Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension.

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules. It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.

Postganglionic, direct axo-axonal contacts on the splenic nerve.

The splenic nerve is made up almost exclusively of adrenergic fibers. Consequently it was used as a model system in the study of autonomic sympathetic neurotransmission. The splenic nerve regulates the vasoconstriction and volume reduction of the spleen. Brain-immune interactions via modulation of the splenic nerve activity may regulate peripheral cellular immunity. An inhibition of noradrenaline release by alpha(2)-adrenoceptor activation has been reported. As we were interested in a structurally detailed distribution of synaptophysin, immunocytochemical methods were applied to splenic nerve axons. In 1 microm plastic sections a network of synaptophysin-positive varicosities could be observed all along the splenic nerve. They were also positive for dopamine-beta-hydroxylase and cytochrome b561. At the ultrastructural level the varicosities were seen to establish direct contact with the splenic axons. In normal morphology the varicosities revealed small synaptic vesicles and several dense granules. It is demonstrated that a network of direct symmetric contacts of adrenergic nature is present all along the nerve. These terminals may have an inhibitory effect on the splenic nerve activity via axonal receptors. This finding opens new perspectives for the study of the splenic nerve in general and more particularly for its role in the regulation of peripheral cellular immunity.

Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve.

Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles.

Retrieved constituents of large dense-cored vesicles and synaptic vesicles intermix in stimulation-induced early endosomes of noradrenergic neurons.

Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-beta-hydroxylase (DbetaH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes, as shown by the colocalization of retrieved DbetaH with the endosomal markers Rab5 and HRP in sucrose density gradients and on confocal microscopical images. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DbetaH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.

The Hermansky-Pudlak syndrome.

The Hermansky-Pudlak syndrome (HPS) associates oculocutaneous albinism with a haemorrhagic diathesis and the accumulation of ceroid-like material in different tissues. HPS is not an uncommon type of albinism as it was diagnosed in 13.5% (8/59) of our autosomal recessive albinos. These eight patients were evaluated ophthalmologically and haematologically. Apart from the symptoms caused by the albinism, accompanying signs vary from ecchymoses to life threatening haemorrhages and death by associated restrictive lung disease. Recognition of this syndrome by the ophthalmologist can be of major importance in this serious and eventually fatal disorder.

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity.

OBJECTIVE: To investigate a method of assisted activation of human oocytes for the treatment of infertility resulting from globozoospermia associated with deficient oocyte activation capacity. DESIGN: The mouse oocyte activation test was used to analyze the oocyte activation capacity of the sperm cells of a patient with globozoospermia. Fresh donor human oocytes were used for determining the most appropriate procedure for oocyte activation. SETTING: Infertility Center, University Hospital of Ghent. PATIENT(S): A couple with infertility resulting from globozoospermia. INTERVENTION(S): Intracytoplasmic sperm injection, assisted oocyte activation, and embryo transfer. MAIN OUTCOME MEASURE(S): Oocyte activation and fertilization rates, implantation, and pregnancy. RESULT(S): Deficiency in oocyte activation capacity was found in the sperm of a patient with globozoospermia. This deficiency was proven by the mouse oocyte activation test and was confirmed further by lack of activation of human oocytes after simple sperm injection. Only human oocytes that were injected with sperm and subjected to calcium chloride and ionophore treatment underwent activation. Transfer of embryos obtained by this procedure of assisted oocyte activation resulted in an ongoing pregnancy. CONCLUSION(S): Assisted oocyte activation of human oocytes is useful when globozoospermia is associated with absence of oocyte activation capacity in the sperm. These cases can be identified by the mouse oocyte activation test.

Ultrastructural evidence for a separate, small synaptic vesicle (SSV) pathway in ligated bovine splenic nerves, incubated in vitro.

In sympathetic nerves the tubules of the axonal reticulum make up the immature elements of the neurosecretory apparatus. The formation of the mature large dense granules occurs via a less dense tubular intermediate, representing the maturing part. At a terminal small synaptophysin-positive vesicles are found intermingled with the dense granules. The biogenesis of these clear, small synaptic vesicles and their relationship with dense granules remains to be determined. In search for the small synaptic vesicles we undertook a careful ultrastructural examination of the axons proximal to a ligation in bovine splenic nerve incubated in vitro for 3 h. The distended axons were crowded with tubules, granulo-tubular elements and dense granules. Occasionally homogeneous clusters of small, uniform vesicles were detected. They were shown to be positive for synaptophysin and were negative for dopamine-beta-hydroxylase, a marker for the granular pathway. The clusters of small vesicles could be found in close spatial relationship with the maturing and mature elements of granular secretion. Our findings argue for the presence of two separate neurosecretory pathways in sympathetic nerves and favour the idea that both small synaptic vesicles and dense granules are a differentiation product of the axonal reticulum. This configuration can explain the biogenesis of small synaptic vesicles and dense granules both in the cell body and at the nerve terminal.

Ultrastructural investigation of M-cells and lymphoepithelial contacts in naso-pharyngeal associated lymphoid tissue (NALT).

The nasopharyngeal and palatine tonsil, two lymphoepithelial structures situated at the entrance of respiratory and gastrointestinal tracts, show striking similarity with lymphoid tissue of bronchus and gut (BALT, GALT). The information obtained by previous investigations in our laboratory on M-cells in the lymphoepithelium of the Peyer's patches (PP) served as a guidance in our search for M-cells in adenoids and tonsils.

Subcellular localization of synaptophysin in noradrenergic nerve terminals: a biochemical and morphological study.

The subcellular localization of synaptophysin was investigated in noradrenergic nerve terminals of bovine vas deferens and dog spleen and compared with membrane-bound and soluble markers of noradrenergic storage vesicles. At the light microscopical level chromogranin A- and cytochrome b561-immunoreactivity revealed an identical and very dense innervation of the entire vas deferens. In the case of synaptophysin, most immunoreactivity was found only in the outmost varicosities closest to the lumen, which were also positive for chromogranin A. Small dense-core vesicles of dog spleen were purified using a combination of velocity gradient centrifugation and size exclusion chromatography. Small dense-core vesicles were enriched 64 times as measured by the noradrenaline content. Enrichments for dopamine-beta-hydroxylase were in a similar range. Synaptophysin-containing vesicles were smaller in size and they did not contain the typical noradrenergic markers dopamine-beta-hydroxylase, cytochrome b561, and noradrenaline. Instead, they might store adenosine triphosphate (ATP). A greater part of synaptophysin immunoreactivity was consistently found at high sucrose densities at the position of large dense-core vesicles. We conclude that in the noradrenergic nerve terminal: (1) small dense-core vesicles have a membrane composition similar to large dense-core vesicles, indicating that the former are derived from the latter, and (2) synaptophysin seems not to be present on small dense-core vesicles. We suggest the possibility that synaptophysin-containing vesicles form a residual population whose role in neurotransmission has been taken over by large and small dense-core vesicles following noradrenergic differentiation.

The organisation of the axonal reticulum at a ligation, in in vitro incubated bovine splenic nerves.

From previous studies we concluded that in noradrenergic neurons the axonal reticulum can be considered to be an extension of the Golgi apparatus, directly involved in the condensation and packaging of neurosecretion. But the precise ultrastructure of the organisation of the axonal reticulum in relation to neurosecretory granule formation remained to be elucidaded. This conversion was studied in ligated bovine splenic nerve incubated in vitro for three hours. The ultrastructure of the material accumulating proximally and distally was examined and its nature was determined by phosphotungstic acid staining and immunocytochemistry on glycolmethacrylate sections. Proximal to the ligation predominantly electron-lucent vesicles and tubules were found. Tubules of intermediate electron density appeared in between. The latter, especially in thicker sections, were seen to form complexes with tubules and granules of high electron density. All those elements were shown to be positive for dopamine-beta-hydroxylase and cytochrome b561. In the distal part multivesicular bodies accumulated and they were also positive for both enzymes. From these findings it is concluded that the different types of structures accumulating proximally belong to a neurosecretory axonal reticulum. At a block the axonal reticulum is triggered to generate a reticular differentiation, in which granular densities of different size are found. This configuration compares well with that in nerve terminals and strongly suggests that granule formation is basically a local process.

Clinical, ultrastructural and biochemical studies in two sibs with Ehlers-Danlos syndrome type VI-B-like features.

Two Turkish sibs with clinical features of Ehlers-Danlos syndrome type VI-B are presented. The hydroxylysine contents of dermis and gel electrophoresis of type I and type III collagen produced by fibroblasts were normal. Ultrastructural studies of skin collagen and elastic fibers showed discrete abnormalities. Other syndromes with similar clinical, biochemical and ultrastructural features are discussed.

Idiopathic inflammatory bowel diseases: immunological hypothesis.

Circumstantial evidence indicates that immunological mechanisms are important in the pathogenesis of inflammatory bowel diseases. In Crohn's disease lesions the naive T cells are reduced and memory T cells are increased in number. Colonocytes of normal mucosa do not express MHC class II molecules but aberrant HLA-DR expression is induced in inflammatory conditions. Also in ileal epithelial cells there is increased MHC class II molecules expression suggesting augmented antigen presentation. Once the initiating event (environmental, dietary, infectious or enterobacterial products) has activated inflammatory cells, an auto-amplifying inflammatory immune cascade is induced by numerous agents such as cytokines, transforming growth factors, leukotrienes, prostaglandins and other mediators. Tissue injury may be caused by oxygen free radicals generated by neutrophils and macrophages. The inflammatory process can be perpetuated by a persistent antigenic stimulus or alternatively, by abnormal regulation of immune and inflammatory responses. This can mean exaggerated activation of inflammatory responses to "normal" stimuli or defective feedback mechanisms of down-regulation. The early lesions of inflammatory bowel disease are patchy necrosis of the surface epithelium, focal accumulations of leukocytes adjacent to crypts and an increased number of intraepithelial lymphocytes. In Crohn's disease the earliest lesions are aphthoid ulcers overlying lymphoid follicles in the terminal ileum. In patients with spondylarthropathy and asymptomatic intestinal inflammation we demonstrated lesions in the top of the follicle associated epithelium. The changes consist of increase in number of membranous (M) epithelial cells, ruptures, fissures and perforations of M cells. Aphthoid ulcers that may occur in the entire gastrointestinal tract are typically found at the M cell level.(ABSTRACT TRUNCATED AT 250 WORDS)

M-cells are damaged and increased in number in inflamed human ileal mucosa.

Ileocolonoscopy and biopsies of patients with spondylarthropathy reveal gut inflammation in 62% of cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy-related gut inflammation, the follicle-associated epithelium was examined. Biopsies from nine controls and 18 patients with spondylarthropathy were studied by electronmicroscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa, M-cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle-associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis, necrotic M-cells, rupture of M-cells and lymphocytes entering the gut lumen were observed. The bursting of M-cells at the top of the lymphoid follicles leads to interruption of the gut epithelial lining and gives the luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.

Differences in the distribution of cytochrome b561 and synaptophysin in dog splenic nerve: a biochemical and immunocytochemical study.

Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotransmitter-containing vesicles, i.e., large (LDVs) and small dense-core vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b561, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b561 coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b561 immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b561, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data clearly illustrate that synaptophysin is absent from LDVs and AR elements in adrenergic axons.

The p185erbB2 protein is localized on cell organelles involved in cell motility.

In a previous paper, we have shown that the c-erbB-2-encoded protein p185erbB2 is localized on the brush border of the proximal tubule kidney cells. In invasive duct cell carcinomas, the labeling was most obvious on the villous plasma membrane protrusions. From these observations the hypothesis was raised that p185erbB2 could play a role in motility. To test this hypothesis, we quantified its distribution on the microvilli and plasma membrane protrusions and on the straight parts of the cell membrane after immunoelectron microscopy. These findings were compared with the localization on p185erbB2 overexpressing SK-BR-3 human breast cancer cells before and after stimulation of motility by treatment with conditioned medium from COLO-16 cells (CM), which is also able to induce chemotaxis of these cells in a Boyden chamber assay. In the invasive duct cell carcinomas, the number of gold particles was nine times higher at the plasma membrane protrusions than at the straight parts of the cell membrane. In untreated SK-BR-3 cells, p185erbB2 was similarly concentrated on the membrane of small microvilli and plasma membrane protrusions. Treatment of SK-BR-3 cells with CM leads to cell spreading, enlargement of the microvilli, formation of pseudopodia and chemotaxis. Aggregation of p185erbB2 at the plasma membrane protrusions and pseudopodia is observed on immunofluorescence light microscopy. The concentration of p185erbB2 is several times higher on these membrane extensions than on the straight parts after immunogold labeling. It is concluded that p185erbB2 is localized on cell organelles involved in motility, and it is suggested that the molecule plays a role in cell movement, providing the capacity of tumor cells to spread and metastasize.

Ultrastructural localization of neuropeptide Y-immunoreactivity in the axonal reticulum elements, accumulating anterogradely in transected rat sciatic nerve.

The detection of dopamine-beta-hydroxylase and cytochrome B561 on the membranes of the axonal reticulum demonstrated that in sympathetic neurons the different compartments of the axonal reticulum participate in the formation of neurosecretory vesicles. In the present study we tried to reveal that the components of the vesicular content are also channeled along the axonal reticulum, by examining whether neuropeptide Y could be localized in elements of the axonal reticulum. Therefore 6 h transected rat sciatic nerve was embedded in glycolmethacrylate and an immunogold labeling was performed. Counterstaining with phosphotungstic acid at low pH selectively contrasted the accumulated axonal reticulum elements and associated granules. In the non-myelinated axons gold labeling was localized on granules and on tubular and granular profiles, demonstrating the presence of neuropeptide Y in the accumulated axonal reticulum elements. This indicates that neuropeptides are indeed transported via the axonal reticulum to the nerve ending and suggests that the accumulation of large dense-cored vesicles at a block is mainly due to local new formation rather than down transport.

M cells are damaged and increased in number in inflamed human ileal mucosa.

Ileocolonoscopy and biopsies of patients with spondylarthropathy revealed gut inflammation in 62% of the cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy related gut inflammation the follicle associated epithelium was examined. Biopsies from 9 controls and 18 patients with spondylarthropathy were studied by electron microscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa M cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis necrotic M cells, ruptures of M cell cytoplasm and lymphocytes entering the gut lumen were observed. The bursts of M cells at the top of the lymphoid follicles lead to interruption of the gut epithelial lining and give luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.

Influence of aging on fluidity and coupling between beta-receptors and G-proteins in rat lung membranes.

We tested the hypothesis that in rat lung membranes, the age-related decline in the percentage of beta-receptors coupled with high affinity to G-proteins, is due to limitation of the diffusion caused by a decrease in membrane fluidity. We measured both parameters simultaneously in a crude membrane preparation from lungs of rat of different age. In contrast to what is found in crude membrane preparations from rat liver and brain, in rat lung fluidity was increased upon aging. We conclude that the age-related alteration in coupling between receptor and G-protein is difficult to explain by alterations of membrane fluidity.