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The biology of cartilage. I. Invertebrate cartilages:Limulus gill cartilage
Abstract
The endoskeletal structure supporting the gill-books of Limulus polyphemus has been investigated by means of light and electron microscopy, chemical analysis and x-ray diffraction. This tissue is a cartilage which has significant correspondences with both vertebrate cartilage and plant tissues. Morphologically, the Limulus cartilage resembles certain cellular vertebrate cartilages with relatively scant matrix, and also certain plant parenchyme, collenchyme and sclerenchyme tissues. Of particular interest, was the observation that during cytoplasmic division, a phragmasome-like structure appears between the daughter cells of the dividing gill cartilage cells. This phragmasome-like structure appears to be a precursor of new matrix (cell-wall) formation between the young chondrocytes, in much the same fashion as its counterpart in plant tissues. Perichondrial cells and underlying chondrocytes contain lipid droplets, abundant glycogen and ribosomes, as do corresponding vertebrate cartilage cells. In some of the Limulus cells, glycogen and ribosomes appear to be admixed with lipid, forming aggregates in which all three materials are in intimate intraparticulate relationship. During molting, the number of ribosomes seen in chondrocytes increases. The tissue contains both hydroxyproline and hydroxylysine, and gives a weak x-ray diffraction pattern.
... The endoskeleton resembles cartilage thought to be present in many invertebrates [31,32,[35][36][37][38][39]. It apparently gives support to the basal portion of at least some of the embryonic appendages in their early stages of development. ...
... The endoskeleton has a thin electron-opaque matrix, and typically many matrix chambers have cell debris or cells with large vacuoles (Figure 13). This suggests the endoskeleton is deteriorating, but vacuolated cells are a common feature of invertebrate cartilage [37][38][39] including the cartilage of adult horseshoe crabs [35]. ...
... Endoskeletal tissue regarded as different types of cartilage is present in numerous invertebrates and at the base of opisthosomal appendages in adult horseshoe crabs [31,32,[35][36][37][38][39]. The branchial endoskeletal matrix in adult horseshoe crabs has elastin, allowing support but also flexibility for the rhythmically moving appendages with attached gills [37]. ...
The transmission electron microscope (TEM) is used for the first time to study the development of book gills in the horseshoe crab. Near the end of the nineteenth century the hypothesis was presented for homology and a common ancestry for horseshoe crab book gills and arachnid book lungs. The present developmental study and the author's recent ones of book gills (SEM) and scorpion book lungs (TEM) are intended to clarify early histological work and provide new ultrastructural details for further research and for hypotheses about evolutionary history and relationships.
The observations herein are in agreement with earlier reports that the book gill lamellae are formed by proliferation and evagination of epithelial cells posterior to opisthosomal branchial appendages. A cartilage-like endoskeleton is produced in the base of the opisthosomal appendages. The lamellar precursor cells in the appendage base proliferate, migrate outward and secrete the lamellar cuticle from their apical surface. A series of external, posteriorly-directed lamellae is formed, with each lamella having a central channel for hemolymph and pillar-type space holders formed from cells of the opposed walls. This repeated, page-like pattern results also in water channels (without space holders) between the sac-like hemolymph lamellae.
The developmental observations herein and in an earlier study (TEM) of scorpion book lungs show that the lamellae in book gills and book lungs result from some similar activities and features of the precursor epithelial cells: proliferation, migration, alignment and apical/basal polarity with secretion of cuticle from the apical surface and the basal surface in contact with hemolymph. These cellular similarities and the resulting book-like structure suggest a common ancestry, but there are also substantial developmental differences in producing these organs for gas exchange in the different environments, aqueous and terrestrial. For scorpion book lungs, the invaginated precursor cells align in rows and secrete rows of cell fragments that are the basis for the internal, anterior-directed air sacs. The hemolymph sacs of book gills are formed by epithelial evagination or outfolding from the posterior surface of the branchial appendages.
... These descriptions of cartilage were based primarily on gross morphological and histological studies. More recent biochemical and electron-microscopic characterizations of invertebrate tissues (Person and Philpott 1967, 1969a, 1969bPhilpott and Person 1970) extended the definition of true cartilage to include several invertebrate cartilaginous tissues. Stemming from these studies, Person (1983, pp 33-34) has defined cartilage as: ..."an animal tissue, usually endoskeletal, but also exoskeletal. ...
... The Limulus branchial cartilage strongly resembles vertebrate hyaline cartilage in gross appearance, but the X-ray diffraction pattern of the matrix is not characteristic of collagen. Amino acid analysis suggests that this tissue contains relatively little collagen (Person and Philpott 1969b). Investigations from our laboratories have further characterized the Limulus branchial cartilage. ...
... Bouin's fixed paraffin sections of the branchial cartilage stain intensively with Verhoeff's reagent (Fig. 4A). The branchial cartilage along with all Person and Philpott (1969b) Most of the information on the cartilage in the Mollusca comes from the cephalopods. However, an odontophore cartilage from a gastropod, the channeled whelk Busycon canaliculatum, has also been characterized. ...
A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.
... The horseshoe crab, known as a living fossil, is an armoured marine arthropod. Despite its solid external skeletal armour, the horseshoe crab has internal cartilaginous structures: the endosternite, supporting the CNS and cartilaginous plates, as well as "branchial cartilage", in the book gills [9,[18][19][20][21][22]. The cuttlefish, a marine cephalopod mollusc, displays a well-developed centralized nervous system that is surrounded by a simple cartilaginous skull providing necessary protection and support [9]. ...
Chrondrocranium, the cartilaginous skull, is one of the major innovations that underlie evolution of the vertebrate head. Control of the induction and shaping of the cartilage is a key for the formation of the facial bones and largely defines facial shape. The appearance of cartilage in the head enabled many new functions such as protection of central nervous system and sensory structures, support of the feeding apparatus and formation of muscle attachment points ensuring faster and coordinated jaw movements. Here we review the evolution of cartilage in the cranial region and discuss shaping of the chondrocranium in different groups of vertebrates.
... Within the tissue supporting Limulus gills (branchial cartilage), the cells are large and spherical and may also contain a large vesicle (or vacuole), making this tissue a vesicular cell-rich cartilage (Cole and Hall, 2004). The entire cartilage organ is surrounded by a PAS-negative perichondrium, indicating low glycosaminoglycan content (Person and Philpott, 1969b;Crowden, 1967). The high cellularity and rigidity of the matrix is reminiscent of Zellknorpel cartilage described by Schaffer (1930) and Benjamin (1990), and therefore we conclude that Limulus branchial cartilages are vesicular Zellknorpel cartilages. ...
Tissues similar to vertebrate cartilage have been described throughout the Metazoa. Often the designation of tissues as cartilage within non-vertebrate lineages is based upon sparse supporting data. To be considered cartilage, a tissue should meet a number of histological criteria that include composition and organization of the extracellular matrix. To re-evaluate the distribution and structural properties of these tissues, we have re-investigated the histological properties of many of these tissues from fresh material, and review the existing literature on invertebrate cartilages. Chondroid connective tissue is common amongst invertebrates, and differs from invertebrate cartilage in the structure and organization of the cells that comprise it. Groups having extensive chondroid connective tissue include brachiopods, polychaetes, and urochordates. Cartilage is found within cephalopod mollusks, chelicerate arthropods and sabellid polychaetes. Skeletal tissues found within enteropneust hemichordates are unique in that the extracellular matrix shares many properties with vertebrate cartilage, yet these tissues are completely acellular. The possibility that this tissue may represent a new category of cartilage, acellular cartilage, is discussed. Immunoreactivity of some invertebrate cartilages with antibodies that recognize molecules specific to vertebrate bone suggests an intermediate phenotype between vertebrate cartilage and bone. Although cartilage is found within a number of invertebrate lineages, we find that not all tissues previously reported to be cartilage have the appropriate properties to merit their distinction as cartilage.
... This suggests the possibility that type II collagen had not yet assumed its role as the major matrix protein of cartilage at the time of the divergence of the lamprey from the vertebrate line, approximately 500 million years ago. The alternative possibility gill cartilage of the invertebrate, the horse-shoe crab (Limu-Note the absence of detectable lamprin mRNA from the lus polyphemus) (14). Amino acid compositions suggest that branchial cartilage. ...
Cartilage structures of the sea lamprey, Petromyzon marinus, have previously been reported to stain by the Verhoeff method for elastin and to have a morphology similar to that of elastic cartilage of higher vertebrates. Earlier we showed that lamprin, the major matrix protein of lamprey annular cartilage, although not identical to elastin, cross-reacted with antibodies to elastin and shared significant sequence and structural similarities. Here we demonstrate that the majority of the skeletal cartilage of the lamprey is noncollagenous in character, remaining intact after treatment of the tissues with cyanogen bromide. However, unlike annular and neurocranial cartilages, which consist mainly of lamprin, evidence from amino acid compositions, immunohistochemistry, and western and northern blotting indicates that the major matrix protein of branchial and pericardial cartilage is not lamprin, but a second cyanogen bromide-insoluble protein or group of proteins with similarities to elastin. These results suggest that the skeletal cartilage of lower vertebrates consists of a family of noncollagenous, elastin-like proteins. The relationships among members of this family of cartilaginous proteins and between these proteins and vertebrate elastins, and the implications of these relationships for understanding the evolutionary lineage of elastin will await further characterization of these proteins.
Pharyngeal arches are a key innovation that likely contributed to the evolution of the jaws and braincase of vertebrates. It has long been hypothesized that the pharyngeal (branchial) arch evolved from an unjointed cartilaginous rod in vertebrate ancestors such as that in the nonvertebrate chordate amphioxus, but whether such ancestral anatomy existed remains unknown. The pharyngeal skeleton of controversial Cambrian animals called yunnanozoans may contain the oldest fossil evidence constraining the early evolution of the arches, yet its correlation with that of vertebrates is still disputed. By examining additional specimens in previously unexplored techniques (for example, x-ray microtomography, scanning and transmission electron microscopy, and energy dispersive spectrometry element mapping), we found evidence that yunnanozoan branchial arches consist of cellular cartilage with an extracellular matrix dominated by microfibrils, a feature hitherto considered specific to vertebrates. Our phylogenetic analysis provides further support that yunnanozoans are stem vertebrates.
Degenerative joint disease affects over 40% of the population over 40 years of age, and the incidence increases dramatically with advancing years. However, most of the basic experimental work carried out on articular cartilage uses animal tissue. Because of fundamental biological differences between human and most laboratory animals, extrapolation from animal work to the human situation is very difficult. Therefore, work on human tissue is vital. A key question in the understanding of articular cartilage is how the extracellular matrix is regulated. In order to address this problem, the synthesis of certain matrix components have been mapped both in whole tissue sections and in isolated chondrocytes grown in culture. Antibodies raised to collagen types I and II, keratan sulphate, dermatan sulphate proteoglycan, hyaluronan binding region and link protein were used, in addition to a biochemical probe to localise free hyaluronan sites. In whole tissue sections, age related differences were found in the localisation of keratan sulphate, dermatan sulphate proteoglycan and hyaluronan. In order to gain insights into the control of matrix synthesis, isolated chondrocytes were grown under two culture conditions, low density monolayer and suspension over agarose. This allowed the effects of anchorage dependence and differences in cell shape to be studied. In suspension culture, cells from the surface region of cartilage formed morphologically distinct clusters from those formed by deep region cells, which remained throughout culture. A relationship was shown to exist between cell shape and synthetic ability for collagen, keratan sulphate, dermatan sulphate proteoglycan, proteoglycan monomer and hyaluronan, but not for link protein. In relation to keratan sulphate, two populations of chondrocytes were isolated and grown in culture, one which preferentially synthesised keratan sulphate, and one which did not. This second population was predominantly located in the surface zone of cartilage. We found that whilst suspension culture promoted the synthesis of keratan sulphate, even in cells which do not normally synthesise this component, culture in monolayer inhibited its synthesis. Our results also suggest that in relation to the study of cell sub-populations, the use of a single phenotypic marker is unacceptable, and a range of indicators is required. The observed modulation in phenotype of chondrocytes maintained in culture is discussed in terms of environmental control of gene expression and possible changes in cell symmetry.
Collagen has been described in several regions of the arthropod body, but this molecule does not perform the ubiquitous connective tissue role as is evident in both Vertebrata and Inverte-brata. The Arthropoda, despite the widespread occurrence of collagen fibrils, are characterized by the relative scarcity of connective elements; there is also a scarcity of works concerning the biochemistry and physiology of collagen. Before examining these points in detail, a number of preliminary remarks are in order:
Morphological, biochemical, molecular biological and biomechanical analysis of cartilages from the Atlantic hagfish, Myxine glutinosa, reveal they are unusual tissues. At least three different cartilages designated Types 1, 2 and 3 have been identified.
Type 1 appears superficially to resemble other vertebrate cartilages; however, ultra-structural analysis reveals that the extracellular matrix (ECM) is non-collagenous. Type 2 bears no morphological resemblance to any known vertebrate cartilage, Type 3 is similar in appearance to Type 1; however, biochemical analysis of Types 1 and 3 cartilages reveal that each is composed primarily of a cyanogen bromide (CNBr) insoluble protein of unique composition. Myxinin, the principle structural protein of Type 1 cartilage, is similar but not identical to lamprin, the main structural protein of lamprey annular cartilage. The major structural protein of Type 3 cartilage has a composition very different from myxinin and as yet has not been named. The CNBr insoluble material from Type 2 cartilage is a minor component with a composition different from myxinin, Type 3 cartilage and lamprin.
Molecular biological studies suggest no homology between hagfish and lamprey cartilages. Comparative biomechanical studies indicate that the modulus of elasticity of hagfish cartilage is about one order of magnitude higher than lamprey and bovine cartilages.
The morphological and biochemical differences between hagfish and lamprey cartilages support the concept that these two agnathans followed long independent evolutionary histories.
Some aspects of the differentiation, growth, and morphogenesis of the tissues within the skeleton are discussed and related to the evolution of the vertebrate skeleton. The tissues considered are bone, cartilage, dentine, and enamel.
The histology of the skeletal tissues of the Ordovician agnatha is reviewed with the conclusion that the skeletal tissues of the first vertebrates were as diverse and as specialized as are those of present-day vertebrates. Phylogenies of skeletal tissues cannot be established. The trend during evolution appears to have been toward reduction in amount of skeletal tissue and in the number of types of tissues present.
The factors which determine when and where a skeletal element develops ontogenetically are reviewed and used to discuss the origin and evolution of jaws, the evolution of membrane bones and the origin of such structures as sesamoid bones. Special importance is attached to epithelial-mesenchymal interactions.
The factors which determine what particular skeletal tissue will form at a particular site within the body are reviewed with especial reference to modulation, germ layer derivation, and the role of epigenetic factors.
The factors which determine size and shape of the skeleton, both ontogenetically and phylogenetically, are reviewed and the directive role of adjacent tissues emphasized.
Connective tissues are responsible for much of the variation in morphology that we see today. Cartilage is a type of connective tissue that is often considered to be restricted to vertebrates, however, cartilaginous tissues are also found within invertebrates. Unfortunately, most definitions and classification schemes for cartilages suffer from a strong vertebrate bias, severely hampering the efforts of those who have attempted to include invertebrate tissues as cartilage. To encompass all types of cartilage, current classification systems need to be expanded. Here we present vesicular cell-rich as a new cartilage classification. Invertebrate cartilages, comparable to vertebrate cartilages at both cell and tissue levels, are composed of similar molecules, yet the extent to which they may be homologous is unknown. One option for studying the evolution of tissues is to adopt molecular phylogenetic approaches. However, the paucity of published molecular data makes addressing the evolution of cartilage using molecular phylogenetic approaches unrealistic at this time. Cartilage likely evolved from a chondroid connective tissue precursor, and may have been independently derived many times. The appearance of cartilaginous tissues of unknown phylogenetic affinities in such a wide diversity of animal groups warrants further investigation.
Bones and Cartilage provides the most in-depth review ever assembled on the topic. It examines the function, development and evolution of bone and cartilage as tissues, organs and skeletal systems. It describes how bone and cartilage is developed in embryos and are maintained in adults, how bone reappears when we break a leg, or even regenerates when a newt grows a new limb, or a lizard a tail. This book also looks at the molecules and cells that make bones and cartilages and how they differ in various parts of the body and across species. It answers such questions as Is bone always bone? Do bones that develop indirectly by replacing other tissues, such as marrow, tendons or ligaments, differ from one another? Is fish bone the same as human bone? Can sharks even make bone? and many more.
The cartilages (or "chondroid" tissue) in tentacles of the polychaete annelid, Sabella melanostigma, have been examined by electron microscopy and a series of histochemical techniques for the demonstration of mucopolysaccharides and protein end-groups. The ultrastructural studies indicated that the cartilages possess an investing layer of dense connective tissue which differs significanly from the matrix material secreted between the chondrocytes. The cartilage matrix was positive for acidic mucins with levels of sulfation above those of mammalian chondroitins A and C. This matrix as well as the investing connective tissue were intensely PAS-positive. Sabella cartilage was also stained intensely by methods for demonstrating tryptophan, tyrosine, side-chain carboxyl groups, disulfide groups, and amino groups. It was not stained by the procedure for sulfhydryl groups. Some evolutionary aspects of cartilage and chondroid tissues were discussed.
Summary The fine structure of the tentacular cartilage cells of the annelidSabella penicillum has been studied by scanning (SEM) and transmission electron microscopy (TEM). The cells contain a large, transparent vacuole centrally, and a thin layer (>0.1 µm) of cytoplasm around. Some granular endoplasmic reticulum, mitochondria and smaller clear vesicles are present in the cytoplasm. The nuclei are dense in the TEM preparations. The surrounding matrix is most dense close to the cells, and form a chondroid matrix, 0.2 to 0.5 µm thick, consisting of a flocculate material. The boundary to the surrounding matrix is condensed in the central cartilaginous cells, but not in the pinnular. The rigid structure of the close chondroid matrix is demonstrated in the SEM. The structure is compared to other invertebrate cartilages so far described, and its functions are discussed.
Summary Between the epimysium of the longitudinal muscle and the posterior aspect of the branchial cartilage ofSabella penicillum, an intermediate layer containing small muscle cells and fibroblasts embedded in an extracellular fibrous material are found. The small muscle cells contain 30 to 140 nm thick filaments with transverse striations at about 14.5 nm intervals, and are supposed to be paramyosin muscles. Their lenght were only a few µm and it could not be determined whether they were single sarcomere “striated” muscles, or true smooth muscles. Both the epimysium and the cartilage had numerous extensions that interdigitated with the muscle cells. Hemidesmosome-like specializations of the muscle cell membrane and its adjoining cytoplasm were characteristic features of the interdigitations. The arrangement with the short muscle cells makes it possible both to move the branchial crown, and to attach it for sustained periods.
Although invertebrate cartilage tissues do not mineralize in nature, it is now reported for the first time that when excised gill cartilage tissue from Limulus (horse shoe crab) is placed in an appropriate incubation medium metastable to hydroxyapatite, mineralization will occur. The mineralization is temperature dependent, and takes place at 37 degrees but not at 20 degrees. Incubations in media metastable to calcite have not produced mineralization. Histologic examination of mineralized tissues showed mineral deposits predominantly within cells, and to a lesser extent in the matrix. X-ray diffraction of the deposited mineral revealed a typical biological hydroxyapatite pattern.
Immunohistochemical and ultra-structural methods were used to examine the distribution of elastin and the fine structure of the trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus. The cells and matrix, as well as the overall organization of these components, in larval and adult trabecular cartilage resemble those of adult annular and piston cartilages (Wright and You-son: Am. J. Anat, 107:59–70, 1983) Chondrocytes are similar to those in hyaline cartilage. Lamprin fibrils and matrix granules, but no collagen fibrils, are found in a matrix arranged into pericellular, territorial, and interterritorial zones. Branchial, pericardial, and nasal cartilages differ from trabecular, annular, and piston cartilages in the organization of their matrix and in the structural components of their matrix and perichondria. Furthermore, immunoreactive elastin-like material is present within the perichondria and peripheral matrices of nasal, branchial, and pericardial cartilages in both larval and adult lampreys. Oxytalan, elaunin, and elastic-like fibers are dispersed between collagen fibers in the perichondrium. The matrix contains lam-prin fibrils, matrix granules, and a band of amorphous material, which is reminiscent of elastin, in the periphery bordering the perichondrium.
The presence of elastic-like fibers and elastin-like material within some lamprey cartilages implies that this protein may have evolved earlier in vertebrate history than has been previously suggested.
Calcified tissues may be defined as exo- and endoskeletons formed both at specific sites and at pathologically induced sites in organisms. These tissues are of either epithelial or mesenchymal origin. Despite the extensive studies on the role of AMPS's and PP complexes in many different calcified tissues, it is not possible at present to state conclusively whether or not AMPS's are actually necessary for biological calcification. It has been generally accepted that matrix formation precedes the onset of calcification. Mineral ions in calcifying solution are deposited and crystallized in vitro even on the surface of inert solid substances such as film and glass; however, if only spaces or lattices available for mineral deposition are present, mineralization may occur. Biologically, however, it may be reasonable that all or most tissues are calcifiable, but some inhibitory factors necessary for their mineralization. Chemically, the presence of both microheterogeneity and polymorphism in CS chains has been increasingly observed. Consequently, a description of the real role of the AMPS'S in biological calcification must await future studies.
In both light and electron microscopes, head cartilage from the squid Loligo pealii strongly resembles vertebrate hyaline cartilage. The tissue is characterized by the presence of irregularly-shaped cells suspended in an abundant matrix. Cell and matrix contents stain metachromatically with cationic dyes such as toluidin blue. Each cell gives off extensions which ramify via a network of channels throughout the matrix. Thereby, a system of inter-connecting canaliculi is established, with many similarities to the intercanalicular systems seen in vertebrate bone and cartilage tissues. In the electron microscope, the squid cartilage cells are seen to have very abundant endoplasmic reticulum and Golgi complex material. Mitochondrial transformations involving loss of cristae, the appearance of filaments in the mitochondrial matrix, and figures suggesting budding, also occur. Nuclear pores are numerous and easily detected. The matrix is characterized by the presence of a system of decussating fibrils which form polygonal figures, with granules usually evident at the points of intersection of fibrils. By chemical analysis the tissue contains 3- and 4-hydroxyproline and hydroxylysine. Preliminary wide single x-ray diffractions show a pattern characteristic for unoriented collagens, with 12 Å (intermolecular) and 2.86 Å (helix) reflections.
Light and electron microscopic observations and biochemical analysis of the lingual cartilages from the Atlantic hagfish, Myxine glutinosa, reveal two different types of cartilage, designated types 1 and 2, respectively. The anterior and medial lingual are type 1, while the posterior lingual cartilage is type 2. Chondrocytes in type 1 cartilage are similar to those found in other vertebrate cartilages. The presence within the Golgi elements of material that resembles a component of the extracellular matrix suggests the involvement of active chondrocytes in the synthesis of the matrix. The matrix of the type 1 cartilage contains fibrils arranged to form concentric lamellae in the territorial matrix and irregularly arranged, branched fibrils in the interterritorial matrix. Biochemical analysis of the type 1 cartilage reveals that it is composed primarily of a cyanogen bromide (CNBr)-insoluble protein of unique composition that we have termed " myxinin ." Myxinin appears to be similar, but not identical, to lamprin . Type 2 cartilage bears no resemblance to any other known vertebrate cartilage. The principal cells are hypertrophied and are characterized by masses of cytoplasmic filaments. The appearance of the organelles in smaller nest cells suggests that nest cells are active in the production of some of the matrix, which consists primarily of collagen. Microfibrils and a basal lamina-like material are also present. Biochemical analysis of the type 2 cartilage reveals that the CNBr-insoluble material is different from myxinin . Comparisons of lamprey and hagfish cartilages prompt the concept that these two agnathans probably followed long-independent evolutionary histories.
The cranial skeleton of the lamprey, a primitive vertebrate, consists of cartilaginous structures that differ from vertebrate cartilages in having a noncollagenous extracellular matrix. Novel matrix proteins found in these cartilages include lamprin in the annular cartilage and an unidentified protein in the branchial cartilages. Both show biochemical similarities to elastin. The inextractability of these proteins, even to chemical cleavage by cyanogen bromide, indicates a polymer with extensive covalent cross-linking. Here we report on the type of cross-linking. Lysyl pyridinoline was found in high concentration in the elastin-like protein of lamprey branchial cartilage at a ratio of 7:1 to hydroxylysyl pyridinoline, the form that dominates in vertebrate collagens. Both forms of pyridinoline cross-link were absent from annular cartilage and desmosine cross-links, which are characteristic of vertebrate elastin, were not detected in either form of lamprey cartilage. Pyridinoline cross-links are considered to be characteristic of collagen, so their presence in an elastin-like protein in a primitive cartilage poses evolutionary questions about the tissue, the protein, and the cross-linking mechanism.
Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid, reproducible, and convenient embedding methods for electron microscopy. The sections are robust and tissue damage is less than with methacrylate embedding.
Heavy metals may be incorporated from solution into tissue sections for electron microscopy. The resulting increase in density of the tissue provides greatly enhanced contrast with minimal distortion. Relative densities of various structures are found to depend on the heavy metal ions present and on the conditions of staining. Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood.
Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO4. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis.
Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO4-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described.
Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.
The cell plate, which separates dividing plant cells in the course of mitosis and subsequently serves as a precursor of the cell wall, originates from Golgi vesicles. Such plates contain cell wall substances, possibly carbohydrates and uronides apt for polymerization, which constitute the amorphous matrix of the future cell wall. The membranes of the coalescing vesicles constitutes the plasmalemma of the new cell surfaces, indicating that Golgi membranes and cell membranes are ontogenetically interrelated.
The use of polyester, as embedding material for ultrathin sections, completely avoids swelling of the tissue fragments and formation of bubbles. We have found Vestopal W to be a very adequate commercial polyester, when used with adequate initiator (tert. butyl perbenzoate) and activator (cobalt naphthonate). The embedding procedure is similar to that used for other embedding materials. Acetone must be used, however, instead of ethanol for the dehydration.Even relatively thick polyester sections provide much higher resolution than methacrylate sections of comparable thickness. This fundamental difference can be ascribed to a different response of these embedding materials when bombarded with electrons: during decomposition and partial volatilization, methacrylate melts while Vestopal does not. As consequences of this melting, surface tension tends to compress the sectioned specimen, altering its spatial arrangement, while capillary effects blur the contours of its structural details. The specimens embedded in polyester, on the contrary, remain undistorted and clearly defined.
The phragmoplast concerned with the formation of cell plate during the cytokinesis appears as a fibrous structure as seen with both the light and the electron microscopes. The vesicles, which form the cell plate by fusion with one another, are associated with the fibrils of the phragmoplast in such a manner that the phragmoplast may be assumed to be involved in the movement of the vesicles toward its equatorial plane.Der Phragmoplast, welcher die Zellplatte whrend der Cytokinese ausbildet, besitzt eine fibrillre Struktur, die im Licht- und Elektronenmikroskop erkannt werden kann. Die Vesikel, welche sich in der quatorialebene des Phragmoplasten ansammeln und dort zu einer Zellplatte zusammenflieen, stehen in solcher Beziehung zu den Fibrillen des Phragmoplasten, da man annehmen kann, da der Phragmoplast die Bewegung der Vesikel in der Richtung nach seiner quatorialebene bewirkt.
Fibrils from certain molluscan muscles, in particular the adductor muscles of the clam Venus mercenaria, were examined with the electron microscope and found to possess periodic variations in structure. In order to make these structural variations visible, it was necessary to treat the fibrils with reagents of high electron scattering power (electron stains). Phosphotungstic acid was found to be particularly suitable. This stain combines with specific regions in the fibrils, forming a remarkably regular geometrical pattern of which the most prominent feature is a regular cross striation, representing a fiber‐axis spacing of about 145A. Within each stained band, the stain is more highly concentrated in spots spaced about 193A from center to center across the band. A line drawn through any such spot parallel to the fiber axis passes through other similar spots, spaced five cross bands apart, making the length of the fiber‐axis period precisely five times the fiber‐axis spacing. X‐ray diffraction data obtained by Bear from the intact muscles are compared with the electron microscope observations. The small angle diffractions are in close agreement with those which would be expected from the observed structure except for the magnitude of the lateral spacing. Electron microscope values for this spacing are significantly smaller than the 325A indicated by the x‐ray data, probably as a result of lateral shrinkage in the vacuum‐dried electron microscope specimens. No significant difference in axis spacing has been observed in fibrils isolated from muscles fixed with alcohol in contracted and extended states.
A simplified scheme for the automatic analysis of amino acids has been developed. It employs a single column, 133 × 0.9 cm, of Dowex 50X12, 25–32 μ (ground), which is eluted with a continuous gradient. The gradient is produced by a nine-chambered device (Varigrad) which allows a wide variety of elution schemes to be constructed. The procedure described allows the complete analysis of a protein hydrolyzate, including collagen, in 24 hr using a single sample. It is also suitable for many other biological amino acid mixtures.
The ascorbic acid method of Ammon and Hinsberg, modified by Lowry and associates, has been applied to the determination of phosphorus in whole blood, plasma, serum, and urine. A sensitivity about eight times that of the aminonaphtholsulfonic acid method permits the use of much smaller samples for measurement in conventional cells (as little as 0.15 γ of phosphorus can be determined in ordinary 3-ml. cuvettes.) A comparison with an accepted procedure on a number of samples showed that the ascorbic acid method gave essentially the same results.
Periodic acid acts upon the 1,2 glycol linkage (-CHOH -CHOH-) of carbohydrates in tissue sections to produce aldehyde (RCHO+RCHO) which can be colored with Schiff s reagent. The method can be used on frozen or paraffin sections and is useful as a reaction for carbohydrates of tissues: glycogen (in paraffin section only), mucin, basement membrane, reticulin, the colloid of the pituitary stalk and thyroid, some of the acidophile cells of the human anterior hypophysis, the granular cells of the renal arteriole, etc.
In abnormal tissues, it colors many of the “hyaline” materials— amyloid infiltrations, arteriolosclerotic hyaline, colloid droplets, mitotic figures, etc.
The histochemical uses of the periodic-acid-Schiff's reagent (PAS) need careful control because of the possibility of attachment of iodate or periodate to tissue constitutents, producing a recoloration of the Schiff's reagent. Whenever possible the positive reacting material should be further identified by other methods since Lison showed other substances besides aldehydes can recolorize SchifFs reagent.
Cross-β X-ray diffraction patterns have been obtained from fibers prepared from the cold, neutral-soluble protein fraction of decalcified, bovine, embryonic enamel matrix. Similar cross-β patterns have previously been reported from the intact organic matrix (8). The structural implications of the unusually high proline content of these proteins are discussed.
The fine structure of Limulus polyphemus skeletal muscle has been studied with the electron microscope. The striations were found to consist of A, I and Z bands only; no M lines, H zones, or other striations were observed. In addition mitochondria, sarcolemma, and sarcoplasmic reticulum were figured and described.
The myofilament structure seemed to consist of large filaments about 160 Å in diameter in the A band, and small filaments, about 30 Å in diameter, in the I band. Small filaments were sometimes observed in the A band when the specimen was prepared under tension. When no tension was applied during fixation, cross-bridging material and large filaments only were observed in the A band. An occasional hexagonal pattern of large filaments was also seen in this muscle.
It was proposed that Limulus striated muscle represents a structural, biochemical, and physiological type intermediate between “classical” smooth muscle and striated muscle. It was proposed that a double filament structure might serve a function in stretch, rather than as a basis for contraction.
Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid, reproducible, and convenient embedding methods for electron microscopy. The sections are robust and tissue damage is less than with methacrylate embedding.
Using the endosperm of Haemanthus katherinae as material and time-lapse cine-micrographic technique, the behavior of granules, the problem of transport of material within the phragmoplast, and the formation of cell plate were analysed.The number of granules appearing in the interzonal region increases and it is concluded that they are synthesized de novo and do not arise by division of already existing ones.Very thin fibrils appear in the phragmoplast (less than 0.3 μ in thickness). Small droplets (“swellings”) are formed on the fibrils close to the plane of the future cell plate as a result of the increase of thickness of the part of the fibril. The droplets slide to the position of the cell plate and fuse, thus forming the cell plate. Some of the droplets move laterally before fusing for a distance up to 12 μ. It was not possible to find out in all cases whether they move together with the fibrils, although some of the fibrils also move laterally.It is also concluded that the elements forming the cell plate are of a different constitution from the small granules found in the phragmoplast.The mechanism of the formation of the cell plate and the relation of granules to the bodies described previously by other authors is briefly discussed.
PREVIOUS biochemical studies of acid mucopolysaccharides of cartilage were largely limited to mammalian tissues1. The principal polysaccharides found were chondroitin sulphate A and chondroitin sulphate C, with keratosulphate a minor component. The wide distribution of these acid mucopolysaccharides among all vertebrate classes, as shown by a preliminary survey2, suggests that their presence may be a systematic characteristic of vertebrate cartilage. Thus far only cartilages of elasmobranchs and lampreys have been found to contain sulphated chondroitin sulphate C type polysaccharides; sulphated keratosulphate is present in elasmobranchs only. These polysaccharides are similar to chondroitin sulphate C and keratosulphate, respectively, except for an excess of ester sulphate, above one mole per mole hexosamine.
- Adamstone
- Sinnott
On the occurrence of chitin as a constituent of the cartilages of Limulus and sepia
- Halliburton W. D.
Uber die ersten Spuren des Knochensystems und die entwicklung der Wirbelsaule in den Tieren
- Schültze C. A. S.
Limulus gill cartilage: a “plant‐like” animal tissue
- Person P.
J.1838Contributions to phytogenesis
- M Schleiden
Anatomische untersuchung eines Limulus mit besonderer Berucksichtigung der Gewebe
- Gegenbaur C.
Studien uber das knorpelgewebe von Wirbellosen
- Nowikoff W.
Über den feinen Bau und die Entwicklung des Knorpelgeweben und uber verwandte Formen der Stutzsubstanz
- Schaffer J.