The role of proteoglycan in the development of sea urchins : I. Abnormal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis

The development of the sea urchins, Hemicentrotus pulcherrimus and Clypeaster japonicus, was examined under conditions which inhibit the synthesis of proteoglycan in various ways. The changes in the developmental fate were compared with the degree of inhibition of proteoglycan synthesis as measured by incorporation of labeled precursors. The inhibition of proteoglycan synthesis was achieved by treating the embryos with (1) aryl-β- -xyloside (inhibition of coupling between protein and polysaccharide moieties); (2) sodium selenate (inhibition of sulfation of polysaccharide); and (3) 2-deoxy- -glucose (inhibition of polysaccharide chain elongation). It was found that the development of embryos ceased at the blastula stage in all cases tested, irrespective of the step of the inhibition. The cessation of development became apparent when the incorporation of the labeled precursors was decreased to a level below 50% compared with the normal level. From these facts, it appears that the synthesis of proteoglycan is necessary for the realization of post-gastrular development.     

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.     

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle‐stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low‐voltage‐activated calcium channel blocker verapamil on both [³⁵S]‐sulfate and [³H]‐glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20‐day‐old rats. Verapamil induced a dose‐ and time‐dependent decrease in labeling of both secreted and cell‐associated proteoglycans, as determined by quantitative solid‐phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse‐chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP‐dependent syntheses such as estradiol production. J. Cell. Biochem. 76:322–331, 1999. © 1999 Wiley‐Liss, Inc.     

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve–muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic cluster of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline–treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline–inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve–muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve-and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve–muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted. © 1995 John Wiley & Sons, Inc.     

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33^oC for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tail-bud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37^oC for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximidetreated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development. © 1995 Wiley-Liss, Inc.     

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan

Primary mesenchyme cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous beta-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchyme cells of a very large, cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mesenchyme cell migration since treatment of actively migrating cells with chondroitinase ABC reversibly inhibited their migration in vitro.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E₂ and F₂α, 6 keto PGF₁α and thromboxane B₂ by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10⁻⁷M – 10⁻³M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E₂ and F₂, and to a lesser extent, 6 keto PGF₁α, but T×B₂ production was only slightly inhibited by the drug in the absenced of arachidonic acid and markedly increased in its presence. Colchicine (10⁻⁷M – 10⁻³M) had the opposite effect, causing an inhibition of T×B₂ and stimulating PGE₂ and 6 keto PGF₁α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A₂ and cyclo-oxygenase, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using ³⁵SO₄ labeling methodology. The results showed that the concentrations tested (10⁻⁵M to 10⁻⁷M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited ³⁵SO₄ incorporation into newly synthesized proteoglycan molecules both in the presence (10⁻⁶M) and absence of exogenous arachidonic acid. In the same concentration range choroquine had no effect.These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Long-term cyclical in vivo loading increases cartilage proteoglycan content in a spatially specific manner: an infrared microspectroscopic imaging and polarized light microscopy study

Understanding the changes in collagen and proteoglycan content of cartilage due to physical forces is necessary for progress in treating joint disorders, including those due to overuse. Physical forces in the chondrocyte environment can affect the cellular processes involved in the biosynthesis of extracellular matrix. In turn, the biomechanical properties of cartilage depend on its collagen and proteoglycan content. To understand changes due to physical forces, this study examined the effect of 80 cumulative hours of in vivo cyclical joint loading on the cartilage content of proteoglycan and collagen in the rabbit metacarpophalangeal joint. The forepaw digits of six anesthetized New Zealand White adult female rabbits were repetitively flexed at 1 Hz with an estimated joint contact pressure of 1 to 2 MPa. Joints were collected from loaded and contralateral control specimens, fixed, decalcified, embedded, and thin-sectioned. Sections were examined under polarized light microscopy to identify and measure superficial and mid zone thicknesses of cartilage. Fourier Transform Infrared microspectroscopy was used to measure proteoglycan and collagen contents in the superficial, mid, and deep zones. Loading led to an increase in proteoglycan in the cartilage of all six rabbits. Specifically, there was a 46% increase in the cartilage deep zone (p = 0.003). The collagen content did not change with loading. Joint loading did not change the superficial and mid zone mean thicknesses. We conclude that long-term (80 cumulative hours) cyclical in vivo joint loading stimulates proteoglycan synthesis. Furthermore, stimulation is localized to cartilage regions of high hydrostatic pressure. These data may be useful in developing interventions to prevent overuse injuries or in developing therapies to improve joint function.     

Heteromorphism for chromosome 1, a requirement for normal development in crested newts

In all adult males and females of T. cristatus most of the long arm of chromosome 1 is achiasmate and heteromorphic with respect to Giemsa C-staining pattern. This heteromorphism correlates well with the heteromorphism seen by previous investigators on the lampbrush chromosomes of adult females. The heteromorphism has nothing to do with sex determination. All those fertile eggs of T. cristatus that develop normally beyond blastula are capable of development through to late tail-bud stage, but 50% of them arrest at late tail-bud and ultimately die. Homomorphism for the long arm of chromosome 1 can only be found in those embryos that arrest at late tailbud. The same findings apply to T. marmoratus, which also shows heteromorphism for chromosome 1, but not to T. alpestris, which has homomorphic chromosomes 1 and a potential 100% developmental success rate.     

Maturation of urinary proteoglycan excretion

Urinary proteoglycan excretion was studied using two newly established methods in subjects aged between 1 and 22 years. Analysis of glycan moieties showed an age-dependent decrease from 9.1±5.5 (SD) g/mol creatinine (n=5) at the age 1–6 years to 1.9±1.3 (n=5,P<0.01) in those aged 16–22 years. Marked qualitative changes in the proteoglycan electrophoretic pattern occurred during the first and second years of life. Two major proteoglycan bands with a molecular weight of 50 kDa decreased in intensity so that the pattern resembled the adult configuration after 6 years of age. The latter consisted of a major band with a molecular weight of 80–100 kDa, the bands corresponding to a molecular weight of 50 kDa and lighter bands of molecular weight around 32 kDa. These changes may be related to functional maturation of the kidney as a whole and to an increase in the number of mature nephrons.     

The effects of trypsin treatment on proteoglycan biosynthesis by bovine articular cartilage.

The effects of mild or severe trypsin treatment of bovine articular-cartilage slices in tissue culture were studied by monitoring the incorporation of [35S]sulphate into proteoglycans. Moderate trypsin treatment caused a subsequent marked inhibition of proteoglycan biosynthesis, which was reversible with time. Analysis on Sepharose CL-2B of the proteoglycan species synthesized showed that, directly after trypsin treatment, there was a 30% increase in the synthesis of the low-Mr proteoglycan (Kav. 0.71), and the total decrease in proteoglycan biosynthesis was reflected in a decrease in the synthesis of the high-Mr proteoglycan species (Kav. 0.31). The small proteoglycan was partially characterized and shown to be a true biosynthetic product and not a breakdown product. Trypsin treatment (20 micrograms/ml per 100 mg of tissue) of cartilage slices also resulted in an increase in the glycosaminoglycan chain size of the large proteoglycan, but not of the small proteoglycan.