Chondroitin/dermatan sulphate promotes the survival of neurons from rat embryonic neocortex

Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.     

Effects of estrogen on gene expression in the chick oviduct. Isolation and fractionation of chromatin non-histone proteins

Non-histones isolated from hen oviduct chromatin have been fractionated by a variety of methods. Chromatin was dissociated in 2 M NaC1, 5 M Urea, 0.1% beta-mercaptoethanol and 0.01 M Tris - HC1, pH 8.3, and the DNA removed by ultracentrifugation. After desalting by gel filtration the chromatin proteins were separated into three distinct fractions by stepwise elution with 0.10 M NaC1, 0.25 M Na C1 and 15% guanidine - HC1 from Bio-Rex 70 columns. Fractions I and II contain only non-histones and Fraction III contains histones plus a small amount of non-histones. Further fractionation of the non-histones was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography for Fraction I and phosphocellulose chromatography and gel filtration on Bio-Gel A-15 m for Fraction II. The histone and non-histones present in Fraction III were separated by gel filtration on Bio-Gel A-0.5 m. All fractionation methods have been used preparatively with reasonable recoveries of protein (greater than or equal to 60%). The fractions have been characterized by acrylamide gel electrophoresis. The integrity of the histones was maintained during the fractionation procedure indicating that proteolytic degradation was unlikely to have occurred. There was no selective loss of chromatin proteins during the ultracentrifugation and desalting steps and the non-histones were separated into distinct fractions with enrichment of some species not apparent prior to fractionation of the chromatin proteins.     

Low molecular weight dermatan sulfate as an antithrombotic agent. Structure-activity relationship studies

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.     

Decorin and glycosaminoglycan synthesis in skin fibroblasts from patients with systemic sclerosis

We investigated the expression of decorin and the synthesis of sulphated glycosaminoglycans (GAGs) in cultured fibroblasts from patients with early-stage systemic sclerosis (SSc). Decorin mRNA levels were 1.8-fold higher in SSc fibroblasts than in control fibroblasts. SSc fibroblasts also produced 2.3-fold more decorin core protein and 2.2-fold more sulphated GAGs including dermatan sulphate and chondroitin sulphate. Newly synthesized GAGs, in the presence of p-nitrophenyl beta-xylopyranoside, which elongates dermatan sulphate and chondroitin sulphate as an initiator, were increased tenfold and were mainly composed of dermatan sulphate and chondroitin sulphate. The rate of stimulation by the beta-xyloside was similar in SSc and control fibroblasts. These results suggest that the increased amount of dermatan/chondroitin sulphate in SSc fibroblasts reflects an enhanced expression of decorin core protein. Type I collagen mRNA levels in SSc fibroblasts were also increased together with its synthesis. Therefore, our results indicate that an altered decorin and collagen production may affect the organization of collagen fibres and the fibrotic process observed in patients with SSc.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography

The development of fibrocartilage in rat lumbar intervertebral discs has been correlated with an immunohistochemical analysis of the changing distribution of extracellular matrix components. Disc anlagen were first recognised by embryonic day 14 as segmental cell condensations. By E16, the notochord formed a series of bulges, each representing a future nucleus pulposus, and the annulus fibrosus had differentiated in the disc anlagen. The inner part of the annulus was composed of cartilage which linked that of adjacent vertebral bodies. The outer part was fibroblastic, with layers of parallel fibroblasts. The long axes of the cells in successive layers lay at an angle of approximately 90 degrees to each other. This criss-cross orientation of cells preceded the oriented deposition of collagen fibres to form the lamellae. Disc anlagen were immunolabelled weakly for types I and III collagen, chondroitin 6-sulphate and dermatan sulphate. Later tissue differentiation was marked by the appearance of type II collagen, chondroitin 4-sulphate and keratan sulphate in the inner annulus. These components also appeared in the outer annulus, but only in adult animals, and indicated metaplastic change in the lamellar fibroblasts. Fibrocartilage in the nucleus pulposus was only seen in old animals, and the origin of the tissue was less clear. However, the fibrocartilage cells appeared to be derived from the cartilage end plate and/or from the inner annulus. We conclude that fibrocartilage in the intervertebral disc is derived from several sources and that the radial distribution patterns of extracellular matrix components in the adult disc are explained by the embryonic origins of its parts.     

Risk factors for premature birth. I. Analysis of selected factors in a group of non-working women

The distribution of glycosaminoglycans in the extracellular matrix of human cementum was investigated in periodontally involved and periodontal disease-free teeth separated into eight different age groups (from 12 to 90 years), to investigate possible changes in the distribution of glycosaminoglycan species associated with ageing and periodontal disease. A standard indirect immunoperoxidase technique was used, with a panel of monoclonal antibodies, 2B6, 3B3, 5D4, and 7D4, that recognize epitopes in chondroitin-4-sulphate/dermatan sulphate (C-4S/DS), chondroitin-6-sulphate (C-6S), keratan sulphate (KS) and a novel sulphated chondroitin sulphate (CS) epitope, respectively. Intense positive staining for C4-S/DS was observed at the margins and lumina of almost all the lacunae and canaliculi in cellular cementum in all sections. Immunoreactivity to C6-S, KS and novel CS epitopes was limited to a proportion of lacunae and canaliculi in all sections, although C6-S and the novel CS epitopes were more widely distributed than KS. In acellular cementum, there was no demonstrable staining for any of the glycosaminoglycans except where periodontal ligament (Sharpey's) fibres insert; periodontal ligament fibres inserting in cellular cementum also demonstrated positive immunoreactivity. In addition, the cementoblasts on the outer root surface, as well as the pericellular areas around a proportion of these cells, demonstrated positive immunoreactivity. These results indicate that glycosaminoglycan species present in human cementum include C4-S, DS, C6-S, and novel sulphated CS epitopes. KS is also present in cementum but is limited to a more restricted proportion of lacunae and canaliculi. Regional differences in the distribution of glycosaminoglycans exist between the two cementum types, but no qualitative differences in that distribution were observed between the various age groups or between periodontally involved and periodontal disease-free teeth. The immunoreactivity observed in a proportion of lacunae after staining for C6-S, KS, and novel sulphated CS epitopes could suggest the existence of different cementocyte subpopulations.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Mode of reconstitution of chicken erythrocyte and reticulocyte chromatin

The mode of reconstitution of chicken erythrocyte and reticulocyte chromatin has been investigated. Chromatin was dissociated in 2 M NaCl, 5 M urea, and 0.01 M potassium phosphate (pH 7.2) and was dialyzed against various NaCl concentrations in 5 M urea and 0.01 M potassium phosphate (pH 7.2). Histone reassociation to DNA occurs with the binding of histone H5 at 0.5 M NaCl in 5 M urea, followed by histone H1 at 0.4 M NaCl in 5 M urea. All the classes of histones are reassociated with DNA at 0.2 M NaCl in 5 M urea and binding of all classes of histones is complete in 0.1 M NaCl and 5 M urea. Nonhistone proteins reassociate with DNA before and at the same time that histones reassociate with DNA. Binding of nonhistone proteins to DNA appears to be complete in 5 M urea and 0.01 M potassium phosphate (pH 7.2). There is also found in both erythrocyte and reticulocyte chromatin a nonhistone protein present in relatively high concentrations, which remains associated with DNA in 2 M NaCl and 5 M urea. This tightly bound protein appears as one major band when chromatographed on sodium dodecyl sulfate-polyacrylamide gels, with a molecular weight of 95 000. This protein is soluble in phenol and sodium dodecyl sulfate but is insoluble in 5 M urea or 4 M guanidine hydrochloride. A fraction of reticulocyte nonhistone proteins was found to bind to DNA-cellulose in 5 M urea. The majority of these proteins elute at 0.15 M NaCl in 5 M urea but a significant fraction elutes at NaCl concentrations at which the bulk of the histones do not bind to DNA. The proteins that bind to free DNA have low molecular weights and do not show species speciificity. Approximatley 50% of the reticulocyte nonhistone protein does not bind to a DNA-cellulose column in 5 M urea and may require histones for complete reassociation.     

Effects of glycosaminoglycans on the development of in vitro-matured and -fertilized porcine oocytes to the blastocyst stage in vitro

We examined the effects of four glycosaminoglycans (GAGs) on the development of in vitro-matured (IVM) and -fertilized (IVF) porcine oocytes to the blastocyst stage. IVM and IVF oocytes were cultured in Whitten's medium supplemented with hyaluronic acid, chondroitin sulfate A, dermatan sulfate, or heparin at 38.5 degrees C in an atmosphere of 5% CO2 in humidified air for up to 6 days. After 2 days in culture, 28-34% of the inseminated oocytes cleaved to the 2- to 8-cell stage, and the GAGs showed no significant effect on development. After 6 days in culture, blastocysts were observed in all groups. The percentage of blastocysts was significantly higher in hyaluronic acid-supplemented medium (14%) than in dermatan sulfate-supplemented (5%), heparin-supplemented (2%), or nonsupplemented (2%) media. In addition, the percentage of blastocysts was significantly higher in chondroitin sulfate A-supplemented medium (11%) than in heparin-supplemented and nonsupplemented media, although the number of blastocysts in chondroitin sulfate A was not significantly different from that in hyaluronic acid- and dermatan sulfate-supplemented media. There were no significant differences in the mean number of nuclei per blastocyst cultured in any group. The effects of hyaluronic acid and chondroitin sulfate A on development to the blastocyst stage was examined at various concentrations. After 6 days in culture, development of IVM and IVF oocytes to the blastocyst stage was best supported in 0.5 mg/ml hyaluronic acid-supplemented (17%) and in 0.1 or 0.5 mg/ml chondroitin sulfate A-supplemented (10% or 9%, respectively) media. It is concluded from these results that hyaluronic acid and chondroitin sulfate A supported the development of porcine oocytes matured and fertilized in vitro to the blastocyst stage.     

Sulphogalactolipid sulphohydrolase activity of arylsulphatase purified from a marine gastropod Charonia lampas

Sulphatide, cerebroside 3-sulphate was hydrolyzed at a considerable rate by arylsulphatase (aryl-sulphate sulphohydrolase, EC 3.1.6.1) purified from a marine gastropod, Charonia lampas. However, it was scarcely hydrolyzed by glycosulphatase (sugar-sulphate sulphohydrolase, EC 3.1.6.3) from the same origin. The same was observed with seminolipid, a sulphoglycerogalactolipid. The enzymatic characteristics of both sulphogalactolipid and sulphohydrolase activities of the arylsulphatase were determined as follows. The enzyme activities are stimulated by the addition of sodium taurodeoxycholate and MnCl2. The pH optimum of sulphatide sulphohydrolase activity was pH 5.0, while seminolipid sulphohydrolase activity had maximum activity at pH 5.5. Both of these pH versus activity curves were broad. The Km value was 6.22-10-5 M for both substrates. However, the V values were sulphatide were lower by a factor of one-third than those with seminolipid. These enzyme activities were inhibited by substrates of the arysulphatase, i.e., p-nitrophenyl sulphate, p-nitrocatechol sulphate, ascorbate 2-sulphate and each other sulphogalactolipid, but not by glucose 6-sulphate. Sulphate and phosphate anions inhibited both of the enzyme activities.     

Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients

During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.     

Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell

The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.     

Modifications of proteoglycans secreted into the growth medium by young and senescent human skin fibroblasts

The properties of proteoglycans (PGs) secreted into the growth medium by normal young and senescent human skin fibroblasts (HFs) were investigated. In both cases, the incorporation per cell of radioactive precursors into total PGs was similar. The polysaccharide chains of PGs from young and senescent HFs were mainly represented by galactosaminoglycuronans and showed a similar range of size distribution. However, galactosaminoglycuronans of PGs secreted by senescent HFs had a lower content of unsulphated disaccharides and a lower proportion of D-glucuronosyl residues. Moreover, senescent HFs released into the growth medium higher relative amounts of small PGs with chondroitin sulphate, dermatan sulphate chains, such as decorin.     

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase

1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.     

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.     

Behavior of leucine aminopeptidase from bovine eye lens in guanidine hydrochloride. Dissociation and reassociation

The dissociation and reassociation behavior of the hexameric leucine aminopeptidase (LAP) in solutions of 0.5 to 6 M guanidine-HC1 (Gu-HC1) was investigated by means of thin layer chromatography on Sephadex G-200 superfine. Up to 0.5 M Gu - hc1 the hexameric LAP-structure remains intact. In 0.75 to 2 M Gu - HC1 the enzyme dissociates nearly completely into its half-molecules (LAP-trimers, MW 160000 +/- 10,000). In 2.5 and 2.75 M Gu - HC1 a mixture of LAP-monomers (subunits) (MW 60000 +/- 5000) and trimers was to be found. Treatment with beta-mercapto ethanol increases the portion of monomers. Only monomers occurred in 3 to 6 M Gu - HC1. Parallel to the dissociation, a loss of the essential zinc (half life: 10.5 and 3.5 hours in 1 M and 4 M Gu - HC1 resp.) and a decrease of the enzymatic activity (30 min after treatment with 1 M and 4 M Gu - HC1 50% and 1% resp. residual activity) of the observed. On the other hand, the enzyme was activated by dilute solutions of Gu - HC1 (0.1 to 0.3 M: max. increase 50%). Removal of the denaturant causes reassociation of all types of fragments to hexameric LAP which was indistinguishable from the native LAP in Ouchterlony immune diffusion test. The reassociation was accompanied by a small increase in activity only.     

Affinity purification and some molecular properties of human liver alkaline phosphatase

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy

A recently developed chitosan-EDTA conjugate, neutralized with sodium hydroxide (NaChito-EDTA), has been tested for possible topical use. The technical properties and microbial stability of NaChito-EDTA have been compared with those of carmellose sodium (NaCMC), hydroxypropylmethylcellulose (HPMC), sodium polycarbophil (NaPCP) and sodium carbopol 980 (NaC980), well established gelatinizing agents. NaChito-EDTA forms stable, colourless, completely transparent hydrogels at a polymer concentration of 0.5%. Of the polymers tested the novel polymer had the lowest incompatibility with multivalent cations and with ethanol, and much the best swelling properties. After 28 days of incubation at room temperature the rates of growth of the complete bacterial spectrum occurring in demineralized water and of Escherichia coli, serving as model strain representative of gram-negative bacteria, were at least 2 log and 5.7 log, respectively, lower in NaChito-EDTA gels than in the other hydrogels. This antimicrobial activity of NaChito-EDTA can be explained by its highest binding affinity towards magnesium, which stabilizes the outer membrane of gram-negative bacteria. However, this antimicrobial effect is insufficient to guarantee microbial stability. Further results showed that the antimicrobially acting polypeptide nisin can be recommended as an alternative novel preservative for NaChito-EDTA gels, because its antimicrobial spectrum could also be increased towards gram-negative bacteria in combination with chelating excipients. NaChito-EDTA seems, therefore, to be a promising novel polymer for topically-used gels, with advantages over well established gelatinizing agents.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.