The distributions of chondroitin sulfates in articular and growth cartilages of human bones

The relative contents of chondroitin 4- and 6-sulfates in cartilages of different human bones are reported. Articular and vertebral body cartilages contain almost exclusively chondroitin 6-sulfate, whereas growth and subarticular cartilages contain nearly equal amounts of chondroitin 4-sulfate and chondroitin 6-sulfate. Adult cartilages, where the calcification process is complete, contain only chondroitin 6-sulfate. These results suggest that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces. A chemical defect of chondroitin 6-sulfate in a new mucopolysaccharidosis, characterized by platyspondyly and irregularities of articular surfaces, is in agreement with these results.     

The distribution of chondroitin sulfates in articular and growth cartilages of human bones.

The relative contents of chondroitin 4- and 6-sulfates in cartilages of different human bones are reported. Articular and vertebral body cartilages contain almost exclusively chondroitin 6-sulfate, whereas growth and subarticular cartilages contain nearly equal amounts of chondroitin 4-sulfate and chondroitin 6-sulfate. Adult cartilages, where the calcification process is complete, contain only chondroitin 6-sulfate. These results that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces. A chemical defect of chondroitin 6-sulfate in a new mucopolysaccharidosis, characterized by platyspondyly and irregularities of articular surfaces, is in agreement with these results.     

Histochemical identification of sulfation position in chondroitin sulfate in various cartilages

The ability of chondrocytes to synthesize chondroitin-4-sulfate (C4S) as opposed to chondroitin-6-sulfate (C6S) is a phylogenetically related phenomenon seen among adult higher vertebrates and developmentally during the embryogenesis of these vertebrates. While the embryonic cartilage may be initially a C6S matrix, C4S synthesis is seen to develop with time. We have histochemically localized these differences in sulfation with the cationic carbocyanine dye, Stains-all, in a spectrum of cartilages that vary in the sulfation position of their chondroitin sulfate. Cartilages from the rat and rabbit that are predominantly C4S stained magenta at pH 4.3, while the C6S-rich cartilage matrices from the regenerating rabbit ear and lamprey cranium stained blue. Embryonic chicken cartilages develop a gradient of magenta matrix with age, with increased concentration toward the articular surface. Both magenta and blue matrices were absent after pretreatment with chondroitinase ABC but were present after Streptomyces hyaluronidase digestion. The magenta staining was a property of the cartilage matrix as a whole, since isolated C4S and C6S stained blue. The differential staining was seen at pH 4.3, but not at pH 8.8, suggesting an interaction between the chondroitin sulfate and the adjacent tissue proteins.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Increase of chondroitin 4-sulfate concentration in the endochondral ossification cartilage of normal dogs

The absolute concentrations of chondroitin 4- and 6-sulfate are compared in articular and endochondral ossification cartilage from normal dogs. In newborn dogs, the absolute concentration of chondroitin 4-sulfate decreases nearly 3-fold from the deeper endochondral ossification cartilage to the articular surface, whereas that of chondroitin 6-sulfate does not change. In cartilage from the articular surface of the epiphysis in adults, where the ossification process is complete, the concentration of chondroitin 4-sulfate is also low. These observations suggest that chondroitin 4-sulfate may be important in the ossification process. The pathogenesis of heritable disorders involving the sulfation of chondroitin sulfate is discussed in view of these findings.     

Sulfated glycosaminoglycans of human aorta: chondroitin 6-sulfate increase with age.

The proportions of chondroitin 4 and 6 sulfates of intima + media layers of normal human aortae vary with age. The two isomers are in approximately equal amounts in aortae of young individuals, while the 6-sulfate is more abundant in those of adult individuals. This increase of chondroitin 6-sulfate is even more pronounced for intima + media obtained from atherosclerotic aortae.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A terminal 6-sulfotransferase catalyzing a synthesis of N-acetylgalactosamine 4,6-bissulfate residue at the nonreducing terminal position of chondroitin sulfate

A soluble enzyme from quail oviduct which incorporates sulfate into position 6 of the nonreducing N-acetylgalactosamine 4-sulfate end group of chondroitin sulfate has been purified. This enzyme (termed "terminal 6-sulfotransferase") was partially separated from a 6-sulfotransferase present in the same tissue which catalyzes the incorporation of sulfate into interior portion of unsulfated chondroitin. The basic requirements for the terminal 6-sulfotransferase reaction were shown to be 3'-phosphoadenylyl sulfate (donor) and chondroitin 4-sulfate (acceptor). The substitution of unsulfated chondroitin (prepared from squid skin) for chondroitin 4-sulfate resulted in a total loss of activity. These results suggest that the organization of the proteoglycan-synthesizing apparatus may well involve hitherto unrecognized mechanisms for the sulfation of chondroitin chains.     

Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta)

The effect of transforming growth factor-beta (TGF-beta, 1 ng/ml) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of 35S-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity (+58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-beta also enhanced the uptake of [35S]sulfate by chondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-beta on the distribution of PG between the medium and the cell layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell-layer associated GAG of TGF-beta-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PG, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-beta, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor: the proportion of chondroitin 6-sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-beta, leading to a decrease of the ratio C6S/C4S (-11 to -22%, P less than 0.01). The present study indicates that TGF-beta promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Mucopolysaccharide-polypeptide interactions: Effect of the position of the sulfate group

The differences in the interaction in solution of poly(l-lysine) with chondroitin 6-sulfate (chondroitin sulfate C) and with chondroitin 4-sulfate (chondroitin sulfate A) have been studied by circular dichroism spectroscopy. Both mucopolysaccharides force the poly(l-lysine) to adopt the α-helix in solution rather than the charged coil form expected at neutral pH. The observed spectra indicates that the polypeptide is at least 80% helical when the 6-sulfate form is present, but only about 20% α-helical in the presence of chondroitin 4-sulfate. Thus chondroitin66-sulfate has a stronger conformation directing effect on poly(l-lysine) than does the 4-sulfate, which is probably due to the different positions of the sulfate group on the polysaccharide c chain.     

Selective hydrolysis of chondroitin sulfates by hyaluronidase

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans.

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.

Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the D1 preparation contained a single component (SO = 23 S), and the A1 preparation contained 30% monomer (SO = 23 S) and 70 percent aggregate (SO = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the A1 preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The A1 preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in A1 preparations from bovine nasal cartilage. When the A1 preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed.