Endocytotic pathways across the human gall-bladder mucosa: permeability studies using horseradish peroxidase

Summary Human gall-bladder epithelium obtained straight from the operating theatre was incubated in an Ussing chamber with the fluid phase marker, horseradish peroxidase (HRP), for up to 60 min. When the marker was presented on the apical surface, within 30 min it had moved readily across the apical cytoplasm in transport vesicles to receptosomes and into the lateral intercellular space, extending across the basement membrane into the lamina propria. When HRP was presented at the basal aspect, within 30 min it had moved through the lamina propria, across the basement membrane and into the lateral intercellular space. By 60 min, only small amounts had been taken up by the epithelial cells and transported to receptosomes. These data indicate a rapid transmucosal endocytotic pathway for blood-or bile-borne macromolecules.     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.     

Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

Surface charges associated with fenestrated brain capillaries. II. In vivo studies on the role of molecular charge in endothelial permeability

We report on the effect of the net charge of a tracer (ferritin) on its permeability in fenestrated capillaries of the brain. Our experiments show that the charge of this tracer actually influences its interaction with the endothelium. Three phases of tracer-endothelial interaction could be discriminated. Anionic and slightly cationic derivatives (pH 4.5-7.8) do not show any affinity to the luminal endothelial membrane. Ferritin derivatives with a pI value between 7.8 and 9.3 result in the labeling of the fenestrae without coating additional luminal plasmalemmal structures (i.e., coated pits and plasmalemmal vesicles). Tracers with a high positive net charge (pI greater than 9.3) led to their endocytotic uptake and extravasation by some transcytotic mechanism. Extravasated cationic ferritin accumulates in the endothelial basement membrane and binds to striated collagen fibrils. It is suggested that the pericapillary collagen fibrils of fenestrated brain capillaries act as a charge filter with respect to macromolecules.     

Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys

Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti- rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti- laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.     

A passionate kiss, then run: exocytosis and recycling of IgG by FcRn

The MHC-class-I-like Fcgamma receptor FcRn recycles immunoglobulin (Ig)G from most cells and transports it bi-directionally across epithelial barriers to affect systemic and mucosal immunity. Recent studies have shown that FcRn rescues IgG from intracellular lysosomal degradation by recycling it from the sorting endosome to the cell surface. Most recycling vesicles fuse completely with the plasma membrane in a classical pattern of exocytosis. Similar to the process seen for neurotransmitter release at synaptic junctions, other vesicles fuse only partially, releasing FcRn-IgG complexes to mix into the plasma membrane in cycles of 3-4s over prolonged periods of time.     

Protein-protein interactions and protein modules in the control of neurotransmitter release

Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release.     

An electron microscopic study of absorption of peroxidase-conjugated immunoglobulin G by guinea pig visceral yolk sac in vitro.

In the guinea pig and some other animals, passive immunity is conferred on the developing fetus by passage of immunoglobulin from mother to fetus across the yolk sac. In order to examine the cytological pathway involved in immunoglobulin transport, guinea pig visceral yolk sacs from late in gestation were exposed in vitro to peroxidase-conjugated guinea pig immunoglobulin G (IgG-HRP). Tissue was then fixed, incubated to show the site of localization of peroxidase reaction product and prepared for electron microscopy. The results suggested that the first step in the uptake of IgG-HRP by yolk sac is attachment of the protein to the surface coats of endocytic invaginations at the apical surfaces of the endodermal cells. The endocytic vesicles then appear to pinch off from the surface and move deeper into the cytoplasm. Some of the small endocytic vesicles fuse with large apical vacuoles, which often contain large amounts of reaction product. Other small endocytic vesicles pinch off from the surface, move deeper into the cytoplasm and fuse with the lateral plasmalemma; their protein content is emptied into the intercellular space by exocytosis. From the intercellular spaces the protein presumably diffuses across the basement membrane and connective tissue spaces and enters the vitelline capillary bed. It is postulated that the latter cellular pathway, involving small vesicles and the intercellular spaces, is utilized by those immunoglobulins which are transferred intact across the yolk sac endoderm.     

The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer

The permeability of the alveolar-capillary membrane to a small molecular weight protein, horseradish peroxidase (HRP), was investigated by means of ultrastructural cytochemistry. Mice were injected intravenously with HRP and sacrificed at varying intervals. Experiments with intranasally instilled HRP were also carried out. The tissue was fixed in formaldehyde-glutaraldehyde fixative. Frozen sections were cut, incubated in Graham and Karnovsky's medium for demonstrating HRP activity, postfixed in OsO4, and processed for electron microscopy. 90 sec after injection, HRP had passed through endothelial junctions into underlying basement membranes, but was stopped from entering the alveolar space by zonulae occludentes between epithelial cells. HRP was demonstrated in pinocytotic vesicles of both endothelial and epithelial cells, but the role of these vesicles in net protein transport appeared to be minimal. Intranasally instilled HRP was similarly prevented from permeating the underlying basement membrane by epithelial zonulae occludentes. Pulmonary endothelial intercellular clefts stained with uranyl acetate appeared to contain maculae occludentes rather than zonulae occludentes. HRP did not alter the ultrastructure of these junctions.     

Receptor mediated endocytosis of hemoglobin-haptoglobin, galactosylated serum albumin and polymeric IgA by the liver

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin. About 45 min after injection to rats, labelled material appears in the bile in amounts corresponding to 3.9% of the injected dose (hem-hap), 2.8% (gal-SA) and 60.1% (p IgA). The molecular weight of the labelled material transferred into the bile has been characterized: it consists almost entirely of intact IgA and for about 60% of intact hem-hap and gal-SA. Cell fractionation experiments indicate that 4 min after injection, the label is associated with components which equilibrate around a density of 1.13 g/cm3 and which dissociate from marker enzymes of Golgi complex, plasma membrane and lysosomes. Longer times after injection (from 20 min for hem-hap and gal-SA to 1 h for p IgA) labelled material appears, within lysosomes. To explain all these data, we suggest that after binding to plasma membrane receptors, the ligands are rapidly interiorized into pinocytic vesicles which fuse with lysosomes. Most of the hem-hap and gal-SA molecules but only part of p IgA would be released and subsequently digested; these vesicles would dissociate from lysosomes and fuse with the biliary membrane where the molecules still bound to the membrane of the vesicles would be detached and excreted into the bile.     

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Summary The circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissner's fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.     

Endocytotic activity in the endometrium during conceptus attachment in the cow

The uptake of horseradish peroxidase tracer injected into the uterine lumen of the cow was studied during the period of conceptus attachment (Days 18-21; Day 0 = oestrus) and also in cyclic animals. Endocytosis occurred in pregnant and non-pregnant cows but was especially marked when circulating progesterone concentrations were high. By 20 min after injection, the tracer was located in apical endocytotic vesicles and in organelles of the lysosomal system. In addition, some of the horseradish peroxidase-containing vesicles were associated with the lateral membranes of the cells and the tracer was also present in the intercellular spaces and beneath the basal membrane, especially in pregnant animals by the time of conceptus attachment. There was no evidence that pinopod-like functions could be attributed to large cytoplasmic protrusions from endometrial cells. Rather, the protrusions seemed to be involved in secretory processes. The presence of clear vesicles among the endocytotic vesicles suggested a coupled secretory-endocytotic activity of the cells, the significance of which remains to be determined.     

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta

Summary Endogenous immunoglobulin-G was localised in ultrathin frozen sections of human term placenta by use of an indirect immuno electron-histochemical methodology. Immunoreactivity of endogenous IgG to rabbit anti-human immunoglobulin-G antibody was visualised by use of protein-A — colloidal gold complex. Gold marked the syncytiotrophoblast in both coated and uncoated regions of the apical plasmalemma, in vesicles and multivesicular bodies, and in vesicles near the basal plasmalemma. Immunoreactivity was also seen in the interstitial space between the trophoblast and the fetal endothelial layer as well as in various types of vesicles within the endothelial cells. No immunoreactivity was seen in the intercellular clefts of the endothelium. The pattern of localisation observed is consistent with receptor-mediated uptake of immunoglobulin-G into the syncytiotrophoblast of the human placenta followed by release into the interstitial space and then vesciular transport through the endothelium.     

Ultrastructure of the coxal gland of the horseshoe crab Limulus polyphemus: Evidence for ultrafiltration and osmoregulation

Electron microscopic examination of the paired coxal glands of the horseshoe crab Limulus polyphemus, focusing on urinary and vascular channels, shows six morphologically distinct regions. Each of four nephridial lobes consists of two cortical layers surrounding a medulla. The outer and inner cortexes contain blood vessels separated by a basement membrane from the urinary space lined by podocytes. Podocyte foot processes are applied to the basement membrane, interdigitate with those from other podocytes, and have a filtration slit diaphragm between them. Cortical morphology demonstrates ultrafiltration of blood, a previously undescribed function of the gland, as well as possible endocytic reabsorption of materials by the podocytes. The medulla drains into the stolon connecting the four lobes. These two areas have urinary tubules of cuboidal epithelium featuring microvillous-like apical projections, cytoplasmic vesicles and vacuoles, elaborate lateral interdigitations with septate junctions, and basal invaginations containing numerous mitochondria. These tubules are closely surrounded by blood channels, lined by a basement membrane containing embedded support cells. The medulla and stolon morphology are suggestive of both ion transport and water movement, in keeping with the gland's role in osmoregulation. The stolon empties into the end sac in the base of the most posterior lobe. It is lined by tall epithelium exhibiting apical overlap, blunt projections into the lumen, apparent endocytic vesicles, and basal plasma membrane infoldings with mitochondria. The end sac drains into the conducting nephric duct, the proximal end of which is lined by a cuticle. J. Morphol. 234:233–252, 1997. © 1997 Wiley-Liss, Inc.     

Production and distribution of entactin and GP-2 in M1536-B3 cells

The endodermally differentiated mouse embryonal carcinoma cell line M1536-B3 produces the basement membrane-associated glycoproteins entactin and GP-2 in cell culture. Immunological techniques coupled with light and electron microscopy were used to study the intracellular distribution and fate of these molecules. The two proteins were localized in cisternae of rough endoplasmic reticulum and in discrete membrane-bound vesicles. There was no evidence for their presence in the Golgi apparatus. The membrane-bound vesicles appeared to fuse with the plasma membrane and thereby transfer their contents to discrete foci on the cell surface. These foci became apparent as cell-to-cell contact was made and were prominent at cell-cell contact sites. They eventually coalesced into continuous densely stained extracellular bands of amorphous material. These bands formed a honeycomb of spaces lined by cells. It appeared that production of the extracellular matrix was probably stimulated by cell-cell contact, and that cell density rather than age of the cell cultures dictated the pattern of extracellular distribution. These results support the hypothesis that these molecules are involved in cell adhesion and multicellular organization.     

Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.     

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

Formation of the acrosome and basal body during spermiogenesis in a marine snail,Nerita picea (Mollusca: Archaeogastropoda)

In Nerita picea the proacrosomal granule is formed basally in the early spermatid from one large cisterna of the Golgi body, with which the other Golgi-derived vesicles fuse. After the proacrosomal granule has attached to the plasma membrane and invaginated to form a cup shape, one cisterna of endoplasmic reticulum inserts into the open end and deposits a granular secretion on the inner surface. Subsequently, the proacrosome migrates along the plasma membrane to the apex of the nucleus, but the Golgi body remains basal, as occurs in other archaeogastropods and also many polychaete annelids. However, the final shape and structure of the acrosome is similar to that of mesogastropods. The annulus attaches the distal centriole to the plasma membrane early in spermiogenesis. The production of the flagellum by the distal centriole not only expands the plasma membrane posteriorly but moves the centriolar complex to the nucleus, causing an invagination of the plasma membrane where it is bound by the annulus. During proacrosome migration, the Golgi body secretes a dense tube around the flagellum, and the mitochondria fuse into two spheres at the base of the nucleus. The nuclear plug that closes off the intranuclear canal until this stage rapidly reorganizes itself into two tubes of material inside the canal. The centrioles continue flagellar production, break away from the annulus, and move deep into the intranuclear canal where they fuse together to form the basal body of the sperm. In the maturing spermatid, the two mitochondria fuse into a single sheath that spirals around the flagellum. The annulus does not migrate posteriorly but remains anterior to the midpiece, which is unusual for a filiform sperm. Spermiogenesis in Nerita picea has features in common with both archaeogastropods and mesogastropods but also has some unique features. These observations lend credence to the idea that the Neritidae are a transitional group between Archaeogastropoda and Mesogastropoda.