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  • Phospholipids  (3)
  • Chondrocytes  (2)
  • 11.10.St  (1)
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 5 (1970), S. 13-20 
    ISSN: 1432-0827
    Keywords: Phospholipids ; Hydroxyapatite ; Bone ; Chelation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé L'association de phospholipides et de la phase minérale de tissus calcifiés est étudiée en équilibrant un mélange de phosphatides avec de l'os anorganique. L'os est élué progressivement avec du chloroforme, chloroforme/méthanol et chloroforme/méthanol/HCl. Dans chaque éluat, les phospholipides sont identifiés par chromatographie quantitative. Les phosphatides acides et neutres sont présents, dans des proportions variables, dans l'éluat chloroformique de l'os anorganique. Ces lipides constituent plus de 80% des phospholipides totaux liés. L'extraction chloroforme/méthanol permit d'isoler un groupe de lipides essentiellement neutres. La composition de cet éluat d'os anorganique est voisin de l'extrait de solvant neutre d'os compact bovin minéralisé. Au troisième stade d'élution, on a pensé que lorsque l'acide du produit d'extraction agit comme un agent de déminéralisation, on pouvait comparer l'éluat avec l'extrait lipidique, obtenu après déminéralisation de l'os compact. Certes il existe des similitudes entre les phospholipides acides de l'extrait obtenu après déminéralisation et le troisième éluat lipidique de l'os. Il est possible que les phospholipides acides soient liés par des liaisons coordonnées aux ions Ca2+ et Mg2+ de la maille apatitique de l'os. En présence d'acide ou d'agent de déminéralisation, ces ions métalliques sont déplacés. Le rôle de ces composés de liaison, en tant que ponts entre les phases organiques et inorganiques des tissus calcifiés, est discuté.
    Abstract: Zusammenfassung Durch Äquilibration eines Gemisches von Phosphatiden mit anorganischen Knochen wurde die Bindung zwischen Phospholipiden und dem Mineralanteil von Hartgewebe untersucht. Der Knochen wurde sukzessive mit Chloroform, Chloroform/Methanol und Chloroform/Methanol/HCl eluiert. Die Phospholipide wurden in jedem der Eluate chromatographisch identifiziert und anschließend quantitativ bestimmt. Sowohl die sauren als auch die neutralen Phosphatide waren in verschiedenen Mengen im Chloroformeluat des anorganischen Knochens vorhanden. Zusammen machten diese Lipide über 80% der gesamtengebundenen Phospholipide aus. Die Extraktion in Chloroform/Methanol entzog dem anorganischen Knochen eine Gruppe von Lipiden mit vorwiegend neutralem Charakter. Die Zusammensetzung dieses zweiten Eluates aus dem anorganischen Knochen war jener ähnlich, wie sie für den Extrakt aus dem mineralisierten kompakten Rinderknochen mit einem neutralen Lösungsmittel beschrieben ist. Bei der dritten Elution wurde erörtert, daß dieses Eluat mit dem nach Demineralisation erhaltenen Lipidextrakt von kompakten Knochen verglichen werden kann, da die Säure des Extraktionsmittels demineralisierend wirkt. In der Tat wurden, was die sauren Phospholipide anbetrifft, Ähnlichkeiten zwischen dem Extrakt nach Demineralisation und dem dritten Lipideluat des Knochens festgestellt. Als Interpretation dieser Resultate wurde vorgeschlagen, daß die sauren Phospholipide koordinativ an Ca2+- und Mg2+-Ionen des Apatitgitters des Knochens gebunden sind. In einem sauren oder einem demineralisierenden Milieu werden die Bindungen zu Metallionen zerstört. Die Rolle solcher koordinativ wirkender Verbindungen als Brücken zwischen organischen und anorganischen Phasen mineralisierter Gewebe wurde besprochen.
    Notes: Abstract The association of phospholipids with the mineral phase of hard tissues was investigated by equilibrating a mixture of phosphatides with anorganic bone. The bone was eluted sequentially with chloroform, chloroform/methanol and chloroform/methanol/HCl. In each of the eluates, the phospholipids were identified chromatographically and were quantitated. Both acidic and neutral phosphatides were present, in variable proportions, in the chloroform eluate of the anorganic bone. Together, these lipids accounted for over 80% of the total bound phospholipids. Chloroform/methanol extraction removed a group of lipids from the anorganic bone which was predominately neutral in character. The composition of this second eluate of anorganic bone resembled the reported neutral solvent extract of mineralized bovine compact bone. At the third elution step, it was argued that as the acid in the extractant acted as a demineralizing agent, this eluate could be compared with the post-demineralized lipid extract of compact bone. With respect to their acidic phospholipids, similarities were noted between the post-demineralized extract and the third lipid eluate of bone. To interpret this finding, it is speculated that acidic phospholipids were bound through co-ordinating bonds to Ca2+ and Mg2+ ions of the apatite lattice of bone. In the presence of either acid or a demineralizing agent, these metal ions were displaced from the ligands. The role of such co-ordinating compounds as bridges between organic and inorganic phases of mineralized tissues is discussed.
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  • 2
    ISSN: 1090-6487
    Keywords: 13.75.Cs ; 11.10.St
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract It is shown that the existence of an N̄N bound state with a mass of approximately 1870 MeV and width 10 MeV follows from analysis of the experimental data on the momentum distribution of recoil protons in the annihilation process p̄d→p s5π.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 5 (1970), S. 30-38 
    ISSN: 1432-0827
    Keywords: Phospholipids ; Bone ; Cartilage ; Mucopolysaccharides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Les tissus squelettiques de poissons téléostéens (Scomber scombrus, etGadus callaria) et d'élasmobranches (Raja batis etScyllium canicula) sont analysés pour leur contenu en lipides. Plus de phospholipides sont extraits des tissus osseux de téléostéens que des tissus cartilagineux d'élasmobranches. Outre la proportion lipidique plus élevée de l'os de téléostéens, des différences de composition lipidique ont été notées entre les deux tissus. Avant démineralisation, les extraits deGadus et Scomber sont surtout constitués de phospholipides neutres. Par contre, les phosphatides des tissus cartilagineux contiennent des quantités importantes de phospholipides acides. Après déminéralisation de l'os et du cartilage de poissions, les solvants pour lipides neutres éluent á la fois les phosphatides acides et neutres de ces tissus. Après extraction des lipides, le traitement des tissus déminéralisés par des solvant acides extrait à nouveau des lipides. Dans cette fraction finale, un grand nombre de phospholipides ont été retrouvés. A ce stade, d'autres phosphatides, que ceux mis en évidence chez les poissons précités, sont extraits à partir de l'os déminéralisé deScomber. Le principal phosphatide de la fraction finale est constitué par la cardiolipine dont des quantités plus importantes, que celles notées au niveau des autres tissus durs, sont retrouvées dans l'os deScomber.
    Abstract: Zusammenfassung Die Skeletgewebe der Knochenfische (Scomber scombrus undGadus callaria) und der Knorpelfische (Raja batis undScyllium canicula) wurden auf ihren Lipidgehalt untersucht. Es ergab sich, daß mehr Phospholipide aus dem Knochengewebe der Knochenfische extrahiert werden konnte, als dies aus dem Knorpelgewebe der Knorpelfische möglich war. Es wurde nicht nur eine größere Lipidmenge im Knochen der Knochenfische festgestellt, sondern auch noch Unterschiede in der Lipidzusammensetzung der beiden Gewebe beobachtet. Die vor der Demineralisation hergestellten Extrakte von Gadus und Scomber bestanden vorwiegend aus neutralen Phospholipiden. Dagegen enthielten die Phosphatide der Knorpelgewebe ansehnliche Mengen saurer Phospholipide. Nach der Demineralisation von Fischknochen und-knorpel wurden durch neutrale Lipidlösungsmittel sowohl saure als auch neutrale Phosphatide aus diesen Geweben eluiert. Wenn die demineralisierten Gewebe, denen bereits Lipide entzogen wurden, noch mit angesäuerten Lösungsmitteln behandelt wurden, konnten erneut Lipide aus Knochen und Knorpel extrahiert werden. Im letzten Extrakt konnten die verschiedensten Phospholipide nachgewiesen werden. In diesem Stadium konnten verhältnismäßig mehr Phosphatide aus dem demineralisierten Knochen von Scomber entzogen werden, als dies bei anderen Fischarten möglich war. Das wichtigste Phosphatid dieses letzten Extraktes war Cardiolipin. Tatsächlich war mehr Cardiolipin in den Knochen von Scomber vorhanden als in allen andern Hartgeweben.
    Notes: Abstract The skeletal tissues of teleost (Scomber scombrus andGadus callaria) and elasmobranch (Raja batis andScyllium canicula) fish were examined for the presence of lipids. It was found that more phospholipid was removed from the bony tissues of the teleosts than was extractable from the cartilaginous tissues of the elasmobranchs. Apart from there being a greater proportion of lipid associated with teleost bone, differences were also noted in the lipid composition of the two tissues. Before demineralization, inGadus andScomber, the extracts were predominantly of neutral phospholipids. In contrast, the phosphatides of the cartilaginous tissues contained appreciable quantities of acidic phospholipids. After demineralization of piscine bone and cartilage, neutral lipid solvents eluted both acidic and neutral phosphatides from these tissues. When the demineralized lipid extracted tissues were treated with acidified solvents, lipid was again removed from the bone and cartilage. In this final extract, a wide variety of phospholipids was detected. At this stage, more phosphatide was extracted from the demineralized bone ofScomber, compared with the other piscine species. The principal phosphatide of this final extract was cardiolipin. More cardiolipin was present in the bone ofScomber than in any other hard tissue.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 5 (1970), S. 21-29 
    ISSN: 1432-0827
    Keywords: Phospholipids ; Bone ; Calcification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Les phospholipides neutres, tels que la sphingomyéline, la lécithine et la phosphatidyléthanolamine, sont les phosphatides principaux de l'os compact bovin. Ces lipides constituent plus de 90% des phospholipides de l'os, avant déminéralisation, et plus de 60% des phosphatides totaux extraits de ce tissu. Après déminéralisation, les solvants lipidiques neutres extraient des phospholipides neutres et acides de la matrice osseuse. Le traitement final de la matrice osseuse avec un mélange de solvants acides permet de récupérer une faible quantité de phosphatides acides et neutres. Pour expliquer la nécessité d'une déminéralisation au cours de l'extraction lipidique, il est possible que les lipides de l'extrait, obtenu après déminéralisation. soient liés à la phase minérale de l'os. Afin d'exclure la possiblité que cette extraction soit dûe simplement aux cristaux d'apatite, pouvant agir comme obstacle à la diffusion du solvant, l'effet de l'extraction lipidique sur des fractions de particules osseuses de diverses tailles a été étudié. La composition des extraits lipidiques n'est pas liée à la taille des particules: le minéral osseux ne parait done pas constituer un obstacle au passage des solvants lipidiques.
    Abstract: Zusammenfassung Die neutralen Phospholipide Sphingomyelin, Lecithin und Phosphatidyläthanolamin sind die wichtigsten Phosphatide des kompakten Rinderknochens. Zusammengenommen machen diese Lipide über 90% der Phospholipide, welche vor der Demineralisation dem Knochen entzogen worden sind und über 60% der aus diesem Gewebe extrahierten totalen Phosphatide aus. Nach der Demineralisation konnten mit neutralen Lösungsmitteln für Lipide ein weiterer Anteil an neutralen und eine beträchtliche Menge an sauren Phospholipiden aus der Knochenmatrix extrahiert werden. Am Schluß wurde die Knochenmatrix mit einem angesäuerten Lösungsmittelgemisch behandelt, und es konnte eine kleine Menge saurer sowie neutraler Phosphatide aus dem Gewebe extrahiert werden. In Anbetracht der notwendigen Demineralisierung bei der Lipidextraktion wurde vorgeschlagen, daß die Lipide aus dem nach Demineralisation gewonnenen Extrakt an die Mineralphase des Knochens gebunden sind. Um die Möglichkeit auszuschließen, daß dieser Extrakt lediglich durch die Apatit-Kristalle bedingt ist, die die Diffusion des Lösungsmittels verhindern, wurde die Wirkung der Lipidextraktion auf Knochenpulver verschiedener Partikelgrößen geprüft. Jedoch besteht keine Beziehung zwischen der Zusammensetzung des Lipidextraktes und der Größe der Partikel. Daraus folgt, daß das Knochenmineral den Durchgang der Lipidextraktionsmittel in das Gewebe nicht verhindert.
    Notes: Abstract The neutral phospholipids sphingomyelin, lecithin and phosphatidylethanolamine were the principal phosphatides of compact bovine bone. Together, these lipids accounted for over 90% of the phospholipids removed from bone, prior to demineralization, and for over 60% of the total phosphatides extracted from this tissue. Following demineralization, neutral lipid solvents removed a further quantity of neutral, and a considerable number of acidic, phospholipids from the bone matrix. A final treatment of the bone matrix with an acidified solvent mixture removed a small number of acidic, as well as neutral, phosphatides from the tissue. To account for the necessity of the demineralization step in the lipid extraction procedure, it was suggested that lipids of the post-demineralized extract were bound to the mineral phase of bone. To exclude the possibility that this extract was merely due to the apatite crystallites acting as a solvent diffusion barrier, the effect of the lipid extraction of bone of different particle size was examined. However, it was found that the composition of the lipid extracts was not related to the particle area; hence, it was inferred that the bone mineral was not obstructing the passage of the lipid extractants in the tissue.
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  • 5
    ISSN: 1432-0827
    Keywords: Hoechst dye ; Chondrocytes ; DNA analysis ; Mineralization ; Alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To overcome these obstacles, we developed a new method of isolating nucleic acids from chondrocytes which facilitates measurement of cell number by DNA analysis. Chondrocytes were isolated from chick embryo sterna and grown continuously without subculturing for 2–3 weeks in monolayer. The cells were treated with triton X-100 and the nucleic acid content of the extract was determined by measuring DNA fluorescence in the presence of Hoechst dye 33258. To minimize background fluorescence due to the triton, we precipitated the DNA with alcohol and then solubilized the nucleic acids in EDTA. This simple procedure removed the detergent and substantially increased the sensitivity of the method. Thus, we could measure with high precision and high recovery, the DNA content of cultures of 10,000–50,000 cells. In a single well containing 0.5–1.0 million cells, sufficient material remained for subsequent measurements of alkaline phosphatase activity and protein and calcium content. As the mineral present in the triton-treated samples was soluble in EDTA, we experienced no problems in measuring the calcium content of the culture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of the triton maintained the catalytic state of alkaline phosphatase. We conclude that this procedure provides a simple and rapid approach to measuring major indicators of chondrocyte maturation and function.
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  • 6
    ISSN: 1432-0827
    Keywords: Thalassemia ; Mineralization ; Alkaline phosphatase ; Deferoximine ; Chondrocytes ; Free radicals ; Ascorbate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract The homozygous form of β-thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine mine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitroblue tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate. We next investigated the possibility that the change in alkaline phosphatase activity was due to the formation of reactive oxygen radicals. Though oxygen radical scavengers and disproportionating agents did not change the activity of the enzyme, α-tocopherol provided complete protection. In conclusion, the deferoximine-ascorbate couple inactivates chondrocyte alkaline phosphatase probably by generation of free radicals. As free radicals can damage cartilage as well as other tissues, clinical regimens that are directed at elevating ascorbate levels in thalassemia need to be carefully reviewed.
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