cholesterol, comprising approximately 5% of the total leech lipid, suggests that H. medicinalis, a blood sucking leech, has adapted itself fully to its mammalian host in terms of its steroid content. It remains to be seen whether lipids are directly transferred from the host to the parasite or whether leeches have evolved mechanisms to synthesize their own steroids.

Animals↗

PubMed1996

Leech photoreceptors project their galectin-containing processes into the optic neuropils where they contact AP cells.

We characterized a subset of leech sensory afferents, the photoreceptors, in terms of their molecular composition, anatomical distribution, and candidate postsynaptic partners. For reagents, we used an antiserum generated against purified LL35, a 35 kD leech lactose-binding protein (galectin); monoclonal antibody (mAb) Lan3-2, which is specific for a mannose-containing epitope common to the full set of sensory afferents; and dye injections. Photoreceptors differ from other types of sensory afferents by their abundant expression of galectin. However, photoreceptors share in common with other sensory modalities the mannose-containing epitope recognized by mAb Lan3-2. Photoreceptors from a given segment project their axons directly into the CNS ganglion innervating the same segment. They assemble in a target region, the optic neuropil, which is separate from the target regions of other sensory modalities. They also extend their axons as an optic tract into the connective to innervate optic neuropils of other CNS ganglia, thereby providing extensive intersegmental innervation for the 33 CNS ganglia comprising the leech nerve cord. Because of its intimate contact with the optic neuropil, a central neuron, the AP effector cell, is a strong candidate second order visual neuron. In confocal images, the AP cell projects its primary axon for about 100 microns alongside the optic neuropil. In electron micrographs, spines emanating from the axon of the AP cell make contact with vesicle laden nerve terminals of photoreceptors. Leech photoreceptors and their second order visual neurons represent a simple visual system for studying the mechanisms of axonal targeting.

Animals↗

PubMed1995

Sequential steps in axonal targeting are mediated by carbohydrate markers.

Mannose and hybrid/complex-type oligosaccharides serve as markers for both the full set of peripheral sensory afferent neurons in the leech and also for disjoint subsets of these neurons. We have shown that these various surface carbohydrates play crucial roles in the multistep process by which afferents meet their synaptic partners in the central nervous system (CNS). The carbohydrate marker common to all these afferents allows their projections (which are fasciculated as they enter the CNS) to disperse and search out target regions. Carbohydrate markers specific for subsets of these afferents subsequently allow each subset to consolidate the position of its projections in appropriate regions of the CNS where it contacts its synaptic partners.

Animals↗

PubMed1995

Kinetics of the inhibition of axonal defasciculation and arborization mediated by carbohydrate markers in the embryonic leech.

We studied carbohydrate interactions that mediate the targeting of sensory afferents in the synaptic neuropil of segmental central nervous system (CNS) ganglia in the embryonic leech. First, we determined the rate of sensory afferent development in vivo, and then we devised a culture system that permits the normal patterning of their projections in the CNS and PNS to proceed at 92% the normal rate. Using this in vitro system, we analyzed the mannose-specific recognition that mediates the defasciculation and arborization of sensory afferents in the CNS neuropil after they have tracked through peripheral nerves as a tight axon bundle. Sensory afferent defasciculation and arborization in the neuropil were inhibited by culturing embryos in Fab fragments directed against the mannose-containing surface epitope of sensory afferents. We demonstrate that the rate at which separately extending axons or their branches are lost from the neuropil can be modeled by a first-order decay process. These kinetic studies indicate that the loss of each separately extending axon or branch is an independent event. This suggests that sensory afferent projections extend autonomously across the target region in the search of their appropriate postsynaptic partners.

Animals↗

PubMed1995

Targeting of neuronal subsets mediated by their sequentially expressed carbohydrate markers.

The targeting of sensory afferent neurons in the leech CNS occurs in two discrete steps that are mediated via different carbohydrate recognitions, as shown by molecular perturbations of cultured embryos. A constitutive carbohydrate marker that is generic to all of these neurons mediates their initial defasciculation and arborization across the entire target region via mannose-specific recognition. Subsequently, two subsets of these same neurons can be differentiated by their expression of other markers that are located on hybrid or complex type carbohydrate chains. These developmentally regulated carbohydrate markers then mediate the target assembly of their respective neuronal subsets into discrete subregions. Thus, by performing opposing functions in a temporal sequence, constitutive and developmentally regulated carbohydrate markers collaborate in the targeting of neuronal subsets in the CNS.

Afferent Pathways↗

PubMed1994

Carbohydrate-binding proteins in the leech: I. Isolation and characterization of lactose-binding proteins.

Three lactose-binding proteins with apparent molecular masses of 16, 35, and 63 kDa [leech lectin 16, 35, and 63 (LL,16, LL35, and LL63, respectively)] were isolated from leech membranes. Polyclonal antibodies raised against LL35 cross-reacted with LL16 and LL63, indicating that all three lectins were immunologically related. These leech lectins, however, can be subdivided into two groups based on their tissue distributions and binding affinities for galactose derivatives. LL16 and LL35 are endogenous to the leech's CNS, whereas LL63 is only present in peripheral organs. LL16 and LL35, found in the CNS, bind both the alpha and beta anomers of methylgalactose, whereas the peripheral lectin LL63 binds only the beta form. LL35 and LL63 also differ in their binding affinities for galactosamine and N-acetylgalactosamine. The binding activity of LL35 was calcium independent and active over a wide pH range. Triton X-100 and 2-mercaptoethanol were necessary to recover LL35 binding activity during extraction. These characteristics strongly suggest that LL35 is another member of the calcium-independent galactose/lactose-specific lectins previously described in vertebrates and recently demonstrated in sponges and nematodes. Because a single leech expresses up to 100 micrograms of LL35, this leech lectin is readily amenable to structural and functional analysis.

Animals↗

PubMed1994

Carbohydrate-binding proteins in the leech: II. Lactose-binding protein LL35 is located to neuronal and muscle subsets and all epithelial cells.

Leech lectin 35 (LL35) is a calcium-independent galactoside-binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium-independent, galactose-specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity-purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.

Animals↗

PubMed1994

Distribution of carbohydrate epitopes among disjoint subsets of leech sensory afferent neurons.

Carbohydrate recognition plays an important role in the development of normal projections of sensory afferent neurons in the leech CNS. Four different carbohydrate epitopes are expressed by sensory afferents on their 130 kDa surface proteins: all sensory afferents share a common carbohydrate epitope (CE0) that helps them to enter and project diffusely across the synaptic neuropil; a restricted expression of three other carbohydrate epitopes (CE1, CE2, and CE3) serves to distinguish three subsets of sensory afferents. We examined the subsets of sensory afferents defined by their subset carbohydrate epitopes in the leech lip, skin, gut, and CNS. We established that the CE1, CE2, and CE3 subset epitopes define disjoint subsets of neurons by double labeling sensory afferents with monoclonal antibodies for different pairs of subset epitopes. We found that CE2 and CE3 afferents populate the lip and skin, but not the gut, and that these two subsets of sensory afferents have convergent projection patterns in the CNS. We found that CE1 afferents populate the gut and skin, but not lips; furthermore, their CNS projections diverge from those of CE2 and CE3 afferents. Our data fit the hypothesis that these carbohydrate epitopes are related to sensory modality of afferent subsets.

Animals↗

PubMed1993

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech.

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). However, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions.

Animals↗