Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human

Polyclonal antibodies against the core protein of the small chondroitin sulfate-dermatan sulfate proteoglycan from human skin fibroblast secretions were used, after affinity-purification, as a probe to study localization of crossreactive material in several human tissues by indirect immunocytochemistry. In contrast to skin, kidney, and the adventitial layer of aorta, positive staining of brain, liver, cartilage, and intimal and medial layers of aorta required pre-treatment of tissue sections with chondroitin ABC lyase. In all tissues investigated, antigenic material was present in the interstitial space. Filamentous structures were perpendicularly oriented towards basement membranes. In liver, specific staining was seen along the sinusoidal walls. Reticular fibers with or without focal condensations were seen in cerebral cortex and cerebellum. The results suggest a role of small chondroitin sulfate-dermatan sulfate proteoglycan in cell-matrix interactions.     

The vascularization of the menisci. Morphological basis for the repair

Degeneration of the knee joint and increase of anterior-posterior tibial displacement are resulting from total meniscectomy, especially in knees with anterior cruciate deficiency. Vascularisation of the meniscus was studied in 12 cadaver knees after latex injection of vessels. Vascularisation of the anterior and posterior horns was found to be much better than that of the body of meniscus. All vessels originated from the popliteal artery, but variably in importance, and formed the perimeniscal and subsynovial network. 11 peripheral meniscus tears (8 freshly injured, 3 ruptures older than 2 months) were repaired by refixation, followed by immobilisation for 6 weeks. Arthroscopy 3 months postoperatively showed complete healing of all tears. Clinical examinations--follow-up for 14 months--showed no signs of rerupture in any of the patients.     

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.     

Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells.

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (peonanin). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.     

Hemagglutination properties of Enterococcus

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.     

Benzoyl cellulose beads in the pure polymeric form as a new powerful sorbent for the chromatographic resolution of racemates

Cellulose-based stationary phases are known to be very efficient and versatile chiral sorbents for the chromatographic resolution of racemates. Except for microcrystalline cellulose triacetate (CTA I), basically all other cellulose-based phases have been prepared by coating of ca. 20% weight polymer on a wide pore silica gel used as a carrier. In this work we describe the preparation of benzoylcellulose (TBC) beads in the pure polymeric form (without inorganic carrier) from an emulsion of the organic polymer. The new material has been fully characterized and used as a chiral stationary phase for the resolution of various classes of racemic compounds such as benzylic alcohols or acetate derivatives of aliphatic alcohols and diols. The structural variety of the separated solutes as well as the irrational influence of the aromatic substituent in different classes of aryl compounds suggest that multiple interaction sites are involved in the complexation, making a prediction of the separation difficult. The benzoyl cellulose beads exhibit a very high loading capacity, which is particularly useful for preparative purposes as demonstrated for selected examples.     

Isolation and properties of glyceraldehyde-3-phosphate dehydrogenase from a sturgeon from the Caspian Sea and its interaction with spin-labeled NAD+ derivatives

Glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was isolated from a sturgeon, Huso huso, from the Caspian Sea. It is closely related to the enzyme from a Pacific sturgeon, Acipenser transmontanus, with respect to amino acid composition, steady-state kinetics and coenzyme binding. The latter, as studied by means of a spin-labeled derivative of NAD+, is negatively cooperative exhibiting a Hill coefficient of 0.84 at 12 degrees C. Two derivatives of NAD+ spin-labeled at N6 or C8 of the adenine ring were found to be active coenzymes with maximum velocities reaching 35 or 45% of the value for NAD+ itself. When more than two equivalents of either spin-labeled NAD+ are bound to the enzyme spin-spin interactions are observed in the ESR spectra. Distances between the nitroxide radicals (8--9 A) calculated from the observed splittings are in excellent agreement with data predicted from the crystal structure of the lobster enzyme when the coenzyme is bound in an anti-conformation of the adenine moiety about the glycosidic bond to all four subunits.