The biology of cartilage. I. Invertebrate cartilages: Limulus gill cartilage

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 fine structure of the cartilage in the annelidSabella penicillum

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 m) 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 m 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.     

The nature and significance of invertebrate cartilages revisited: distribution and histology of cartilage and cartilage-like tissues within the Metazoa

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.     

Studies on rickets

Summary Observations with the light and electron microscope on the epiphyseal plate of rickety rats demonstrate several differences between the uncalcified cartilage and bone matrix. Uncalcified cartilage matrix is less refractile than bone matrix when it is viewed in polarized light. Electron microscopy shows that the fibrils of epiphyseal cartilage matrix are delicate and do not reveal the regular asymmetrical periodic structure that is so characteristic of collagen. The uncalcified bone matrix consists of fibrils with the regular fine structure of collagen. No unusual features could be found in the periodic banding of the collagen fibrils of the rickety osteoid, but some variations in their diameter and array were observed.This work was supported by research grant A 706 C-3, United States Public Health Service, National Institutes of Health (Arthritis and Metabolic Diseases).Markle Scholar in Medical Science, Electron Microscope Laboratory, Department of Pathology, Pathological Institute, McGill University Faculty of Medicine, Montreal, Canada.     

Cytochemical studies on cytoplasmic RNA-associated basic proteins in oocytes,somatic cells,and ribosomes

Summary Cytochemical studies on oocytes from representatives of several animal groups, of somatic tissues known to possess high levels of cytoplasmic RNA, and of isolated ribosomes indicated that basic proteins associated with the ribosomes may be stained by the methods currently in use for demonstrating histones. These proteins were resistent to weak acid extraction in fixed material and were labile to acetylation, but pretreatment with hot 5% TCA caused a measurable increase in staining with a modified Sakaguchi reaction. Such proteins were not confined to oocytes, but could be demonstrated with the TCA-alkaline fast green method in mouse pancreas and liver cells, and by the ammoniacal-silver method in all cells with high levels of cytoplasmic RNA. Microspectrophotometric studies onAscidia nigra andClavelina picta oocytes indicated that the highest concentration of cytoplasmic basic proteins was found in smaller oocytes, but the concentration of alkaline fast green stainable cytoplasmic RNA-associated basic proteins decreased in a pattern that differed significantly from that of stainable levels of cytoplasmic RNA. The implications of these findings to current concepts in developmental biology were discussed.Nucleoplasmic basic proteins with high levels of arginine which are not associated with nucleic acids were encountered in oocytes of all the echinoderm species studied and in the mouse.This work was supported by a U. S. Public Health Service grant HD-1499-04 and a Career Development Award 5-K3-6176-04.Contribution number 382 of the Bermuda Biological Station.     

Cardioinhibitory peptides from Limulus polyphemus: modulation of the neurogenic heart

Four neuropeptides have been isolated and sequenced from acetone extracts of brains of the horseshoe crab Limulus polyphemus. They belong to a newly discovered peptide family in invertebrates. A possible role of the four peptides from Limulus as cardioregulatory neurotransmitters has been tested on the isolated Limulus heart. Three of the peptides (DEGHKMLYFamide, GHSLLHFamide, and PDHHMMYFamide) produce dose-dependent decreases in both amplitude and rate of the heart contractions, whereas DHGNMLYFamide reduces only the amplitude of the heartbeat. All four peptides differ in threshold, potency and duration of their effects.Abbreviations DIC diisopropylcarbodiimide - DTT dithiotreitol - Fmoc 9-fluorenylmethyloxycarbonyl - GABA gamma amino butyric acid - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - IBMX 3-isobutyl 1-methyl-xanthine - Lip-HP Limulus head peptide - LP Limulus peptide - TBTU 2-(1H-benzotriazole-1yl)-1,1,3,3,tetramethyluronium tetrafluoroborate - TFA trifluoroacetic acid - TLC thin-layer chromatography     

Tissue channel morphology in Octopus

Summary The morphology of tissue channels in muscle and neural tissues of Octopus was investigated, at the ultrastructural level, with a technique involving the precipitation of ferrocyanide ions. The numbers, sizes and conductivities of the channels were estimated from quantitative data. No evidence was gained to indicate that the low microvascular density in Octopus is coupled to an especially extensive network of extravascular channels. The tissue channel system in Octopus appears to be broadly comparable with the mammalian system; a lack of information prevents more appropriate comparisons with marine fishes. Probable functions of tissue channels in Octopus and mammals, and reasons for apparent similarities and differences in the channel organization of these divergent groups, are discussed.     

An Immunohistochemical Study of Matrix Proteins in the Craniofacial Cartilage in Midterm Human Fetuses

Immunohistochemical localization of collagen types I, II, and X, aggrecan, versican, dentin matrix protein (DMP)-1, martix extracellular phosphoprotein (MEPE) were performed for Meckel’s cartilage, cranial base cartilage, and mandibular condylar cartilage in human midterm fetuses; staining patterns within the condylar cartilage were compared to those within other cartilaginous structures. Mandibular condylar cartilage contained aggrecan; it also had more type I collagen and a thicker hypertrophic cell layer than the other two types of cartilage; these three characteristics are similar to those of the secondary cartilage of rodents. MEPE immunoreactivity was first evident in the cartilage matrix of all types of cartilage in the human fetuses and in Meckel’s cartilage of mice and rats. MEPE immunoreactivity was enhanced in the deep layer of the hypertrophic cell layer and in the cartilaginous core of the bone trabeculae in the primary spongiosa. These results indicated that MEPE is a component of cartilage matrix and may be involved in cartilage mineralization. DMP-1 immunoreactivity first became evident in human bone lacunae walls and canaliculi; this pattern of expression was comparable to the pattern seen in rodents. In addition, chondroid bone was evident in the mandibular (glenoid) fossa of the temporal bone, and it had aggrecan, collagen types I and X, MEPE, and DMP-1 immunoreactivity; these findings indicated that chondroid bone in this region has phenotypic expression indicative of both hypertrophic chondrocytes and osteocytes.Key words: condylar cartilage, human fetus, extracellular matrix, MEPE, DMP-1     

Comparison of protein structure and genomic structure of human, rabbit, and limulus C-reactive proteins: Possible implications for function and evolution

The primary structures of human, rabbit, and Limulus C-reactive proteins (CRPs) have been compared by a computer program. Based on these data, a PAMs matrix (accepted point mutation per 100 residues) was constructed to generate topologies for the three proteins. Five trees with the shortest absolute length were generated, but only one positive tree was found. Using the relatively well-established distance between human and rabbit of 150 million years, we calculate that human and Limulus CRPs diverged at least 500 million years ago. The data indicate that the amino acid sequence indentity between Limulus CRPs and their mammalian counterparts is about 25%, strongly suggesting that human CRP, rabbit CRP, and Limulus CRPs share common ancestral genes. There are two highly conserved regions in the primary structures among the CRPs. Residues 52–67 in Limulus CRP and residues 51–66 in human CRP show identity in 10 of 16 positions, with 3 additional conservative replacements. This region of the molecule is thought to be involved in the binding of phosphorylcholine ligand. Residues 139–153 in Limulus CRP and residues 133–147 in human CRP show identity in 9 of 15 positions, with 5 additional conservative replacements. The biological function of this stretch of amino acid sequence is thought to be associated with the CA²⁺ binding of the CRPs.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Ligand displacement reactions of oxyhemocyanin: comparison of reactivities of arthropods and molluscs.

The ligand displacement reactions of oxyhemocyanin have been compared over a series of arthropods and molluscs. The arthropods (with the exception of $\text{Limulus}$) are found to be more reactive than the molluscs, (k_cancer = .04 hr^?1, k_busycon = .002 hr^?1, k_limulus ? 10^?4hr^?1 N₃^? reactions). Correlation of the spectral properties of the oxy sites require these to be extremely similar with small differences being associated with shifts in the dd transition energies. The met produced by ligand displacement contains variable amounts of EPR detectable, group 2 exogenous ligand damaged sites (arthropods 25–35%, molluscs 3–9%, $\text{Limulus}$ <1%). A parallel (arthropod > mollusc ～ $\text{Limulus}$ ～ 0) group 2 ligand damaged site is also reported for the half met derivatives.     

The effects of Clostridium histolyticum collagenase on amino sugar-containing compound-collagen complexes

Summary Clostridium histolyticum collagenase has been used on fetal cartilage and bone in an effort to determine its effects on amino sugar-containing-compound collagen complexes. After enzyme treatment it has been found that the staining for acid glycosaminoglycans and glycoproteins in cartilage was abolished only after previous hyaluromdase digestion. The interaction dye-substrate in bone was, instead, readily suppressed after collagenase treatment. These findings suggest a complex formation between some amino sugar-containing compounds and collagen.Supported by Research Grant DE-01952 (04) of the National Institutes of Public Health, Bethesda, Md.     

Akute Vitamin A-Intoxikation der Maus

Summary Isolated fixed liver nuclei of Rana pipiens were stained at varied pH by the acid dye, fast green FCF. The amount of nuclear protein, as determined by cytophotometric measurement of the relative absorption of the stained nuclei, was found to increase in proportion to nuclear size when the pH of the dye solution was below 4.0. Relative absorption was found to be independent of nuclear size, but proportional to the pH of the staining solutions in the pH range between 5.0 and 7.0, These data suggest a change in composition and/or structure of the nuclear protein with increasing nuclear size.This investigation was supported by a grant from the U.S. Public Health Service, GM-10003-04.Post-Doctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, NIH-GM-1142-03.Supported by a Career Development Award from the National Institute of Child Health and Human Development, K3-HD-6176-04.     

Mapping of neuropeptide Y expression in Octopus brains

Neuropeptide Y (NPY) is an evolutionarily conserved neurosecretory molecule implicated in a diverse complement of functions across taxa and in regulating feeding behavior and reproductive maturation in Octopus. However, little is known about the precise molecular circuitry of NPY-mediated behaviors and physiological processes, which likely involve a complex interaction of multiple signal molecules in specific brain regions. Here, we examined the expression of NPY throughout the Octopus central nervous system. The sequence analysis of Octopus NPY precursor confirmed the presence of both, signal peptide and putative active peptides, which are highly conserved across bilaterians. In situ hybridization revealed distinct expression of NPY in specialized compartments, including potential “integration centers,” where visual, tactile, and other behavioral circuitries converge. These centers integrating separate circuits may maintain and modulate learning and memory or other behaviors not yet attributed to NPY-dependent modulation in Octopus. Extrasomatic localization of NPY mRNA in the neurites of specific neuron populations in the brain suggests a potential demand for immediate translation at synapses and a crucial temporal role for NPY in these cell populations. We also documented the presence of NPY mRNA in a small cell population in the olfactory lobe, which is a component of the Octopus feeding and reproductive control centers. However, the molecular mapping of NPY expression only partially overlapped with that produced by immunohistochemistry in previous studies. Our study provides a precise molecular map of NPY mRNA expression that can be used to design and test future hypotheses about molecular signaling in various Octopus behaviors.     

Über Farbwechsel und Farbensinn von Cephalopoden

Zusammenfassung Sepia und Octopus passen sich in Helligkeit und Farbton, in gewissem Maß auch in dem Helligkeitsmuster an ihre Umgebung an. Bei Sepia unterscheidet sich Farbton und Grauverhüllung auf unbunten Helligkeiten stark von den Farbtönen und Verhüllungsgraden auf bunten Untergründen. Die Farbtöne der Sepien auf blauen und grünen Untergründen weichen in entgegengesetztem Sinn von der Färbung der Sepien auf unbunten Untergründen ab wie auf gelben und roten Untergründen. Die Färbung der Sepien ist auf den bunten Untergründen gesättigter (weniger grauverhüllt) als auf unbunten Untergründen verschiedener Helligkeit.Bei Octopus sind die Gegensätze nicht so ausgesprochen, doch ist das Aussehen von Octopus in blauer und in roter Umgebung ebenso gesichert von der Erscheinung in unbunter Umgebung im selben Sinne wie bei Sepia verschieden.Die verschiedene Färbung der Haut wird bei Sepia und Octopus durch ein System von schwarzen, gelben und orangefarbigen Chromatophoren und von Iridozyten (Reflektorzellen) bewirkt. Einer Umgebung, die kurzwelliges Licht blauer und grüner Bereich) zurückwirft, bleiben die bunten Chromatophoren mehr kontrahiert als in einer Umgebung, die langwelliges Licht reflektiert (gelber und roter Bereich). In roter Umgebung werden die orangefarbigen Chromatophoren maximal ausgebreitet.Diese Farbenanpassungen beweisen, daß von Sepia und Octopus Lichter verschiedener Wellenlänge nicht nur nach ihrem Helligkeitswert unterschieden werden. Das wird auch durch die Dressurversuche an Octopus bestätigt.In einer aus weißen und schwarzen Feldern bestehenden Umgebung wird Octopus meist unregelmäßig gefleckt.     

The Mitochondrial Sequences of Heptathela hangzhouensis and Ornithoctonus huwena Reveal Unique Gene Arrangements and Atypical tRNAs

We have sequenced the complete mitochondrial genomes of the spiders Heptathela hangzhouensis and Ornithoctonus huwena. Both genomes encode 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. H. hangzhouensis, a species of the suborder Mesothelae and a representative of the most basal clade of Araneae, possesses a gene order identical to that of Limulus polyphemus of Xiphosura. On the other hand, O. huwena, a representative of suborder Opisthothelae, infraorder Mygalomorphae, was found to have seven tRNA genes positioned differently from those of Limulus. The rrnL–trnL1–nad1 arrangement shared by the araneomorph families Salticidae, Nesticidae, and Linyphiidae and the mygalomorph family Theraphosidae is a putative synapomorphy joining the mygalomorph with the araneomorph. Between the two species examined, base compositions also differ significantly. The lengths of most protein-coding genes in H. hangzhouensis and O. huwena mtDNA are either identical to or slightly shorter than their Limulus counterparts. Usage of initiation and termination codons in these protein-coding genes seems to follow patterns conserved among most arthropod and some other metazoan mitochondrial genomes. The sequences of the 3 ends of rrnS and rrnL in the two species are similar to those reported for Limulus, and the entire genes are shortened by about 100–250 nucleotides with respect to Limulus. The lengths of most tRNA genes from the two species are distinctly shorter than those of Limulus and the sequences reveal unusual inferred tRNA secondary structures. Our finding provides new molecular evidence supporting that the suborder Mesothelae is basal to opisthothelids.Reviewing Editor Dr. Rafael Zardoya     

Acidic glycosaminoglycans in developing sterno-costal cartilage of the hydrocephalic (ch+/ch+) mouse

The sterno-costal cartilage of the hydrocephalic mouse carrying the autosomal recessive gene (ch+/ch+) has 40 ± 3% of the acidic glycosaminoglycan concentration of the normal control containing the satin marker (+sa/+sa). The acidic glycosaminoglycan concentration of the sterno-costal cartilage in the heterozygous mouse (ch+/+sa) is significantly higher (114 ± 8%) than the normal control. The distribution of the acidic glycosaminoglycans in the sterno-costal cartilage is similar in the normal, heterozygous and homozygous mice at all stages of development studied, (prenatal, newborn and postnatal) being 78 ± 4% chondroitin 4(6)-sulfate and 22% hyaluronic acid and/or keratan sulfate. The concentration of acidic glycosaminoglycans in the sterno-costal cartilage decreases as development progresses in all three gene types of mice. The reduced level of acidic glycosaminoglycans in the sterno-costal cartilage of the autosomal recessive mouse, ch+/ch+, is associated with a defect in the formation of the sternum. The higher than normal acidic glycosaminoglycan concentration in the sterno-costal cartilage of the heterozygous mouse ch+/+sa is associated with delayed calcification of the sternum. This study characterizes the molecular locus of a defect in the extra-cellular matrix of a mouse carrying a lethal gene and may help in understanding proteoglycan disorders (mucopolysaccharidosis) in the human.     

Karyometric and cytophotometric study of hepatocyte nuclei of frogs exposed to cold and prolonged starvation

Summary Prolonged starvation of Rana pipiens at room temperature results in a decrease in the volume of the hepatocyte nuclei from that of recently fed individuals. A converse increase in the volume of the liver nuclei over the normal was noted in frogs exposed to low temperature for 10 days. The present work was conducted in an effort to determine which nuclear fraction is responsible for the observed changes in nuclear size.The amount of DNA, RNA and protein-bound sulfhydryl groups per nucleus was measured cytophotometrically and was found to be independent of changes in nuclear size. Total nuclear protein content, on the other hand, was found to vary in proportion to nuclear volume. It was concluded that the variation in nuclear size resulting from starvation and cold-treatment is due to differences in non-chromosomal protein content and cannot be attributed to changes in ploidy.Dedicated to Prof. Berta Scharrer in honor of her 60th Birthday. — Supported by U.S. Public Health Service Grant HD-1499-04 to Dr. Cowden.Work completed while a Postdoctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, NIH-GM-1142-03.Supported by a Career Development Award from the National Institute of Child Health and Human Development, K 3-HD-6176-04.     

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Lectin and other histochemical studies of the articular cartilage and the chondro-osseous junction of the normal human knee joint

There are few studies on normal, adult diarthrodial joints which look in detail at the histochemical properties of the chondro-osseous junctional region. This study of the normal human knee joint was performed using lectin and other histochemical techniques. There were differences in the reactions of mineralised cartilage compared to those of hyaline cartilage with the former demonstrating more collagen and less glycosaminoglycans. Lectin histochemistry revealed more accessible terminal 2-deoxy,2-acetamido-α-d-galactose and more N-acetyllactosamine but less fucosyl and α-2,6-linked-sialyl termini in the mineralised cartilage. The hyaline cartilage chondrocytes stained for N-glycans but those of mineralised cartilage did not. The staining patterns of prolongations and islands of uncalcified cartilage running through the calcified layer to abut bone and marrow spaces were distinct, resembling the patterns of the hyaline cartilage but with some unique features. A possible relationship was revealed between the presence of the Maclura pomifera ligand (Galβ1,3GalNAcα1-) and mineralisation. Subchondral bone had a markedly restricted glycoprofile.     

Involvement of ATP,increase of intracellular calcium and the early expression of <Emphasis Type="Italic">c-fos</Emphasis> in the repair of rat fetal articular cartilage

To compare the potential of adult and fetal animals to repair articular cartilage, we investigated the early process after creating superficial defects in the femoral knee cartilage in rat models. In fetuses at 19 days of gestation, both chondrocytes and the extracellular matrix responded notably by 48 h after artificial injury. Staining patterns with safranin O revealed that, by 1 h after injury, some components of the extracellular matrix around the wound were modified, and the change spread from the limited region to the entire knee cartilage within 24 h. The chondrocytes in the area surrounding the wound transiently expressed increased level of c-fos from 1 h to 6 h. The wound remained 1 day after birth, i.e., 72 h after injury, but was completely repaired 10 days after birth. In contrast, neither visible responses nor transient c-fos expression was observed in 12-week-old adult articular cartilage 48 h after injury. We also examined the relationships between the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the induction of c-fos expression in the cartilage. Applications of ATP or Ca²⁺ ionophore A23187, both of which increase [Ca²⁺]_i, induced immediate expression of c-fos in primary cultured chondrocytes: 1 M ATP elicited an increase of [Ca²⁺]_i in chondrocytes in fetal cartilage slices, but 1 mM was required in adult cartilage slices. Our findings show the presence of a signaling pathway that is apparently active in the repair of fetal but not adult articular cartilage and that involves the intercellular transfer of ATP, increase of [Ca²⁺]_i, and expression of c-fos in cartilage.This study was supported in part by Health Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology to M.O. from the Japanese Ministry of Health, Labor and Welfare