MUCOPOLYSACCHARIDES SYNTHESIZED BY CULTURED GLIAL CELLS DERIVED FROM A PATIENT WITH SANFILIPPO A SYNDROME

—Glial cells were cultured from brain tissue obtained at autopsy of a patient with Sanfilippo A syndrome. Mucopolysaccharides were labeled by culturing the cells in the presence of [³⁵S]sulfate. After proteolysis, intracellular and media-elaborated mucopolysaccharides were fractionated by Dowex 1 chromatography. One fraction, identified as heparan sulfate by chromatographic, electrophoretic, and enzyme susceptibility properties, accumulated in Sanfilippo glial cells in greater amounts than in controls. Heparan sulfate was also excreted into the culture media by both Sanfilippo and normal cultures, and it constituted a major fraction of the sulfated mucopolysaccharides synthesized by glial cells. Sanfilippo and normal fibroblasts were also included in these studies for comparative purposes. Sanfilippo fibroblasts accumulated significantly increased amounts of heparan sulfate as compared to normal fibroblasts. Heparan sulfate was excreted into the culture media by Sanfilippo and normal fibroblasts in equivalent amounts, but in contrast to glial cells, it was only a minor component of the sulfated mucopolysaccharides produced. Cultured glial cells should provide a useful system for investigating the role of heparan sulfate in glial cell function.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Sulfolipid metabolism in chlorella

When S-deficient cells of Chlorella cllipsoidea were incubated in radio-sulfate in light or in aerobic darkness for 1 hour, equal amounts of radioactivity were found in sulfolipid and glutathione but none was detected in sulfoquinovosyl glycerol which is one of the major S-compounds in this alga. No assimilation of radiosulfate was observed under anaerobic darkness.

To elucidate the function of sulfolipid in algal cells uniformly ³⁵S-labeled Chlorella cells were transferred to S-deficient culture medium or unlabeled normal culture medium and the changes of radioactivity in sulfolipid and the related compounds were followed. A) On incubating ³⁵S-labeled algal cells in S-deficient medium under photosynthetic conditions, the amounts of radioactivity in sulfate, sulfoquinovosyl glycerol and sulfolipid decreased rapidly. B) When ³⁵S-labeled cells were cultured photoautotrophically in unlabeled medium, no decrease of radioactivity was observed in sulfoquinovosyl glycerol and sulfolipid. C) A decrease of ³⁵S-sulfolipid and an increase of ³⁵S-sulfoquinovosyl glycerol were observed when the uniformly ³⁵S-labeled algal cells were illuminated in CO₂-free air.

When S-deficient Chlorella cells were incubated in ³⁵S-sulfolipid under photosynthetic conditions, significant radioactivity was found in the insoluble fraction of the cells. A similar result was observed when normal Chlorella cells were incubated in ¹⁴C-sulfolipid and CO₂-free air.

It is inferred from these observations that sulfolipid is a reservoir of sulfur and carbon compounds.

In order to ascertain if the sulfolipid is involved in the mechanism of photosynthetic oxygen evolution, the rate of photosynthesis was measured during the incubation of ³⁵S-labeled cells in a S-deficient medium. Parallelism was not observed between the rate of photosynthetic activity and the decrease of sulfolipid.

     

A conditioned medium (CM) factor produced by chondrocytes that promotes their own differentiation

This report presents evidence demonstrating that chick embryo chondrocyte cultures release into the culture medium a factor(s) which itself can act on chondrocytes to promote their own differentiation. Conditioned medium (CM) stimulates the synthesis of both sulfated mucopolysaccharides, as shown by increased incorporation of ³⁵SO₄ or glucose-¹⁴C into hyaluronidase-sensitive material, and collagen. However, protein synthesis, DNA synthesis, and cell number are not affected. While the identity of the factor is not yet known, it is nondialyzable, trypsin-and heat-sensitive. The factor is evidently a specialized product of chondrocytes, because it is not made by unexpressed chondrocytes or differentiated pigmented retina cultures. CM works rapidly on test cultures and has a significant effect on ³⁵SO₄ incorporation after 2 hr of treatment. In addition, the effect is relatively stable and is not reversed when CM is replaced with fresh medium. The results are significant in that they demonstrate that chondrocytes produce a factor that promotes their own differentiation, as defined in terms of the synthesis of two distinct specialized products.     

Early evaluation of mixed leukocyte interactions in mice and guinea pigs using measurements of protein synthesis

A rapid microassay for mixed lymphocyte interactions (MLI) in the mouse and the guinea pig has been developed. This assay is based on the stimulation of protein synthesis (SPS) that occurs as soon as 11 hr after initiation of the culture and can be measured with high precision. The cells are cultured in leucine-free medium without serum in microtiter plates in 0.2 ml volumes using 2 × 10⁶ cells of each cell type. Stimulation of protein synthesis as measured by increased incorporation of a pulse of ³H-leucine into protein occurs in allogeneic MLI's in which cells differ at major and minor, as well as at only minor histocompatibility loci, but does not occur in xenogeneic MLI's. Unidirectional MLI's can also be performed using either Xirradiated cells or cells from F₁-hybrid animals. The sum of the two one-way MLI's in each case equals that of the two-way MLI. The advantage of this assay are: (1) it can be completed in less than 24 hr; (2) serum is not required in the culture medium; (3) relatively few cells and small amounts of reagents are required; (4) it is highly reproducible.     

Autoradiographic detection of mucopolysaccharide accumulation in single fibroblasts

Synopsis A method is described for localizing acid mucopolysaccharides autoradiographically in cultured cells. Normal fibroblasts and fibroblasts, from patients suffering from Mucopolysaccharidosis II disease (MPS II), were cultured for six days in the presence of³⁵SO₄ and one day in unlabelled medium. The cultured cells were transferred to a plastic film dish and, after settling, they were rapidly quenched, freeze-dried, fixed in osmium tetroxide vapour and embedded in Epon. Grain counting after autoradiography in 2 m sections revealed a significant difference (P>0.001)iin³⁵SO₄ incorporation in the perinuclear cytoplasm of MPS II cells and control cells grown under the same conditions. Autoradiography was also performed after mixing MPS II cells and control fibroblasts in a ratio 11. 8 prior to freezing and the same ratio was found between labelled and unlabelled fibroblasts. These results demonstrate the feasibility of the present autordiographic technique for the detection of the acid mucopolysaccharide storage at the single cell level.     

Transferrin is an autocrine growth factor secreted by reuber H-35 cells in serum-free culture

Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [³⁵S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59±0.12×10⁵ to 3.35±0.34×10⁵ after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×10⁵ cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [³⁵S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1±1.2 ng of transferrin was secreted per 10⁶ cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin. This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA 16463-14 from The National Institutes of Health, Bethesda, MD.     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Software sensors for the monitoring of perfusion cultures: Evaluation of the hybridoma density and the medium composition from glucose concentration measurements

New software sensors based on the Extended Kalman Filter technique have been developed for the monitoring of animal cell perfusion cultures. They use a kinetic model describing the growth, death and metabolism of hybridoma cells as a function of the medium composition. The model was initially validated on a batch culture and found to correctly predict the continuous perfusion culture kinetics, except for the production of ammonia and lactate. Using the measurement of a single component in the culture medium, in this case glucose, the Extended Kalman Filter provides an excellent evaluation of the time variation of the concentrations of living and dead cells, of glutamine and antibodies, during the whole perfusion culture for a retained cell density rising from 1 to 11×10⁶ cells.ml^–1 inside the reactor.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

BONE FORMATION INDUCED IN MOUSE THIGH BY CULTURED HUMAN CELLS

Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. ³⁵S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible ³⁵S utilization and secretion. FL cells, labeled in vitro with thymidine-³H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.     

Growth modulation and proteoglycan turnover in cultured mesangial cells

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with ³⁵SO₄^2? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (K_d of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (K_d = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with K_d values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (K_d = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular ³⁵S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [³H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G₁/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc.     

An estrogen responsive primary amphibian liver cell culture system

Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [³H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [³H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited.Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation.The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.     

Production of sulfated proteoglycans by human breast cancer cell lines: Binding to fibroblast growth factor-2

The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [³⁵S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (K_av 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (K_av 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind ¹²⁵I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties. J. Cell. Biochem. 64:605–617. © 1997 Wiley-Liss, Inc.     

Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein-Isothiocyanate-Labeled Lectins

In the coccolithophorid microalgae acidic polysaccharides are considered to be involved in the formation of the calcified scale, coccolith. Characteristics of the acidic polysaccharides extracted from the cell surface of the coccolithophorid Pleurochrysis haptonemofera were analyzed. The acidic polysaccharides on the cell surface can be detected by measuring fluorescence of cells after fluorescein-isothiocyanate-labeled lectin staining by flow cytometry. Flow cytometric analyses revealed that the acidic polysaccharides remained on the cell surface even after CaCO₃ in the coccolith was dissolved by lowering pH, but they were extracted by subsequent EDTA or EGTA treatment, suggesting that they are bound not into the CaCO₃ crystals of the coccolith, but onto the surface via Ca²⁺. Analyses of the acidic polysaccharides by anion exchange chromatography, colloidal precipitation with divalent cations, and polyacrylamide gel electrophoresis (PAGE) revealed that P. haptonemofera has 3 types of acidic polysaccharides (Ph-PS-l, -2, and -3). The PAGE patterns suggested that Ph-PS-2 has a repeated structure with a broad range of molecular weight, as in Pleurochrysis carterae, while Ph-PS-1 and -3 contain several minor components in addition to a major component, respectively. The minor components in Ph-PS-1 and -3 that have not been found in P. carterae might be characteristic of P. haptonemofera. Analyses of both the cell surface treated by various concentrations of EDTA and EGTA and the extracts suggested that Ph-PS-2, which is distinguishable by a higher affinity to concanavalin A, is bound onto the coccolith surface more intensely than the other two types of acidic polysaccharides.     

Sugar compositions of extra- and intracellular polysaccharides produced by candida lipolytica (4124) grown on n-hexadecane

The free monosaccharide content of C. lipolytica (strain 4 124) cells grown on n-hexadecane was identified and found to be only glucose. The chromatographic analysis of the hydrolysate of intracellular cell wall polysaccharides indicated the presence of glucose: mannose: galactose: xylose in a ratio of 1 : 1.32 : 1.07 : 0.35. Paper and dise electrophoresis of extracellular polysaccharid from the culture broth was found to be heterogeneous. Ethanol fractionation separated it to a major component F (I) 81.99% and a minor one F (II) 13.04%. Analysis of the major fraction showed that it consisted of galactose and mannose only while the minor polysaccharide consisted of galactose, glucose and mannose. Thus it was concluded that the predominant sugar in both extracellular and intracellular polysaccharides was mannose. Xylose was detected in the intracellular polysaccharide only.     

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[³H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate