Golgi Preparations Research Articles

Intact Golgi synthesize complex branched O-linked chains on glycoside primers: evidence for the functional continuity of seven glycosyltransferases and three sugar nucleotide transporters.

We examined the functional co-localization and continuity of glycosyltransferases and sugar nucleotide transporters in the Golgi of two Chinese hamster ovary (CHO) cell lines that synthesize different types of O-linked oligosaccharides. CHO cells normally synthesize primarily Sia2,3Galbeta1,3GalNAc- on glycoproteins. CHO cells transfected with core-2 GlcNAc transferase (Core 2) can synthesize glycoproteins containing branched O-linked oligosaccharides with poly-N-acetyllactosamines. CHO lines incubated with [(3)H]galactose and GalNAc-alpha-phenyl (GAP) as a primer, synthesize labeled glycoside products that faithfully resemble those found on the endogenous acceptors: CHO cells make Sia2,3[(3)H]Gal(beta)1,3GAP, while CHO Core2 cells synthesize GAPs with complex branched chains including poly-N-acetyllactosamines. To determine if isolated Golgi preparations make similar products, we prepared Golgi by established homogenization methods, documented their intactness, and added tracer UDP-[(3)H]Gal, unlabeled sugar nucleotides, and GAP. CHO Golgi preparations synthesized only Sia2,3[(3)H]Gal(beta)1,3GAP. CHO Core2, also made this product and a small amount of Core-2 GlcNAc transferase-dependent products. No endogenous glycoproteins were labeled. However, when either cell line was gently permeabilized with streptolysin-O or given hypo-osmotic shock, both GAP and endogenous acceptors were efficiently glycosylated within an intact functional Golgi lumen and remained there. Significantly, Golgi from CHO Core2 cells made mostly branched GAP products including some with poly-N-acetyllactosamines as complex as those made and secreted by living cells incubated with GAP. These results suggest that the lumen of the Golgi apparatus is functionally continuous or interconnected. Once glycosides diffuse into the Golgi lumen, they have access to all the sugar nucleotide transporters and glycosyltransferases used for complex GAP-based products without requiring metabolic energy or inter-vesicular transport. Glycosylation of artificial acceptors could be used to track the functional continuity or co-localization of multiple glycosyltransferases and transporters under conditions where Golgi morphology disintegrates and/or reappears.

Development of the mammillothalamic tract in normal and Pax-6 mutant mice

The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17–E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron. J. Comp. Neurol. 419:485–504, 2000. © 2000 Wiley-Liss, Inc.

Development of the mammillothalamic tract in normal andPax-6 mutant mice

The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17-E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron.

The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex

The present study has revealed that OC axons gave rise to a number of thin collaterals. Due to the abundance of these non-CF thin collaterals, it seems better to make a distinction between the terms CFs and OC axons, as was done in the present paper. The present findings on the innervation of PC dendrites by CFs are basically similar to those in previous reports (Ramón y Cajal, 1911; Palay and Chan-Palay, 1974). The number of swellings on a single CF in the present study (n = 250) is comparable to a previously measured value in the rat (n = 288; Rossi et al., 1993) and larger than a value in the frog (n = about 100 beads; Llinás et al., 1969). The average number of CFs per OC axon in this study was close to the number (n = about 7) inferred in the rat by counting the total number of IO neurons and PCs (Schild, 1970). Contact of interneurons by some swellings of CFs in the molecular layer was emphasized by Scheibel and Scheibel (1954) in their study with Golgi staining. Despite the contact of CF terminals on interneurons, the formation of a synaptic structure between them has been excluded in an electron-microscopic study (Hámori and Szentàothai, 1980). On the other hand, electrophysiological studies have demonstrated a weak excitatory effect of CFs on some interneurons (Eccles et al., 1966). Terminals in the granular layer were originated either from thin collaterals of OC axons or from retrograde collaterals of CF terminal arborizations. The former was the main source of swellings in the granular layer. The morphology of the thin collaterals in the present study was consistent with "globose varicosities connected by a fine thread" as described in Golgi preparations and electron micrograms (Chan-Palay and Palay, 1971). Swellings of thin collaterals (about 1.7% of the total number of swellings per OC axon) were most abundant in the upper portion of the granular layer just underneath the PC layer, in which Golgi cells are usually located. Furthermore, some of these swellings were observed to touch presumed Golgi cells in the present study, which is consistent with electron-microscopic findings on the innervation of somata of Golgi cells by thin collaterals (Hámori and Szentàothai, 1980; Chan-Palay and Palay, 1971). Inferior olive stimulation has been shown electrophysiologically to have a weak direct excitatory effect on Golgi cells (Eccles et al., 1966). Ninety-one percent of the OC axons examined had nuclear collaterals; since the possibility of insufficient staining could not be excluded, this percentage may be an underestimation. The ratio of swellings in the cerebellar nuclei versus those of CF terminal arborizations was about 0.036 in individual OC axons in the present study. However, since the volume of the cerebellar nuclei is much smaller than that of the cerebellar cortex, and significant convergence of input from OC axons to cerebellar nucleus neurons is present (Sugihara et al., 1996), cerebellar nucleus projection of OC fibers can still be functionally important. Some swellings seemed to make contact with the soma and the proximal portions of dendrites of large neurons in the present study, which is consistent with the steep rising phase of postsynaptic excitatory potentials in cerebellar nucleus neurons following IO stimulation (Kitai et al., 1977; Shinoda et al., 1987). Although intracellular potentials were presumably recorded only from large output neurons in the cerebellar nuclei, the present study suggested that small neurons were also innervated by OC axons. The present study revealed that virtually all reconstructed LRN axons projected not only to the Cx as mossy fibers, but also to the DCN including the VN by their axon collaterals. None of the LRN neurons specifically projected to the DCN without projecting to the Cx, namely all axon terminals of LRN neurons in the DCN and VN belonged to axon collaterals of mossy fibers projecting to the Cx. (ABSTRACT TRUNCATED)

Synthesis of α-D-Glucopyranosyl-(1-3)-α-D-Mannopyranosyl-(1-7)-4-Methylumbelliferone, A Fluorogenic Substrate for Endo-α-1,2-Mannosidase

ABSTRACT α-D-Glucopyranosyl-(1-3)-α-D-mannopyranosyl-(1-7)-4-methylumbelliferone (Glc-Man-Muf) was synthesized as a potential fluorogenic substrate for endo-α-1,2-mannosidase. The synthesis was designed in a convergent way. The glucose donor ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-glucopyranoside and the mannose acceptor 1,2:4,6-di-O-isopropylidene-ß-D-mannopyranose were coupled in the presence of N-iodosuccinimide and trifluoromethane-sulfonic acid to yield the corresponding disaccharide derivative. After conversion into peracetylated α-D-glucopyranosyl-(1-3)-α-D-mannopyranose the disaccharide was attached to 4-methylumbelliferone using the Helferich method. After separation of the desired isomer, deacetylation yielded the title compound. Glc-Man-Muf was used as a substrate in endomannosidase assays with rat liver Golgi preparations as an enzyme source (in the presence of the α-glucosidase inhibitor deoxynojirimycin). The degradation of Glc-Man-Muf was linear with protein up to 300 μg and with time up to 2 h. Vmax and Km were determined to be 0.17 nmol/mg x h and 3.7 mM, respectively.

Recycling of Apolipoprotein E in Mouse Liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.

Identification of a new Pyk2 target protein with Arf-GAP activity.

Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

Temporal dispersion windows in cortical neurons.

A temporal dispersion window is the time required for a volley of action potentials on presynaptic axons to cross the dendritic arbor of a postsynaptic neuron. The volley produces a series of unitary postsynaptic potentials (PSPs) on the postsynaptic neuron. Temporal dispersion is, thus, one factor that can influence the integration of unitary PSPs and the production of action potentials in cortical neurons. Temporal dispersion windows for neurons in the visual cortex of the freshwater turtle, Pseudemys scripta, were estimated by characterizing geniculate afferents and the morphology of neurons in the visual cortex. Horseradish peroxidase injections in the thalamus revealed thin and unmyelinated terminal arbors that run horizontally from lateral to medial across the cortex, forming en passant synapses across the dendrites of cortical neurons. Axons with two calibers were seen, one with diameters between 0.5 and 2.0 microm, and a second with diameters below the resolution limit of the light microscope. The conduction velocity of geniculate afferents in the cortex was measured at 0.18 m/sec +/-0.04 using the latency of extracellular field potentials evoked by electrical stimulation of the lateral forebrain bundle. The positions and dendritic arbors were characterized in Golgi preparations. Seven morphologically distinct neuron types were positioned to intersect the geniculate afferents in Golgi preparations. The spatial overlap between the dendritic arbors of these cells and the geniculate afferents varied from 128 to 850 microm. Temporal dispersion windows for the seven cell types ranged from 0.7 to 4.7 msec, estimated using a geniculate fiber conduction velocity of 0.18 m/sec. Estimated conduction velocities of 0.04 m/sec for small-caliber fibers produce temporal dispersion windows of 3.2 to 21.3 m/sec.

In Vitro Synthesis of Sulfated Glycosaminoglycans Coupled to Inter-compartmental Golgi Transport

We have used isolated rat liver Golgi membranes to reconstitute the synthesis of sulfated glycosaminoglycans (GAGs) onto the membrane-permeable, external acceptor xyloside. Biosynthetic labeling of GAGs with [35S]sulfate in vitro is shown to have an absolute requirement for ATP and cytosolic proteins and is inhibited by dismantling the Golgi apparatus with okadaic acid or under mitotic conditions suggesting that inter-compartmental transport between Golgi cisternae is a prerequisite for the successful completion of the initiation, polymerization, and sulfation of GAGs. Accordingly, we show that in vitro synthesis of 35S-GAGs utilizes the same machinery employed in Golgi transport events in terms of vesicle budding (ADP-ribosylation factor and coatomer), docking (Rabs), targeting (SNAREs), and fusion (N-ethylmaleimide-sensitive factor). This provides compelling evidence that GAGs synthesis is linked to Golgi membrane traffic and suggests that it can be used as a complementation-independent method to study membrane transport in Golgi preparations from any source. We have applied this system to show that intra-Golgi traffic requires the function of the Golgi target-SNARE, syntaxin 5.

The Golgi architecture and some EM observations on the avian nucleus dorsolateralis anterior thalami: cell types, fibres and synapses

In Golgi preparations of the chicken diencephalon, various types of cells and fibres were studied in the nucleus dorsolateralis anterior thalami (DLA). Two groups of neurons were found: projection neurons with long axons and interneurons with locally branching axons. The projection neurons varied in the different areas of the DLA. In the medial part the neurons are large cells with long, moderately spiny dendrites, and in the dorsal part there are small cells with short, wavy and moderately spiny dendrites. These neurons differ completely from those found in the rostral and lateral parts, where the neurons have medium-sized cell bodies and curving, spiny dendrites, which branch tuft-like or with bifurcations. In the rostral and lateral parts of the DLA, thick afferent fibres were impregnated which developed their terminal branchings among the neurons. Owing to their terminal branching pattern and the shape of the terminals they are thought to be optic fibres. The terminal pattern of these fibres is similar to the optic terminals in the LGB of the mammalian brain. The interneurons are GABA positive, as attested by immunostaining in light microscopic and EM specimens. There are rather few of them. The HRP-filled projection neurons and the surrounding neuropil were investigated under EM: synaptic connections around the large terminals and/or around dendrites were identified, but this synaptic arrangement does not display the characteristics of a synaptic glomerulus.

Golgi and Electron-Microscopic Golgi-GABA Immunostaining Study of the Avian Optic Tectum

The neuronal structures and their possible connections in the optic tectum of chicks were studied by using Golgi impregnation, in light microscopy, and Golgi-GABA immunogold staining in electron microscopy. The terminals of the optic fibers displayed different patterns and orientation in various laminae of the avian optic tectum. The shape of the branching and the terminals of the optic fibers conformed to the postsynaptic neuronal structures. In Golgi preparations, the terminals in tectal layers 4 and 5 seemed to be located rather densely, and they contact, among other things, the ‘dendritic terminal sections’ of large ganglion cells of layer 13 of the tectum. This connection could be responsible for direct optic transmission via ganglion cells to the nucleus rotundus. Additionally, the contacts established by optic terminals in these layers with the tectal neurons arranged with their dendrites parallel to the surface of the tectum, could provide the structural basis for an inhibitory system. The terminals of isthmic nuclei were found in this optic lamina, too. The optic terminals in layer 7 contacted the dendritic side branches of some radial tectal neurons.

Effects of neonatal serotonin depletion on the development of rat dentate granule cells

The appearance of serotonergic (5-HT) neurons and projections early in central nervous system (CNS) development has resulted in the hypothesis that 5-HT is an important factor in neuronal differentiation and synaptogenesis. Studies of the effects of 5-HT on the development of molluscan and mammalian neurons in vitro support this hypothesis, but mammalian in vivo studies have produced equivocal results. The present study reinvestigated the role of 5-HT in CNS development using the dentate granule cell as a model. Dentate granule cells were chosen for this study of the effects of 5-HT depletion on neuronal development because they are generated in the early postnatal period. Thus, 5-HT depletion could be effected by the treatment of rat pups with either parachloroamphetamine (PCA) or 5,7-dihydroxytryptamine (5,7-DHT) thereby avoiding problems inherent in maternal treatment paradigms. The morphology of Neurobiotin-filled granule cells was studied on P14, P21, P60 and P120 (PO = day of birth). The parameters measured were total dendritic length, number of dendritic segments and dendritic spine density (number of spines/50 μm dendritic length). Granule cells from vehicle-treated controls were similar to those previously reported in studies of normal granule cell development in all respects. In particular, the decrease in dendritic spine density from P14 to P120 observed in Golgi preparations was verified in our population of intracellularly filled granule cells. Transient depletion of 5-HT by neonatal PCA treatment resulted in a decrease dendritic length that was not statistically different from control values. However, dendritic spine density was reduced by about 27% at all ages studied. 5,7-DHT treatment produces a permanent, severe depletion of 5-HT. Spine densities in granule cells from 5,7-DHT-treated pups were also about 38% lower than controls. Total dendritic length in cells from 5,7-DHT-treated rats was reduced to a degree comparable to that observed in PCA-treated pups. The number of granule cell dendritic segments was also less than that observed in control and PCA-treated rats but this difference was not statistically significant. These observations suggest that reduction of 5-HT in the early postnatal period can result in changes in the morphology of dentate granule cells, particularly at the level of the synapse as reflected by the permanent reduction in synaptic spine density. The comparison of results from cases with permanent and transient reduction of 5-HT indicates that the developmental influence of 5-HT is most important during the first three postnatal weeks.

Dynamics of Cell Migration from the Lateral Ganglionic Eminence in the Rat

From previous developmental studies, it has been proposed that the neurons of the ventrolateral cortex, including the primary olfactory cortex, differentiate from progenitor cells in the lateral ganglionic eminence. The objective of the present study was to test this hypothesis. The cells first generated in the forebrain of the rat migrate to the surface of the telencephalic vesicle by embryonic day (E) 12. Using [3H]thymidine, we found that most of these cells contributed to the formation of the deep layer III of the primary olfactory cortex. To study the migratory routes of these cells, we made localized injections of the carbocyanine fluorescent tracers Dil and DiA into various parts of the lateral ganglionic eminence in living embryos at E12-E14 and subsequently maintained the embryos in a culture device for 17-48 hr. After fixation, most migrating cells were located at the surface of the telencephalic vesicle, whereas others were seen coursing tangentially into the preplate. Injections made at E13 and in fixed tissue at E15 showed that migrating cells follow radial glial fibers extending from the ventricular zone of the lateral ganglionic eminence to the ventrolateral surface of the telencephalic vesicle. The spatial distribution of radial glial fibers was studied in Golgi preparations, and these observations provided further evidence of the existence of long glial fibers extending from the ventricular zone of the lateral ganglionic eminence to the ventrolateral cortex. We conclude that cells of the primary olfactory cortex derive from the lateral ganglionic eminence and that some early generated cells migrating from the lateral ganglionic eminence transgress the cortico-striatal boundary entering the preplate of the neocortical primordium.

A new approach to mapping co-localization of multiple glycosyl transferases in functional Golgi preparations.

We have developed a new method to co-localize multiple glycosyl transferases in different Golgi compartments. The approach relies on the proven ability of intact, sealed rat liver Golgi preparations to concentrate exogenous labeled sugar nucleotides into the lumen where they glycosylate either endogenous or artificial acceptors. The premise is that if two glycosyl transferases are co-localized within the same compartment, they will compete for the limited amount of transported donor. If the donor is consumed in glycosylating a permeable artificial glycoside within a Golgi compartment, it will be unavailable to glycosylate endogenous products within that same compartment. The greater the degree of transferase co-localization, the greater the potential decrease in glycosylation of endogenous acceptors. We provide an example consistent with these predictions. Adding 1 microM UDP[3H]Gal to Golgi preparations followed by a chase with a cocktail of unlabeled sugar nucleotides labels mostly endogenous N-linked oligosaccharides containing both beta 1,3- and beta 1,4[3H]Gal residues with and without sialic acid. Addition of increasing amounts of 4-methylumbelliferyl-beta-xyloside (Xyl beta MU) produces [3H]Gal1 beta, 4Xyl beta MU and leads to a reciprocal decrease in labeling of a restricted set of the endogenous acceptors. This decrease is preferential for [3H]Gal beta 1-->3GlcNAc beta 1-->R and, to a lesser extent, [3H]Gal beta 1-->4GlcNAc beta 1-->R structures in neutral and mono-sialylated oligosaccharides; synthesis of these structures in di- and tri-sialylated oligosaccharides was unaffected. These preferential decreases are not seen in detergent permeabilized, sugar nucleotide transport-independent Golgi incubations, and are not due to inhibition by the Gal beta 1,4Xyl beta MU product. These results argue that there is significant overlap in the functional co-localization of sialyl and galactosyltransferases in rat liver Golgi preparations and that GAG chain core specific Galactosyltransferase I is co-localized with subsets of N-glycan Gal beta 1,3 and Gal beta 1,4 transferases. This approach can be used with other glycosides and sugar nucleotides to map and co-localize other glycosyl transferases. The functional compartments defined by this approach may or may not correspond entirely with morphologically defined Golgi domains.

Trimeric G Protein α Subunits of the Gs and Gi Families Localized at the Golgi Membrane

The occurrence of individual G proteins within Golgi membranes from liver and from adrenal medulla were analyzed by Western blotting. Two splice variants of Gαs and also Gαi-3 were found in both tissues. Additionally, Gαi-2, its 43-kDa splice variant and Gαo were strongly labeled in Golgi of adrenal medulla. Golgi preparations of liver contained comparable quantities of Gαi-2 and a second variant of 46 kDa, but no Gαo was detected. Immunoelectron microscopic studies of the above Golgi preparations showed that both Gαi-2 and Gαi-3 are localized at multivesicular structures identified as Golgi complexes. The occurrence of two stimulatory G proteins (Gαs-S and Gαs-L) and three or four α subunits of the Gi/Go type as components of the Golgi membrane support the notion that formation of different vesicle types might be regulated by individual stimulatory and inhibitory G proteins.

A Novel Method to Co-localize Glycosaminoglycan-Core Oligosaccharide Glycosyltransferases in Rat Liver Golgi: CO-LOCALIZATION OF GALACTOSYLTRANSFERASE I WITH A SIALYLTRANSFERASE

4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with Xyl beta MU and 1 microM UDP-[3H]Gal and then chased with 5 microM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Sia alpha 2,3Gal beta 1,4Xyl-beta MU and Sia alpha 2,8Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Ga1 transferase I and II are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.

Three‐dimensional organization of neuronal and glial processes: High voltage electron microscopy

Neurons and glia cells in the mammalian central nervous system have many complicated processes. They are too fine for light microscopic study and too complicated and widely spread for thin section electron microscopy. High-voltage electron microscopic (HVEM) stereo observation of thick Golgi preparation provides detailed 3-D images of their processes. Three-dimensional fine structures of astocytic processes in the neuropile and on the surface of neuronal somata, and those of the ruffed cell axon initial segment and thorny excrescences of CA3 pyramidal cell dendrites, are elucidated with the aid of HVEM stereoscopy of thick Golgi preparations. In addition, some results obtained by 3-D morphometrical analysis of dendritic spines using HVEM stereo images are shown. The examples presented here clearly show the usefulness of high-voltage electron microscope stereo observation of thick specimens for detailed morphological and morphometric study of the central nervous system.

α-(2 → 3)- and α-(2 → 6)-Sialyltransferase activities present in three variants of Ehrlich tumor cells: identification of the products derived from N-acetyllactosamine and β- d-Gal-(1 → 3)-α d GalNAc-(1 → O)-Bn

We compared several sialyltransferase activities related to synthesis of O-linked and N-linked sialylglycoproteins in Ehrlich ascites tumor cells that grow normally in murine ascites, but are not adherent nor grow in tissue culture (na-EAT cells), with those in cells that were selected to grow in tissue culture and adhere to extracellular matrices (a-EAT cells). Crude Golgi preparations from both cell types contained predominantly β- d-Gal-(1 → 3)- d-GalNAc α-(2 → 3)-sialyltransferase activity. Sialylation of N-acetyllactosamine, lacto- N-tetraose, and benzyl α- d-Ga1NAc occurred at from 1 to 4% of that activity. Analysis, by ion-exchange HPLC at high pH, of sialylated N-acetyllactosamine showed that na-EAT cells sialylated β- d-Gal-(1 → 4)- d-GlcNAc mostly by α-(2 → 3)-sialyltransferase, whereas β- d-Gal-(1 → 4)- d-GlcNAc α-(2 → 6)-sialyltransferase activity was prominent in a-EAT cells. In addition, preparations from na-EAT cells formed significant quantities of an unknown tritiated product from CMP-[9- 3H]sialic acid, suggesting at least one other difference in enzyme levels between the cell types. a-EAT cells reestablished in murine ascites for 11 passages retained the sialyltransferase levels characteristic of a-EAT cells. When viable cells were labeled with

Phase specific morphological changes induced by social experience in two forebrain areas of the zebra finch

We examined the changes of spine density in Golgi preparations of two different areas of the forebrain of the zebra finch, the ANC (Archi-Neostriatum caudale) and MNH (medial Neo-Hyperstriatum) during development, after transferring male birds from isolation to a social condition (exposure to a female for 1 week), and after a second isolation period. MNH and ANC are two of four brain regions which are strongly activated if a male bird is exposed to a female after some time of isolation. The results of our study can be summarized as follows. 1: a peak-decline trend is observed in ANC, but not in MNH. 2: rearing conditions do not affect the development of both areas until day 70. 3: from 80 days of age, isolation leads to reduced spine density within ANC, but to enhanced spine density within MNH. 4: short social contact after isolation diminishes or eliminates the effects of isolation by an enhancement of spine density in ANC and a reduction of spine density within MNH. 5: the effects of short social rearing after isolation are reversible within ANC, but not within MNH. We presume that the alterations of spine density, which are induced by changes in social conditions, are restricted to ages older than 70 days by hormonal factors. We propose that the complexity of the ANC neuronal net follows the complexity of the social environment, and that the level of arousal is the most important factor influencing the complexity. We further suppose that the reduction of spines within MNH is the anatomical manifestation of an imprinting process, which has been shown to occur in the same experimental situation as we used it in our study.

Neuropathology of Rett Syndrome: Case Report With Neuronal and Mitochondrial Abnormalities in the Brain

Neuronal changes in the brain of a Rett syndrome patient were examined in a frontal lobe biopsy performed at age 3 years and in the postmortem brain at age 15 years. In the brain biopsy, frontal cortex contained numerous scattered pyramidal neurons with cytoplasmic vacuolation and increased cytoplasmic density, with no neuronophagia or inflammation detected; electron microscopy showed these neurons to have large, lucent-appearing mitochondria, very abundant ribosomal content, and some lipofuscin granules. Postmortem brain 12 years later showed scattered neurons in frontal cortex, substantia nigra, and cerebellar folia, with increased electron density of the cytoplasm, stacks of ribosomal endoplasmic reticulum, and large amounts of disorganized membranous material, including autophagic-type organelles. Mitochondria of these neurons contained electron-dense, finely granular matrix inclusions; in the substantia nigra, some spherical mitochondrial inclusions completely filled the matrix space. Golgi preparations of (autopsy) frontal cortex and cerebellar folia showed truncation and thickening of dendrites and a degenerate appearance of cortical pyramidal neurons, similar to changes found in aged brain. Synaptophysin immunohistochemistry indicated that the density of synapses was not greatly altered compared to controls in frontal cortex and cerebellum. The patient also had a second genetic defect, severe combined immunodeficiency with thymic aplasia, which may be X-linked.