The sulphur content of chondrocyte nuclei

Summary The sulphur content as a measure of glycosaminoglycan content of growth plate cartilage was determined by energy dispersive x-ray analysis on fresh freeze dried unstained, unfixed ultra thin sections of rat growth plate. In the resting and proliferative zones, quantities of sulphur were found in the nuclei equal to that of the matrix. Less sulphur was present in the cytoplasm. In areas of cell degeneration, nuclear and cytoplasmic content of sulphur fell to levels a fraction of that seen in the matrix. It was presumed that most of the sulphur was in glycosaminoglycans. Although glycosaminoglycans have been reported in small amounts in the nuclei of cells, no study of the glycosaminoglycan content of chondrocyte nuclei has been reported. The use of freeze dried unstained, unfixed sections presumably prevented the migration of sulphur and glycosaminoglycans from compartment to compartment.Supported by grants from the Medical Research Council and the Shriners of North America     

Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification

Summary This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine, which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.     

Correlation between Alcian Blue staining of glycosaminoglycans of cat nucleus pulposus and TEM X-ray probe microanalysis

Synopsis The nucleus pulposus of cat intervertebral disc was examined after staining glycosaminoglycans with Alcian Blue and the results correlated with TEM X-ray probe microanalysis. In unstained sections a difference in copper levels between tissues and resin was detected. In tissue stained with Alcian blue before embedding, the copper levels were slightly increased and the morphological appearance of the intercellular material was amorphous. In sections restained after cutting, the relative levels of copper in the resin were considerably increased and tissue levels were significantly higher than in the resin. Moreover, the morphology of the intercellular material was altered from a rather amorphous material to a network. Sulphur levels behaved in similar manner to copper levels but any correlation between the elements was due to factors unrelated to glycosaminoglycan staining and probably resulted from contaminating sulphur.     

Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage     

Electron microscopical microprobe analysis of freeze dried and unstained mineralized epiphyseal cartilage

Summary Electron micrographs have been taken of unfixed, freeze dried, unstained epiphyseal cartilage. In the mineralized long septa round to elliptic clusters (up to 0.6 m in diameter) consisting mainly of dots and needles could be observed. The clusters were surrounded by microareas with a low contrast consisting mainly of ribbon plate-like crystallites. With the aid of scanning mode (STEM) of a transmission electron microscope, equipped with a Si(Li)detector system, both regions were analyzed for calcium and phosphorus by electronprobe X-ray microanalysis. In ten series of 106 measurements in each region, it could be determined by registration of the CaK and PKX-ray counts that the mineral content in the clusters was in the range of 30–100 % higher than in the light regions. The question of the sequence of the epiphyseal plate mineralization is discussed and whether the dense clusters represent the mineralized matrix vesicles.The authors thank the Deutsche Forschungsgemeinschaft for financial supportDedicated to Professor Dr. G. Pfefferkorn on the occasion of his 65th birthday     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Localisation of Vanadium, Sulphur and Bromine Within the Vanadocytes of Ascidia mentula Müller: A Quantitative Electron Probe X-Ray Microanalytical Study

Levels of vanadium, sulphur and bromine within the vanadopohores of Ascidia mentula vanadocytes have been determined by quantitative X-ray microanalytical techniques. Values obtained for vanadophore vanadium in thin sections correspond to 2.5-3.25% or 0.5-0.65 molal aqueous solutions in vivo. Quantitation of sulphur in thin sections gives levels an order of magnitude lower than for vanadium, but it is likely that major losses of sulphur have occurred, since analysis of unfixed, air dried vanadocytes gives V/S atomic ratios close to unity. This 1: 1 ratio may have implications for the structure of the vanadium complex. Quantitation of bromine is less reliable, but levels of this halogen in vanadocytes are significant. The possibility that bromine, in the form of brominated metabolites, together with vanadium and acid, contribute to a multicomponent chemical defense role in ascidians is discussed.     

FACTORS INFLUENCING THE FREQUENCY OF MESOSOMES OBSERVED IN FIXED AND UNFIXED CELLS OF STREPTOCOCCUS FAECALIS

Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.     

Accumulation of collagen in ovarian benign tumours

Extracellular matrix components of benign ovarian tumours (cystadenoma, adenofibroma, cystadenofibroma) were analysed. The investigated tumours contained twice as much collagen than control ovarian tissues. Significant alterations in mutual quantitative relationships between collagens of various types were observed. The proportion of type I collagen decreased and that of type III collagen increased. The accumulation of collagen was accompanied by a reduction in sulphated glycosaminoglycan content whereas the amount of hyaluronic acid was not changed. Dermatan sulphate was the most abundant glycosaminoglycan component. It is suggested that the accumulation of collagen (natural barrier to the migration of tumour cells) and underexpression of glycosaminoglycans/proteoglycans (binding some growth factors and interleukins) may exert an inhibitory effect on tumour growth.     

Energy dispersive X-ray microanalysis of sulfated glycosaminoglycans in cartilage matrix stained with alcian blue 8GX

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.     

Energy dispersive X-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments

Summary Isolated cells of matrix fragments of freeze fractured and freeze dried growth plate from the four species was analyzed by EDX. Cells were removed from the tissue by stereoscopic microdissection using an SEM and mounted on thin film supports on TEM grids: this approach eliminated specimen X-ray background and reduced instrumental and support back-ground levels to insignificant proportions. Chondrocyte and matrix fragments were dissected and analyzed. Cell Ca reaches EDX detectable levels in hypertrophic cells close to the mineralization front. At all stages of maturation, the cells exhibit high P; however, matrix Ca levels are elevated before P. This data suggests that the early cartilage matrix is accumulating Ca and that the cells' role in this process may be to elevate the matrix Ca and P concentration. All cells showed clear S peaks although these are reduced in late hypertrophic cells. With mineralization, matrix S levels fall, indicating a loss of sulfated proteoglycans. Matrix before mineralization contains more K than would be expected from data of previous studies. It is suggested that while this K may be bound to fixed anionic sites in the matrix, reported values for cartilage lymph and extracellular fluid should be reviewed.     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures

Synopsis Disaggregated foetal mouse brain tissue cultures were examined for glycosaminoglycans using Alcian Blue and periodic acid-Schiff staining techniques. It was found that spongioblasts (neuron and glial cell precursors) were rich in sulphated glycosaminoglycans, while astrocytes contained little or no sulphated polymers. The chief acid glycosaminoglycans of the brain reportedin vivo, hyaluronic acid, chondroitin sulphate and sialic acid-bearing polymers, were not demonstrated in the mouse brain cultures. There was a decline in glycosaminoglycan content over two weeks in culture, but during the corresponding periodin vivo an increase has been reported. These deficiencies are possibly correlated with the failure of the cultures to myelinate.     

Fetal breathing, sleep state, and cardiovascular responses to graded hypoxia in sheep

We studied changes in glycosaminoglycan content and concentration during postresectional compensatory lung growth in adult male rats. After right trilobectomy, left lung dry weight was normal at 4 days, increased 74% between 4 and 7 days, and more slowly over the next week. Total glycosaminoglycan content per milligram dry lung weight increased early and rapidly, reaching 189% of the control value at 4 days postresection. The magnitude and temporal pattern of increase was different for different glycosaminoglycan subtypes. Hyaluronate and chondroitin sulfate content were increased by 198 and 113%, respectively, at 4 days, with no further increases subsequently. Heparan sulfate content increased more slowly and steadily, and dermatan sulfate concentrations did not change. At 4 days, the percent of total glycosaminoglycans that was hyaluronate was almost doubled, whereas the percent that was heparan sulfate was decreased; by day 7 the percent compositions had returned to normal. We conclude that changes in glycosaminoglycans occur early in postresectional lung growth and speculate that they may play a facilitatory role.     

A STAINING TECHNIQUE FOR AUTORADIOGRAPHS OF NONDIFFUSIBLE ISOTOPES

Nondeparaffinized radioactive tissue sections are stained with hematoxylin and eosin by being floated on aqueous solutions for 1 hr each. The sections are then thoroughly washed, dried and exposed to autoradiographic plates or emulsions for predetermined periods of time. When desirable, both stained and unstained adjacent tissue sections can be mounted on a single slide of autoradiographic plate for exposure. Kodak DK-19 and 30% Na₂S₂O₃.5H₂O solutions are used for subsequent developing and fixing. The finished autoradiographs show excellent resolution and cytologic detail, since the gelatin remains unstained while the tissue retains its stain. Stains other than hematoxylin and eosin can be applied to this technique, provided they withstand the developmental and fixation processes.     

Forskolin- and dibutyryl cyclic AMP-mediated inhibition of chondrogenesis

The regulatory role of cyclic AMP in various cellular activities is well known. It has been documented that both the notochord and extracellular matrix materials (ECM) induce somite chrondrogenesis. We believe that the ECM modulates the intracellular cAMP level during chondrogenic differentiation. The studies indicated that notochordal induction, which resulted in somite chondrogenesis (reflected by increased sulfated glycosaminoglycan synthesis) reduced the intracellular cAMP level in somites. Addition of forskolin and dibutyryl cAMP resulted in increased intracellular cAMP levels and decreased synthesis of sulfated glycosaminoglycans (decreased chondrogenesis). In the case of dibutyryl cAMP, the inhibition of sulfated glycosaminoglycan synthesis was related to the length of exposure time. Thus, the inverse relationship between cAMP content and enhanced chondrogenesis supports the theory that, in somites, a decrease in the intracellular cAMP level may be necessary to trigger chondrogenic differentiation.     

Density-dependent changes in the amount of sulfated glycosaminoglycans associated with mouse 3T3 cells.

The relative amount of sulfated glycosaminoglycans associated with the cell layer of parent and SV40-transformed Swiss mouse 3T3 cells was determined from the incorporation of labeled sulfate (35SO4) into macromolecular material. In cultures of SV40-transformed cells, the glycosaminoglycan content per cell was constant over a wide range of densities. In cultures of parent 3T3 cells, the glycosaminoglycan content per cell increased directly with density, the highest values being found in contact-inhibited cultures. At high cell densities, the glycosaminoglycan content of 3T3 cells was several-fold higher than that for SV40-transformed cells. Most of the density-dependent increase in glycosaminoglycans of 3T3 cells was accounted for by chondroitin sulfate (dermatan sulfate) which was over 6-fold higher in confluent cultures than in low density cultures.     

Measurement of colloidal iron binding at low pH in cartilage using the proton microprobe

Summary Quantitative micro-PIXE analysis was performed on mouse embryo epiphyseal cartilage and on the rib cartilage of mature animals after incubation of sections with colloidal iron at pH 1.8. The iron content as well as that of sulphur and phosphorus and Fe/S, Fe/P ratios were determined. It was found that colloidal iron content was higher in the cartilage than in other tissues. The cartilage also displayed the highest content of sulphur. The Fe/S ratio was however not constant, being highest in the degeneration zone close to the mineralization front, where the binding of iron was strongest while the amount of sulphur decreased. This indicates that factors other than number of sulphate groups influence the binding of positively charged molecules to glycosaminoglycans. This is confirmed by differences in the results obtained for embryonic and mature rib cartilage.     

Glycosaminoglycans bind factor Xa in a Ca2+-dependent fashion and modulate its catalytic activity

Recent studies have demonstrated the existence of a Ca(2+)-dependent heparin-binding site on factor Xa. To characterize this heparin-binding site, the extrinsic fluorescence of fluorescein-labeled, active site-blocked factor Xa was monitored as it was titrated with glycosaminoglycans of various sulfate content and chain length. The binding of glycosaminoglycans to factor Xa appears to be charge-dependent because affinity is correlated with degree of glycosaminoglycan sulfation. All glycosaminoglycans bind factor Xa with higher affinity in the presence of Ca(2+) than in its absence. In contrast, when Gla-domainless factor Xa was substituted for factor Xa, glycosaminoglycans bound with similar affinities in the absence and presence of Ca(2+). These results support the hypothesis that the anionic Gla domain impairs glycosaminoglycan binding in the absence of Ca(2+). The changes in fluorescence intensity of factor Xa when titrated with glycosaminoglycans suggest that glycosaminoglycans induce conformational changes in the active site environment of factor Xa. To explore the consequences of these conformational changes, the effect of glycosaminoglycans on the catalytic activity of factor Xa was examined. Glycosaminoglycans influenced the ability of factor Xa to cleave chromogenic substrates and attenuated the capacity of factor Xa to activate factor VII. The potency of glycosaminoglycans in these assays reflected their affinity for factor Xa. These studies suggest that glycosaminoglycan binding perturbs exosites on the surface of factor Xa, potentially modifying interactions with cofactors or substrates.