Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Interactions between extracellular matrix components and proteoglycans released from monocytesin vitro

³⁵S-labelled chondroitin sulfate proteoglycans isolated from conditioned media of cultured human monocytes (day 1in vitro) and monocyte-derived macrophages (day 6in vitro) were chromatographed on columns of immobilized fibronectin and collagen, respectively. The elution profiles prior to and after alkali treatment were compared with those of standards chondroitin 4-sulfate and chondroitin sulfate E and heparin. The day 6³⁵S-proteoglycans have a higher sulfate density than the day 1 species, but this difference did not affect the elution profiles after chromatography on collagen-Sepharose, whereas the day 6 proteoglycans bound more firmly than the day 1 fraction to fibronectin-Sepharose. The elution patterns obtained for these distinct proteoglycans closely resembled those of heparin and oversulfated chondroitin sulfate E standards, and clearly demonstrated the importance of sulfate density both for the affinity to fibronectin and collagen. Neither day 1 nor day 6³⁵S-proteoglycans were found to interact with hyaluronate.Abbreviations used CSPG chondroitin sulfate proteoglycan - GAG glycosaminoglycan - CS chondroitin sulfate - CS-E chondroitin 4,6 disulfate - MDM monocyte-derived macrophages     

A molecular linkage map of rye

A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F₂ population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.     

The expression of proteoglycans in phorbol ester induced U-937 cells

U-937 monoblastic cells were differentiated into macrophage-like cells in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Control cells and differentiated cells were labeled with³⁵S-sulfate and were both found to produce exclusively chondroitin sulfate proteoglycan. No differences in glycosaminoglycan structure or macromolecular properties of the proteoglycans produced in the two different cell systems could be observed. However, the differentiated cells were found to have a lower capacity for chondroitin sulfate proteoglycan synthesis, both under ordinary experimental conditions, and when exposed to stimulators of glycosaminoglycan biosynthesis such as -d-xylosides.Abbreviations SDS sodium dodecyl sulfate - TPA 12-O-tetradecanoylphorbol-13-acetate - PG proteoglycan - GAG glycoaminoglycan - CS chondroitin sulfate - CSPG chondroitin sulfate proteoglycan - NASDAE naphthol AS-D acetate esterase     

A linkage map of rye

A linkage map of rye (Secale cereale L.) is presented which comprises 60 loci including RFLPs, RAPDs, isozyme, morphological and physiological markers. The genetics and linkage relationships of these markers were investigated in several inbred lines of rye. For the RFLP mapping a genomic library of PstI-digested DNA was constructed from which 50 size-selected clones were analysed. The portion of single-copy and multi-copy DNA and the frequency of polymorphic DNA was determined. The markers are unequally distributed over the seven chromosomes of rye. Many of them exhibit a distorted segregation. The main region of deviating segregation ratios could be localized near the self-incompatibility loci.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F₂ population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.     

Amino acids and sugars in nectar,and their putative evolutionary significance

Individual amino acids and sugars from flower nectar of 32 plant species with different pollination systems were quantified and compared. Data show that there is no correlation between sugar and amino acid concentration. Furthermore there is no correlation between composition and concentration of amino acids and evolutionary advancement, nor any direct relation with pollination systems. However, higher sugar concentrations are often linked with more advanced morphological characters. Nectars from pierced or damaged flowers or nectars contaminated with pollen exhibit modifications and increases in amino acid composition. The presence of proline probably indicates such pollen contamination. Most pollinating animals depend on flower nectar in their energetic requirements, yet innumerable alternative amino acid and protein sources exist. Future research has to consider the relationship between nutritional requirements of pollinating animals and dependence on flower nectars.Dedicated to Prof. Dr.L. van der Pijl, Den Haag, in honour of his 80th birthday.     

Ureolytic nitrification at low pH by Nitrosospira spec.

An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.     

A chromosomal linkage map of Azotobacter vinelandii

Summary A chromosomal map of Azotobacter vinelandii strain UW was constructed. The map was based on measures of cotransfer of various markers mediated by plasmids R68.45 and pJB3JI, on results obtained from conjugal experiments with R-primes, and on recombinants obtained by chromosomal transfer mediated by RP4/Tn5-Mob.     

Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat

Retrotransposon-based molecular markers have been developed to study bread wheat ( Triticum aestivum) and its wild relatives. SSAP (Sequence-Specific Amplification Polymorphism) markers based on the BARE-1/ Wis-2-1A retrotransposons were assigned to T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are distributed among all wheat chromosomes, with the lowest proportion being assigned the D wheat genome. SSAP markers for BARE-1/ Wis-2-1A and three other wheat retrotransposons, Thv19 , Tagermina and Tar1, are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both showing approximately 13% of bands polymorphic in a mapping population, Tagermina showing approximately 17% SSAP band polymorphism and Tar1 roughly 18%. This suggests that Tagermina and Tar1 have been more transpositionally active in the recent evolutionary past, and are potentially the more useful source of molecular markers in wheat. Lastly, BARE-1 / Wis-2-1A markers have also been used to characterise the genetic diversity among a set of 35 diploid and tetraploid wheat species including 26 Aegilops and 9 Triticum accessions. The SSAP-based diversity tree for Aegilops species agrees well with current classifications, though the Triticum tree shows several significant differences, which may be associated with polyploidy in this genus.Communicated by M.-A. Grandbastien     

A linkage map with RFLP and isozyme markers for almond

Inheritance and linkage studies were conducted with seven isozyme genes and 120 RFLPs in the F₁ progeny of a cross between almond cultivars Ferragnes and Tuono. RFLPs were detected using 57 genomic and 43 cDNA almond clones. Eight of the cDNA probes corresponded to known genes (extensin, prunin (2), -tubulin, endopolygalacturonase, oleosin, actin depolymerizing factor and phosphoglyceromutase). Single-copy clones were found more frequently in the cDNA (65%) than in the genomic libraries (26%). Two maps were elaborated, one with the 93 loci heterozygous in Ferragnes and another with the 69 loci heterozygous in Tuono. Thirty-five loci were heterozygous in both parents and were used as bridges between both maps. Most of the segregations (91%) were of the 11 or 1111 types, and data were analyzed as if they derived from two backcross populations. Eight linkage groups covering 393 cM in Ferragnes and 394 in Tuono were found for each map. None of the loci examined in either map was found to be unlinked. Distorted segregation ratios were mainly concentrated in two linkage groups of the Ferragnes map.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.     

A rapid and sensitive method for the screening of DNA intercalating antibiotics

An improved, rapid and inexpensive gel mobility shift assay was developed for the screening of anthracycline antibiotics. The assay based on the intercalation activity of these molecules into dsDNA was used to assess the activity of partially purified antibiotics. Detection limits were of 0.1 ng ml^–1 with an average run time of 2 h. The assay is potentially useful for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.     

Normapolles plants: a prominent component of the Cretaceous rosid diversification

The Normapolles complex, characterised by its oblate and triaperturate pollen, constitutes an important and diverse element of many Late Cretaceous and Early Cainozoic floras of the Northern Hemisphere. Based on the dispersed pollen record alone it has been difficult to assess systematic affinities, but relationships with Fagales have been proposed. Over the past twenty years several exquisitely preserved Late Cretaceous reproductive structures with Normapolles type pollen in situ have been described. In this study we provide a summary and new information of these floral structures. Further, a new genus, Dahlgrenianthus, is described from the Late Cretaceous of southern Sweden. The genus includes the type species Dahlgrenianthus suecicus, a number of reproductive structures referred to Dahlgrenianthus sp., and Dahlgrenianthus trigonus (Knobloch et Mai) comb. nov. from the Maastrichtian flora of Walbeck, Germany. Dahlgrenianthus comprises small flowers with pentamerous perianth and androecium and a tricarpellate gynoecium. It is distinguished from all other Normapolles floral structures in its hypogynous floral organisation. All Normapolles floral structures described so far are thought to be related to various members of the core Fagales, but the group is obviously not monophyletic. The stratigraphic range of the Normapolles taxa and other fagalean fossils strongly suggests that all major fagalean lineages were present by the Cenomanian or earlier.     

Heterotrophic growth of Thiobacillus A2 on sugars and organic acids

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and -linked disaccharides, but not on -linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occurred on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.     

A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.