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1.
The ultrastructure of the rat primary decidual zone   总被引:1,自引:0,他引:1  
The rat primary decidual zone (PDZ) is a transitory, avascular region of transformed fibroblasts surrounding the implanting embryo. Tracer studies have indicated that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight of the tracer. To clarify the morphological basis of the permeability barrier, we have studied the ultrastructure of the PDZ with particular emphasis on the intercellular features and cellular junctions. The cells of the PDZ were large and tightly packed; their apposed membranes showed extensive interdigitations in some regions, but elsewhere they were relatively straight. Tight junctions, gap junctions, and desmosomelike junctions were observed between decidual cells. The tight junctions usually consisted of one or two points of membrane fusion, and they were oriented both parallel and perpendicular to the long axis of the PDZ. These junctions were frequently associated with gap junctions. Scattered pockets of dilated extracellular space between decidual cells contained collagen fibrils and an amorphous, dense material. These extracellular components were also sequestered by the decidual cells in deep invaginations of the cell surface that were continuous with the extracellular space. Decidual cells also exhibited flangelike processes that penetrated the basal laminae of the adjacent epithelium and capillary endothelium. Our present observations indicate that decidual cells are connected by tight junctions, and a previous study demonstrated that macromolecules up to 40 kDa readily cross the PDZ; hence, the tight junctions appear to be discontinuous. We suggest that the structures restricting the movement of large macromolecules (66 kDa and larger) across the PDZ from blood vessels to the embryo may include discontinuous tight junctions, membrane interdigitations, and amorphous intercellular material.  相似文献   

2.
Summary Horseradish peroxidase (HRP) was used to study macromolecule permeation into the guinea-pig placenta perfused in situ. When tissue culture medium 199 (TC 199) was used as fetal-side perfusate, the tracer reaction product was found only lining the fetal endothelium. When a longer period of perfusion with HRP in TC 199 was used, a small amount of reaction product was found in the subendothelial space and syncytiotrophoblastic vesicles, but not in maternal lacunae. In similar experiments using a Krebs bicarbonate Ringer (KRBG) as perfusate the tracer was found (i) lining the fetal endothelium, (ii) in the lateral intercellular spaces of the endothelium, (iii) in the subendothelial space, and (iv) in the maternal lacunae.It is therefore evident that the vehicle influenced the permeability of the guinea-pig placenta to horseradish peroxidase. As other studies have shown that perfusion of the fetal side with salt solution increases pore size, the results with TC 199 are regarded as more representative of the situation in the intact animal. It is therefore suggested that the fetal endothelium of the guinea-pig placenta may be largely impermeable to molecules of the size of horseradish peroxidase (4 nm) or larger.  相似文献   

3.
Junctional complexes between the epithelial cells in the four distinct regions of the glow-worm Malpighian tubule were investigated by electron microscopy using thin sectioning, freeze-fracturing, osmotic disruption and tracer techniques. The lateral plasma membranes of all four cell types are joined by smooth septate junctions but the extent of the complex across the cell depth varies in the four different regions. The width of the septa, the interseptal spacing and the separation between the outer leaflets of the adjacent plasma membranes are different for each cell type. Gap junctions were identified only in the junctional complex between Type IV cells and were intercalated amongst large lateral sinuses. In oblique sections of lanthanum infiltrated tissue, the electron-lucent septa at the basal side of the junction are outlined by the tracer as it penetrates. In the Junctional complexes of all four regions the septa appear as short, distinct, linear bars. In tangential sections of gap junctions between Type IV cells, the junctions appear as a hexagonal array of intermembrane particles with a centre to centre spacing of 18 nm. Horseradish peroxidase did not penetrate the junctional complexes very far but readily passed through the basal lamina into the spaces between extracellular invaginations of the basement membrane of the cells. Junctional complexes in all four areas of the tubule have similar freeze-fracture faces. In freeze-fracture replicas of fixed tissue continuous ridges of fused particles are seen on the P face and complementary furrows are found on the E face. Junctional response to osmotically adjusted Ringer solutions was similar in all four cell types. Distortion or ‘blistering’ of the intercellular space between the septa of the junction occurred when the tissue was bathed in or injected with a hypertonic Ringer solution. The structure of these junctions, visualized by the different techniques, and the role of the septate junction in a transporting epithelium, are discussed.  相似文献   

4.
Summary Selected lobules of human term placentae were extracorporeally perfused for a recovery period of 20 min, fixed by perfusion and mordanted with ferrocyanide prior to processing for transmission electron microscopy. The lateral membranes of the endothelial cells of the terminal villous capillaries were found to be separated by paracellular clefts of mean width 15.6 nm. At tight junctional regions (1–4 sites per cleft) the two membranes approached each other more closely and frequently appeared to fuse. However, tilting of the sections in the electron microscope stage showed that the membranes were separated by a gap of mean width 4.1 nm in at least 94% of tight junctional profiles. When individual tight junctions were studied by a combination of serial sectioning and goniometric tilting, they were seen to widen abruptly within a distance of three to seven consecutive thin sections, indicating they were not continuous throughout the axial length of the capillaries. The wide regions of the clefts usually showed linkers, strands of glycocalyx-like material spanning the gap. Linkers may contribute to cell adhesion and possibly form part of a filter within the tortuous paracellular pathway provided by the discontinuous network of tight junctional strands. Human term placental capillaries appear to resemble closely other continuous non-brain capillaries.  相似文献   

5.
Endothelial cell membranes of rabbit carotid arteries were examined by the freeze-fracture technique. In the normal endothelium the mean densities of membrane-bound vesicles were 75 vesicles/microns 2 on the luminal cell membrane and 102 vesicles/microns 2 on the abluminal membrane. Whilst the vesical openings on the luminal membrane were randomly distributed those on the abluminal membrane were typically ordered in a macular pattern with lines free of vesicles. Tight and gap junctions between endothelial cells were numerous. After stimulating the carotid arteries with weak electrical impulses, a technique used to induce enhanced endothelial permeability and the formation of atheromatous plaques after repeated stimulations (Betz et al. 1985), vesicle openings were reduced to 78 vesicles/microns 2 on abluminal membranes. Membranes on the luminal side and intercellular tight and gap junctions remained unchanged.  相似文献   

6.
Summary Haem proteins of different molecular sizes were perfused into the foetal circulation of the guinea-pig placenta to study the permeability of the foetal endothelium.The smallest molecules tested, microperoxidase (ae 1.0 nm) and cytochrome C (ae 1.5 nm), readily penetrated the endothelium; tracer-reaction product was found in the subendothelial space of the capillaries. However, there was no uptake of these two tracers into the syncytiotrophoblast layer of the placenta. An intermediate-sized molecule, myoglobin (ae 1.7 nm), produced only a weak reaction product in the subendothelial space even when perfused at high concentration. The largest molecule tested, haemoglobin (ae 2.8 nm), did not penetrate the foetal endothelium at any of the concentrations employed.The foetal capillary endothelium thus provided a barrier to protein penetration from the foetal circulation, dependent on molecular size. There was evidence that the site of this barrier was located in the lateral intercellular spaces between the endothelial cells.The syncytiotrophoblast of this haemomonochorial placenta provided an almost absolute barrier to protein penetration from the foetal circulation. As other workers have described maternal-to-foetal transmission of proteins across this layer in the guinea-pig, a working hypothesis of the role of endothelium and syncytiotrophoblast in maternal/foetal protein exchange is discussed.  相似文献   

7.
The junctional complex in the gill epithelium of the freshwater mussel (Elliptio complanatus) consists of an intermediary junction followed by a 2–3 µ long septate junction. Homologous and heterologous cell pairs are connected by this junction. After fixation with 1% OsO4 containing 1% potassium pyroantimonate, electron microscopy of the gill reveals deposits of electron-opaque precipitate, specifically and consistently localized along cellular membranes. In both junctional and nonjunctional membrane regions, the precipitate usefully outlines the convolutions without obliterating the 150 A intercellular space, which suggests the rarity or absence of either vertebrate-type gap or tight junctions along the entire cell border. The precipitate appears on the cytoplasmic side of the limiting unit membranes of frontal (F), laterofrontal (LF), intermediate (I), lateral (L), and postlateral (PL) cells. The membrane surfaces of certain vesicles of the smooth endoplasmic reticulum, of multivesicular bodies, and of mitochondrial cristae contain precipitate, as does the nucleolus. In other portions of the cell, precipitate is largely absent. The amount of over-all deposition is variable and depends on the treatment of the tissue prior to fixation. Deposition is usually enhanced by pretreatment with 40 mM NaCl as opposed to 40 mM KCl, which suggests that the precipitate is in part sodium pyroantimonate. Treatment with 0.2 mM ouabain does not enhance deposition. Regional differentiation of cell membranes with respect to their ability to precipitate pyroantimonate is found in at least three instances: (a) between the ciliary membranes and other portions of the cell membrane: the precipitate terminates abruptly at the ciliary base, (b) between the LF and I cell borders: the precipitate is asymmetric, favoring the LF side of the junction, and (c) between the septate junctional membrane and adjacent membrane: the precipitate occurs periodically throughout the septate junction region with the periodicity corresponding to the spacing of the septa. This suggests that different regions of the cell membrane may have differing ion permeability properties and, in particular, that the septa may be the regions of high ion permeability in the septate junction.  相似文献   

8.
The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.  相似文献   

9.
Intercellular junctions were often found between macrophages in sinuses of regional lymph nodes of the rat after injection of large doses of cholesterol, cortisone acetate, and estrone at the footpad. They were identified by subplasmalemmal densities, 20-50 nm in width, beneath the plasma membranes of apposed macrophages. No distinct filamentous structures were visible in those dense regions. Electron-dense amorphous materials are lined up at the center of the intercellular space in the junctional regions. Some macrophages form clusters with intercellular junctions. No significant difference in the effect of cholesterol, cortisone acetate, and estrone on the number of intercellular junctions between macrophages was found.  相似文献   

10.
Estimates of capillary permeability for hydrophilic solutes are generally interpreted in terms of Pappenheimer's pore theory. The intercellular clefts of the capillary endothelium are considered a likely structural equivalent to the postulated system of small hydrophilic pores. However, correlation of permeabilities and cleft structure requires more knowledge of the detailed structure of the tight junctions which appear to obliterate the clefts. In this study the organization of tight junctions in endothelium of rat heart capillaries has been investigated by serial-section electron microscopy. Cross-sectioned intercellular clefts were photographed in a series of 190 consecutive sections (average thickness approximately equal to 40 nm) and in a series of 16 consecutive sections (average thickness approximately equal to 12.5 nm). Seventy-one junctional segments, each extending over 5-32 consecutive sections, were reconstructed. The endothelial junctions were organized as irregular networks of lines of contact between neighboring cells. Six pathways circumventing the lines of contact were followed through the entire junctional region of the clefts providing a tortuous pathway connecting the luminal and abluminal aspects of the clefts. Moreover, the individual lines of contact were provided with discrete discontinuities, apparently 4 nm wide. The observations support the notion that the paracellular pathway in capillary endothelium is permeable not only to small solutes but also to certain macromolecules.  相似文献   

11.
Gap junctional structures containing a 16-kDa intrinsic membrane protein have been isolated from the hepatopancreas of the crustacean Nephrops norvegicus. These structures are double membranes 14-15 nm thick and composed of hexagonal arrays of particles which have a central pore that is penetrated by a cationic negative stain. Membrane preparations have also been isolated from the hepatopancreas and these contain similar gap junctional regions of uniform width. Affinity purified antibodies to the 16-kDa protein bind principally to these gap junctional regions. Antiserum raised against the isolated gap junctional structures binds strongly to the lateral surfaces of the columnar epithelial cells and in particular to gap junction-like regions.  相似文献   

12.
Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.  相似文献   

13.
The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.  相似文献   

14.
We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.  相似文献   

15.
Cell movements in Fundulus blastoderms during gastrulation were studied utilizing time-lapse cinemicrography and electron microscopy. Time-lapse films reveal that cells of the enveloping layer undulate and sometimes separate briefly but remain together in a cohesive layer. During epiboly, the marginal enveloping layer cells move over the periblast as it expands over the yolk sphere. Movement occurs as a result of ruffled membrane activity of the free borders of the marginal cells. Deep blastomeres become increasingly active during blastula and gastrula stages. Lobopodia project from the blastomeres in blastulae and adhere to other cells in gastrulae, giving the cells traction for movement. Contact specializations are formed by the lateral adjacent plasma membranes of enveloping layer cells. An apical junction is characterized by an intercellular gap of 60–75 A. Below this contact, the plasma membranes are separated by 120 A or more. In mid-gastrulae, cytoplasmic fibrils occur adjacent to some apical junctions, and small desmosomes appear below the apical junction. Septate desmosomes also appear at this time. A junction with an intercellular gap of 60 A occurs between marginal enveloping layer cells and periblast. Contacts between deep blastomeres become numerous in gastrulae and consist of contacts at the crests of surface undulations, short areas of contact in which the plasma membranes are 60 or 120 A apart, and long regions characterized by a 200-A intercellular gap. Lobopodia contact other blastomeres only in gastrulae. These junctions contain a 200-A intercellular space. Some deep blastomeres are in contact with the tips of periblast microvilli. The mechanism of epiboly in Fundulus is discussed and reevaluated in terms of these observations. The enveloping layer is adherent to the margin of the periblast and moves over it as a coherent cellular sheet. Periblast epiboly involves a controlled flow of cytoplasm from the thicker periblast into the thinner yolk cytoplasmic layer with which it is continuous. Deep cells move by adhering to each other, to the inner surface of the enveloping layer, and to the periblast.  相似文献   

16.
Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.  相似文献   

17.
mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/gamma IFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.  相似文献   

18.
The microvillar and lumenal plasma membrane P-face of Ascaris intestinal cells is shown to be covered by relatively large (13 nm) particles at a fairly high density (1000/μm2), while the E-face has virtually none. The P-face of the lateral cell membranes, those separating the cells, have fewer and smaller (8 nm) particles. The intestinal cells are also shown to be connected by an apical complex of smooth septate and tricellular junctions similar to those found between some insect midgut cells. A periodic layer of tannic acid staining material is found on the cytoplasmic sides of the smooth septate junction, and when the intercellular space is filled with lanthanum, smoothly curved, 10 nm wide septal walls can be seen. Below the belt of septate junctions are a large number of gap junctions. These have closely packed arrays of particles on the P-face with some particle aggregates adhering to the closely packed pit arrays on the E-face.  相似文献   

19.
Gupta AP  Han SS 《Tissue & cell》1988,20(4):629-634
Granulocytes (GRs) and/or plasmatocytes (PLs), the two major immunocytes in arthropods, participate in cellular encapsulation of foreign tissue. Although gap and desmosome junctions have been reported in insect capsules, smooth septate junctions are being reported for the first time by both thin section and freeze-fracture techniques in Blattella germanica. In 7-day-old capsules, the septa are 23 nm thick, faintly 'scalloped' and slightly curved in appearance; the interseptal space has a periodicity of about 5 nm. In freeze-fractured capsules, the septa are associated on both sides with the corresponding intramembranous structures, belonging to the plasma membranes of the two junction-forming GRs. The intercellular space is 27 nm wide. There are 36-40 septa/1 mum junctional length. The junctions show furrows on the extracellular fracture face (E) and the complementary regular rows of intramembranous particles on the cytoplasmic face (P). The septate junctions often occur in the region of the capsule that also shows the presence of gap junctions, but only rarely that of desmosomes. The septate junctions are in close proximity with mitochondria. It is suggested that the function of these junctions is to produce compact capsules.  相似文献   

20.
On gap junction structure   总被引:4,自引:2,他引:2       下载免费PDF全文
We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.  相似文献   

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