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The elasmobranch renal corpuscle: fine structure of Bowman's capsule and the glomerular capillary wall.

The fine structure of the renal corpuscle of the marine elasmobranch fish, the little skate (Raja erinacea), and two species of dogfish sharks, the spiny dogfish (Squalus acanthias) and the smooth dogfish (Mustelus canis), was studied by light microscopy and by transmission (thin sections, freeze-fracture replicas) and scanning electron microscopy. Bowman's capsule was lined by ciliated cells, similar to those of the first part of the tubule, at the urinary pole and squamous cells in the zone between urinary and vascular poles. At the vascular pole the visceral epithelial cells had some closely apposed cuboidal cell bodies with a few processes inserted along the basement membrane, but foot processes were absent. These cuboidal cells were continuous with podocytes, which had primary, secondary and tertiary processes from which the pedicels arose. An inconsistently present slit membrane bridged the pedicels at varying distance from the urinary space. Small maculae and large fasciae occludentes joined the podocytes and/or their processes among which gap junctions were observed. In the skate kidney, the podocyte plasma membrane facing the basement membrane contained orthogonal arrays of particles. The epithelial basement membrane of the glomeruli in all elasmobranchs was consistently thick. The mesangial cells were numerous and partially enveloped in a basement membrane; their long processes almost completely circumscribed the capillary walls. The mesangial matrix was abundant and consisted of loosely arranged collagen fibrils, microfibrils and occasional anchoring fibrils. The endothelial cells had irregularly distributed fenestrations of various sizes and lay on their discontinuous basement membrane which was separate from that under the epithelial cells. The filtration apparatus resembles that of immature mammals and lower vertebrates.

Animals↗

Spatial disorder of collagens in the great vessels, associated with congenital heart defects.

Surgical ablation of the cardiac neural crest from the chicken embryo results in persistent truncus arteriosus (PTA) and a change in the elastic laminae of the great vessels, wherein elastin and the elastin microfibril show significant spatial disorder. The purpose of this study was to test the hypothesis that the interstitial collagens would also be disordered in the elastic laminae of chicken embryos with PTA. The birefringence characteristics of interstitial collagen were examined to evaluate spatial ordering. The results showed that collagen in the elastic laminae assumed an orderly configuration of well-defined fiber bundles in the great vessel walls of control embryos, whereas vessels from embryos with PTA lacked any distinct spatial order. Collagens type I and III were localized in the vessel walls. Type III collagen was the principal collagen of the elastic laminae, but was absent from the intima of all vessels. In the elastic laminae of vessels from control embryos, collagen type III showed well-defined fiber bundles whereas embryos with PTA had diffuse collagen type III in poorly defined laminae that were not separated by discrete layers of smooth muscle cells. Collagen type I was a minor component of the elastic laminae but formed robust pericellular fiber bundles throughout the media and intima. Collagen type I fibers appeared to be coarsened and less uniform in the vessels from embryos with PTA.

Animals↗

Extracellular matrix of the developing heart in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum.

As part of an ongoing study of heart development in normal and cardiac lethal mutant axolotls (Mexican salamanders) we examined the extracellular matrix (ECM) by microscopical methods. With scanning electron microscopy we are unable to detect ECM on the apical surface of cells of the early cardiogenic mesoderm. During the period of lateral plate migration, which coincides with the period of cardiogenic induction of mesoderm by anterior endoderm, there is little ECM, aside from some microfibrils, on the basal surface of the endoderm or mesoderm of the pharyngeal region. Later, a basal lamina (BL) is found on the endoderm and along portions of the developing endocardial and myocardial tubes. By the time of heartbeat initiation the BLs are complete and invested with striated collagen-like fibrils that are sparsely distributed in the "cardiac jelly" of normal and mutant hearts. Striated fibril deposition, which increases with time, is generally random in orientation, with the exception of some regions where there is a preferred directionality. During the post-hatching period striated fibrils appear in the subepicardial space. In addition, branching fibers that are probably elastin appear in the bulbus arteriosus. In these later stages the density of fibrils in the cardiac lethal mutant heart is much less than normal. Indirect immunofluorescent microscopy reveals laminin and fibronectin in the basal laminae of the endocardial and myocardial tubes of both normal and cardiac lethal mutant hearts. In addition, punctate and fibrillar staining for fibronectin, and punctate staining for laminin are found in the cardiac jelly. These matrix proteins are not abundant at the apical (exterior) surface of the myocardium until the epicardium appears.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambystoma mexicanum↗

Elastic fibers in the duct system of the rat submandibular salivary gland.

The submandibular salivary gland originates from the floor of the mouth whose mucosa contains elastic fibers. Therefore, such fibers were sought in the duct system of the derivative organ. In adult rats, light microscopy has indeed revealed fine, circumferential, elastic fibers near the basement membrane of the duct epithelium. In the larger extralobular ducts, they were separated from several layers of longitudinal elastic fibers by a capillary-rich zone sparse in elastic fibers except for fine angular ones. More peripherally, larger angular-appearing fibers were frequently present near the submandibular parasympathetic ganglia in the duct wall. As duct diameter decreased, elastic fiber size and number diminished. Intralobularly, the smaller striated ducts, granular and intercalated ducts, and acini generally lacked such fibers. Electron microscopy of the extraglandular portion of the main duct revealed fibrils extending from both fibroblasts and elastic fibers that were close to the epithelium. Microfibrils coursed from them toward the lamina densa. Anchoring filaments joined the lamina densa to the basal plasma membrane of the epithelium. Elastic fibers also appeared to connect to both capillaries and collagen via finer intermediate structures. These associations might permit dynamic interactions of fibroblasts, fibers, smaller fibrillar components, vasa, and the duct epithelium. This interplay could occur during feeding and grooming when tongue protrusion and neck extension stretch the submandibular duct and the gland itself. As a result, the tensile forces engendered could modify cell geometry and the calibers of the larger ducts' lumens and intercellular spaces, thus affecting the flow and composition of salivary secretion.

Animals↗

Vascularization of the pineal complex in the lizard Tiliqua rugosa.

The vascularization of the pineal complex in the lizard Tiliqua rugosa was investigated by vascular corrosion and latex casting techniques. The fine structure of the pineal capillaries was also studied by transmission electron microscopy. The pineal complex in T. rugosa consists of an elongated pineal gland proper and a separate, distinct parietal eye. The pineal complex derives an abundant blood supply from branches of the middle and posterior cerebral arteries. Scanning electron microscopy of vascular corrosion casts revealed a dense and extensive pineal capillary bed which drains ultimately into a wide longitudinal sinus suggesting an efficient pathway for the rapid removal of substances secreted by the gland. The parietal eye, which receives a unilateral left-sided blood supply from the unpaired anterior pineal artery, is shown to be a highly vascularized structure. The close morphological relationship between the pineal gland and dorsal sac, where the two structures apparently share the same blood vessels, suggests a functional relationship between them. The pineal capillaries are fenestrated with tight junctions between adjoining endothelial cells. Podia-like abluminal extensions of the endothelial cells were observed in close relation to unmyelinated nerve bundles. The basal margin of the pineal parenchyma is highly invaginated with thin finger-like cytoplasmic protrusions into the pericapillary space. Distinct bands of microfibrils form "struts" anchoring the pineal parenchyma to the endothelial wall. These features may have a role in the transfer of materials between the pineal gland and the blood stream.

Animals↗

Ultrastructure and development of a thick basement membrane-like layer in the anchoring villi of macaque placentas.

BACKGROUND: Anchoring villi and cytotrophoblastic cell columns are important structural components involved in placental morphogenesis. We have previously described the presence of an unusual basement membrane-like layer (BMLL) that separates these placental compartments. The purpose of the present study was to identify developmental changes in the ultrastructure of the BMLL and to assess its changes in extracellular matrix composition over the course of gestation. METHODS: Conventional techniques were used to examine macaque placental tissue by transmission electron microscopy. Standard immunoperoxidase methods were used to identify type IV collagen, laminin and fibronectin in paraffin sections. RESULTS: Until day 35 of gestation the BMLL was 70-100 nm thick and appeared similar to basement membranes seen in other regions of the villus, although it usually lacked a lamina lucida along the surface adjacent to the cytotrophoblast cells. Immunohistochemistry revealed the presence of laminin and type IV collagen in the BMLL. By 53 days of gestation the BMLL had hypertrophied at the junction of the anchoring villus and cell column, measuring 2,000-5,000 nm in thickness. The BMLL retained immunoreactivity for laminin and type IV collagen. Ultrastructural examination revealed the presence of a new component in the form of 10 nm microfibrils. By 89 days of gestation the BMLL was not reactive for laminin or type IV collagen but otherwise maintained the structural organization seen at 53 days. No additional changes were observed in the BMLL during late pregnancy. CONCLUSIONS: The BMLL is a distinct extracellular matrix region that separates the distal aspect of the anchoring villus from the proximal portion of the cell columns. Evidence indicates that adjacent cytotrophoblast plays a prominent role in the production of the BMLL. The BMLL may serve to organize this complex tissue by separating fetal mesenchyme from cytotrophoblast cells that are proliferating, differentiating, and migrating. Modifications to the composition of the BMLL may indicate changes in the role this matrix plays in the development of the placenta.

Animals↗

Renal corpuscle of the sturgeon kidney: an ultrastructural, chemical dissection, and lectin-binding study.

The sturgeon is an ancient species of fish that thrives in a wide range of ecological environments, from freshwater to seawater. Basic in this process of adaptation is the ability of the kidney to control fluid filtration and urine formation. However, the morphological basis of this process is mostly unknown. The aim of the present study was to use microdissection techniques (scanning electron microscopy (SEM), transmission electron microscopy (TEM), and lectin-binding histochemistry) to examine the structure of the renal corpuscle of the sturgeon Acipenser nacarii in order to reveal morphologic features that could be related to function, phylogeny, and habitat. The renal corpuscles are aligned along the intrarrenal arteries. The urinary pole shows a siphon-like neck segment (NS) in 92% of the nephrons, whose structural characteristics are different from those of other fish. The podocytes have cuboidal cellular bodies, intercellular contacts, and poorly developed cell processes. The podocyte glycocalyx contains N-acetylglucosamine and lacks sialic acid. The structural and lectin-binding patterns are similar to those found in the immature mammalian kidney. The glomerular basement membrane (GBM) is very thick and consists of three layers: a lamina rara externa, a lamina densa, and a thick subendothelial lamina. The latter contains tubular microfibrils, collagen fibers, and long mesangial cell processes. Frequently, the podocyte bodies attach directly to the GBM, and the area occupied by the filtration slits is very small. Furthermore, the GBM shows a glycosylation pattern different from that observed in most vertebrates. Contrary to what would be expected in sturgeons living in freshwater, the A. nacarii renal corpuscle morphology suggests a low glomerular filtration rate.

Acetylglucosamine↗

Lepidopteran peritrophic membranes and effects of dietary wheat germ agglutinin on their formation and structure.

Peritrophic membrane (PM) structure and the effects of dietary wheat germ agglutinin (WGA) on PM formation were studied in larvae of the European corn borer (ECB), Ostrinia nubilalis, and the tobacco hornworm (THW), Manduca sexta. Growth of ECB was strongly inhibited by low amounts of WGA in the diet (0.05%), whereas THW was not affected by amounts of up to 2%. In ECB larvae, chitin microfibrils were secreted to form an orthogonal network within the apical region of the anterior midgut microvilli. The network then moved to the tips of the microvilli where proteinacious matrix was added prior to delamination of a single PM into the lumen to enclose the food bolus. Multiple PMs rapidly appeared as the food moved posteriorly and some of these became greatly thickened in the middle and posterior regions of the midgut. WGA in the diet caused hypersecretion of unorganized PM in the anterior midgut lumen, disintegration of microvilli, and cessation of feeding. It was also shown to bind to both the chitinous network and to several PM proteins, perhaps causing voids in the PM and sparse matrix material. This allowed the passage of food particles through a defective PM into the ectoperitrophic space and penetration into the microvillar brush border. Stimulation of PM secretion and cessation of feeding may have been a response to damage to the brush border. Unlike ECB, the chitinous network of THW is a randomly organized felt-like structure embedded in a proteinaceous matrix. This PM is secreted as a thin multilayered structure in the anterior region of the midgut, but multiple and thickened PMs occur in the middle and posterior lumens of the midgut. THW tolerated high amounts of WGA in its diet with no disruption of PM formation or inhibition of growth. WGA did accumulate as large masses embedded in the PM, but caused no voids that would allow the penetration of food particles and subsequent damage to the brush border. Therefore, differences in PM formation and structure between ECB and THW appeared to affect how WGA interacts with chitinous and proteinaceous components of the PM and subsequent effects on larval feeding and growth.

Animals↗

Ultrastructure of the mouse synovial membrane. Development and organization of the extracellular matrix.

The synovial membrane of the mouse knee joint was examined by electron microscopy and electron microscopic histochemistry, with special reference to the development of the extracellular matrix. In the embryonic synovium, the intercellular spaces were filled with hyaluronate and chondroitin sulfate. The formation of the early joint cavity appeared to be initiated by accumulation of hyaluronate and chondroitin sulfate in the synovial primordium. At the postnatal stage, the synovial primordium differentiated into a true synovial intima that could be easily identified by the presence of two distinct lining cells: fibroblast-like cells (B cells) and phagocytic cells (A cells). Simultaneously, the synovial intima provided the specialized extracellular matrix that was characterized by organized structures of microfibrils, collagen fibers, and fibrous long spacing fibers embedded in a large number of glycoproteins.

Animals↗

High exogenous homocysteine modifies eye development in early chick embryos.

BACKGROUND: Homocysteine is a nonessential aminoacid whose increase is related to the appearance of neural tube defects in humans. In chick embryos, high levels of homocysteine produce neural tube defects and alteration of neural crest cell migration. METHODS: In our study, 8 microl of L-homocysteine thiolatone (20 micromol) was added to chick embryos of Stages 3-8/10 (Hamburger and Hamilton, 1951), (1238 hr of incubation). Three days later, 50 embryos, externally normal or carrying isolated spinal neural tube defects, were sectioned and stained by hematoxilin-eosin or anti-fibrillin-1 antibody. RESULTS: The eye showed alterations of the optic cup as microphthalmia, or lens dislocation. In both cases, the incidence of alterations diminished with the age of the homocysteine-increased embryos. Optic cup modifications are probably associated with central nervous system alterations, because most of the affected embryos exhibited isolated spinal neural tube defects and had altered neural crest cells. We have shown for the first time that high exogenous homocysteine during early development could produce a caudally-displaced lens axis before the zonule is formed. Fibrillin-1 is the main component of elastic microfibrils, and in the adult human it is seen as a protein particularly susceptible to homocysteine attack. CONCLUSIONS: Antibody staining against fibrillin-1 showed no evident morphological differences in distribution between experimental and control embryos in the lens, suggesting that fibrillin-1 was not the cause, and malformations may be attributed to other mechanisms.

Abnormalities, Drug-Induced↗

Fibulins in development and heritable disease.

Fibulins are a family of five extracellular glycoproteins found in a variety of tissues in association with diverse supramolecular structures, including elastic fibers, basement membrane networks, fibronectin microfibrils, and proteoglycan aggregates. Studies of the developmental expression patterns have indicated that several fibulins are prominently expressed at sites of epithelial-mesenchymal transformations during embryogenesis; among these sites, the cardiovascular system has been analyzed in more detail. Gene targeting of fibulins in mice has provided important insights into their biological roles, and has led to the identification of gene mutations in a congenital disorder of humans, cutis laxa. Genetic linkage and molecular studies have also associated several fibulin genes with various human heritable disorders that affect a wide range of organs, including limb, eye, blood, and arteries. In this review, we discuss the role of fibulins in development, with an emphasis on the cardiovascular system, and their involvement in human genetic disease.

Amino Acid Sequence↗

Plant cell enlargement and the action of expansins.

Plant cells are caged within a distended polymeric network (the cell wall), which enlarges by a process of stress relaxation and slippage (creep) of the polysaccharides that make up the load-bearing network of the wall. Protein mediators of wall creep have recently been isolated and characterized. These proteins, called expansins, appear to disrupt the noncovalent adhesion of matrix polysaccharides to cellulose microfibrils, thereby permitting turgor-driven wall enlargement. Expansin activity is specifically expressed in the growing tissues of dicotyledons and monocotyledons. Sequence analysis of cDNAs indicates that expansins are novel proteins, without previously known functional motifs. Comparison of expansin cDNAs from cucumber, pea, Arabidopsis and rice shows that the proteins are highly conserved in size and amino acid sequence. Phylogenetic analysis of expansin sequences suggests that this multigene family diverged before the evolution of angiosperms. Speculation is presented about the role of this gene family in plant development and evolution.

Amino Acid Sequence↗

A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection.

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.

Caulimovirus↗

Ultrastructure of the mouse leptomeninx.

An electron microscopic study was made of the mouse leptomeninx with special attention to the ultrastructure of the subarachnoid space. The arachnoid membrane was divided into outer and inner layers. The outer layer was composed of elongated cells in contact with one another with numerous tight junctions. The cells in the inner layer had intricate cytoprocesses , some of which embraced the connective tissue matrix containing collagen fibers. Beneath the arachnoid membrane, there were a loose meshwork of arachnoid cells and a single layer of pial cells. No arachnoid trabeculae were found. The intercellular space of the leptomeninx except in the outer layer of the arachnoid membrane was filled with Ruthenium Red-positive flocculent material and particles, in which various amounts of the collagen fibers and microfibrils were embedded. Elastic fibers were not identified. The subarachnoid space, which is believed to be a specialized channel exclusively containing the cerebrospinal fluid, was not recognized. These observations indicate that the cerebrospinal fluid circulates through the broad intercellular space, which contains extracellular matrix, of the leptomeninx .

Animals↗

Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database.

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were first described in the heritable connective disorder, Marfan syndrome (MFS). FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS, called "type-1 fibrillinopathies." In 1995, in an effort to standardize the information regarding these mutations and to facilitate their mutational analysis and identification of structure/function and phenotype/genotype relationships, we created a human FBN1 mutation database, UMD-FBN1. This database gives access to a software package that provides specific routines and optimized multicriteria research and sorting tools. For each mutation, information is provided at the gene, protein, and clinical levels. This tool is now a worldwide reference and is frequently used by teams working in the field; more than 220,000 interrogations have been made to it since January 1998. The database has recently been modified to follow the guidelines on mutation databases of the HUGO Mutation Database Initiative (MDI) and the Human Genome Variation Society (HGVS), including their approved mutation nomenclature. The current update shows 559 entries, of which 421 are novel. UMD-FBN1 is accessible at www.umd.be/. We have also recently developed a FBN1 polymorphism database in order to facilitate diagnostics.

Animals↗

Clustering of fibrillin (FBN1) missense mutations in Marfan syndrome patients at cysteine residues in EGF-like domains.

The Marfan syndrome is an autosomal dominant heritable disorder of connective tissue with prominent involvement of the ocular, skeletal, and cardiovascular systems. The gene on chromosome 15 encoding fibrillin (FBN1), a 350-kDa glycoprotein component of the extracellular microfibril, is the site of defect in most, if not all cases. Complementary DNA sequence reveals a gene composed largely of epidermal growth factor-like repeats, each containing six predictably spaced cysteine residues. To date, two FBN1 gene missense mutations have been reported. Here we describe the identification of three new missense mutations in the FBN1 gene in patients with the Marfan syndrome. All of the 5 characterized missense mutations occur within the epidermal growth factor-like repeats of the FBN1 gene. In addition, 4 of 5 involve the substitution of cysteine residues and 3 of 5 substitute the third cysteine in the epidermal growth factor-like motif consensus sequence. These data suggest that defined residues within EGF-like domains of FBN1 have particular significance and, when altered, play a pivotal role in expression of the Marfan phenotype.

Amino Acid Sequence↗

Identification of 29 novel and nine recurrent fibrillin-1 (FBN1) mutations and genotype-phenotype correlations in 76 patients with Marfan syndrome.

Marfan syndrome (MFS) is an autosomal-dominant disorder of the fibrous connective tissue that is typically caused by mutations in the gene coding for fibrillin-1 (FBN1), a major component of extracellular microfibrils. The clinical spectrum of MFS is highly variable and includes involvement of the cardiovascular, skeletal, ocular, and other organ systems; however, the genotype-phenotype correlations have not been well developed. Various screening methods have led to the identification of about 600 different mutations (FBN1-UMD database; www.umd.be). In this study we performed SSCP and/or direct sequencing to analyze all 65 exons of the FBN1 gene in 116 patients presenting with classic MFS or related phenotypes. Twenty-nine novel and nine recurrent mutations were identified in 38 of the analyzed patients. The mutations comprised 18 missense (47%), eight nonsense (21%), and five splice site (13%) mutations. Seven further mutations (18%) resulted from deletion, insertion, or duplication events, six of which led to a frameshift and subsequent premature termination. Additionally, we describe new polymorphisms and sequence variants. On the basis of the data presented here and in a previous study, we were able to establish highly significant correlations between the FBN1 mutation type and the MFS phenotype in a group of 76 mutation-positive patients for whom comprehensive clinical data were available. Most strikingly, there was a significantly lower incidence of ectopia lentis in patients who carried a mutation that led to a premature termination codon (PTC) or a missense mutation without cysteine involvement in FBN1, as compared to patients whose mutations involved a cysteine substitution or splice site alteration.

Amino Acid Sequence↗

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.

Amino Acid Sequence↗