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[Mechanism of the WNT family in vertebrate skeleton development].

Skeletal elements of vertebrate limb are derived from mesenchymal cells. Initially, these cells differentiate into chondrocytes which form the initial anlage element. Subsequently, chondrocytes in the middle of the cartilage element exit the cell cycle and are eventually replaced by bone through endochondral ossification. Recently, it is shown that the secreted signaling molecules encoded by members of the wnt gene family and their related elements play pivotal roles in the process of the skeleton system development. These molecules ensure the chondrocytes can differentiate continuously at proper speed in the definitive space by the negative and positive control effect on different steps of the chondrocyte differentiation and form the matural skeleton system possessing normal function and structure. However, both the members of WNT family and their related molecules in the signaling pathway expressing abnormally can disrupt the balance remained by the WNT family and result in skeleton malformation. Understanding the roles of the WNT family is helpful to explore the mechanism of vertebrate skeleton development.

Animals↗

Osteopenic mice: animal models of the aging skeleton.

While our understanding of the developmental biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics, we still know very little, in molecular and genetic terms, about skeletal physiology. Thus, among the many questions that are largely unexplained are the following: why is osteoporosis mainly a women's disease? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? Molecular genetics has emerged as a powerful tool to study previously unexplored aspects of the physiology of the skeleton. Among mammals, mice are the most promising animals for this experimental work. This has been previously demonstrated e.g. through the tremendous impact of the different osteopetrotic models on our molecular understanding of osteoclastic bone resorption. Until recently the only way of studying bone loss situations and osteoporosis in mice was by using ovariectomy with all its limitations. Today, however, we have access to more sophisticated osteoporotic mouse-models from four different origins: Transgenic mice (HSV-TK), knock-out mice (OPG), inbred-strains (SAMP6), and through physiological modulation (icv application). These new models have already taught us several important lessons. The first is, that bone remodeling is more than just an autocrine/paracrine process. Multiple experimental evidence has demonstrated that the latter regulation exists, but genetics prove that there is no functional cross-control between resorption and formation. The second lesson is, that remodeling is, at least in part, subject to central regulation. Thus, osteoporosis is partly a central or hypothalamic disease. However, the most dramatic change and the most important advantage we feel is, that today we have models to test a new hypothesis regarding the etiology of osteoporosis before it turns to dogma. Taken together, mouse-studies may lead to a shift in our physiological understanding of skeleton biology and to the emergence of novel paradigms. These, in turn, should help us to devise new treatments for degenerative diseases of the skeleton such as osteoporosis and its associated clinical problems.

Journal Article↗

Trace elements and the European skeleton through 5000 years.

For our research, one thousand forty-four samples were taken from the femurs of 522 skeletons from 25 sites in Europe from the Neolithic Age (4000-5000 B.C), from La Téne Period, the Roman Era (500 B.C-400 A.D.), the Middle Ages and from contemporary cadavers. We found the following distribution of elements in the longitudional axis of long bones (the femurs and the tibias). The elements Zn, Fe, Ni, Cr, Pb, Mn, Co and Sn cumulated in the epiphysis. On the other hand, we found that Ca, Sr, Na and K prevailed in the central part of the diaphysis. In the central parts of the cross-section the highest concentration of the metal element Pb was in the external layer from the historical femurs. It was the same with cadavers of the recent population. A specific shift from Neolithic farming to agricultural intensification in the Roman Era was also apparent in the skeletons. Special sources of the above mentioned elements were found both in Celtic and Germanic tribes. Meat is the main source of zinc. Zinc is also important for the growth of the skeleton. When we investigated the development of the human skeleton during the last 5000 years we found the highest concetrations of Zn in communities with a good supply of animal food, whereas the lowest concentrations were paralleled with well-developed agriculturists. We assume the Neolithic gracilization, which is in the background for the increase of agricultural populations, is directly linked with the concentration of Zn and other elements essential for growth (Cu, Fe and others). The individuals most vulnerable to zinc deficiency include infants, adolescents during rapid growth phases and women during pregnancy and lactation. Trace elements in the bones of the La Téne period designate two areas of Celtic diet patterns--a "French one" (Roulier, Mont Trote and Acy Romance) and a "Czech one" (Karlov, Radovesice and Jenisův Ujezd). At Czech sites levels of zinc increased westward towards the Germanic region. Over the Germanic territory in the region of the Saala River there are similar supply trace element sources for bones of the Germanic tribes for a period of more than 1000 years. The dietary customs and environment that formed this development were preserved from 400 B.C. to the period of the Merovingians. A specific ratio of Zn and Sr can be found not only in men, but also in woman and children. At the beginning of our era lead emerged as a civilization element. This element influenced the diet until the 20th century. Since the beginning of the 20th century it occurs much less in the population. On the contrary, we have found significant presence of tin in human bones. Cadavers of the modem population indicate a high content of tin, considerably higher than those in the populations from the beginning of our era (as much as 40 microg.g(-1) bone).

Bone and Bones↗

Response to complete and skeleton photoperiods in subtropical male house sparrow, Passer domesticus (Linnaeus).

To examine the importance of the inductive light period of a skeleton photoperiod in relation to the endogenous circadian rhythm of photoinducibility mediating photoperiodic induction, P. domesticus were exposed for 28 weeks to a series of skeleton photoperiods, viz. 6L:4D:1L:13D, 6L:6D:1L:11D. 6L:8D:1L:9D and 6L:14D:1L:3D. The inductive effects of 1 hr light pulse at night varied depending on the time of its placement. To compare the inductive effects of complete and its corresponding skeleton photoperiods, birds in the second experiment were subjected for 20 weeks to 12L:12D and 6L:5D:1L:12D given daily or interposed on alternate days with constant darkness (12L:12D/DD and 6L:5D:1L:12D/DD). There was a difference in the rate and magnitude of response between the complete and skeleton photoperiods. It appears that the subtropical house sparrow uses photoperiodic strategy in regulation of its seasonal testicular responses similar to that is reported for its temperate population.

Animals↗

GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate and is a component of a membrane skeleton subcellular fraction.

To ascertain the subcellular localization of the growth-associated protein GAP-43 in growth cones, we isolated growth cones from the forebrains of neonatal rats. Anti-GAP-43 immunoreactivity in these isolated growth cones (IGCs) resembled that seen in cultured growth cones in 2 respects: First, in substrate-attached IGCs, as in cultured growth cones, immunoreactivity was intracellular, punctate, and extended throughout the IGCs and their processes. Second, when IGCs were dislodged from this substrate, patches of membrane that were highly immunoreactive for GAP-43 were left behind. Extracting IGCs in nonionic detergents revealed that almost all the particulate GAP-43 colocalized with a membrane skeleton fraction: It was not present in the cytoskeleton. The association of GAP-43 with the membrane skeleton was not due to nonspecific aggregation, nor was it calcium dependent. Examination of this fraction under the electron microscope showed it to consist of membrane fragments associated with amorphous material that could be visualized with tannic acid. Immunoelectron microscopy showed that anti-GAP-43 immunoreactivity was localized to this amorphous material. Fodrin, talin, and a-actinin immunoreactivity could be detected in the membrane skeleton fraction, and actin and tubulin were also identifiable from SDS gels. The association of GAP-43 with the membrane skeleton, which by analogy with other cell types is involved in the dynamic regulation of cell shape, implies that GAP-43 in growth cones may be involved in this function.

Animals↗

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes.

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.

Anion Exchange Protein 1, Erythrocyte↗

[Reconstruction of the facial skeleton using Pallacos R].

Defects of the facial skeleton cause frequently severe aesthetic facial changes with negative repercussions also on the patient's mental state. The skeleton may be affected by injury, a neoplastic disease and iatrogenically when it is necessary to remove a part of the skeleton during operation. One possible way how to replace and reconstruct the skeleton is to use bone cement Pallacos R. This method was used in 8 patients with skeletal affections. The use of Pallacos R has the advantage that it can be used quickly, that it is easy to work with directly at the site of the defect and that its tolerance is satisfactory. It permits also to preserve the physiological shape of the paranasal sinuses. The method is one approach to individually indicated reconstruction.

Bone Cements↗

Separation of the lipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts.

The membrane skeleton, a protein lattice at the internal side of the red cell membrane, is principally composed of spectrin, actin and proteins 4.1 and 4.9. We have examined negatively stained red cell ghosts and demonstrated, on an ultrastructural level, a separation of the lipid bilayer from the membrane skeleton during echinocytic transformation. The electron micrographs of discoidal red cell ghosts suspended in hypotonic buffer revealed a filamentous reticulum that uniformly laminated the entire submembrane region. transformation of the discoidal ghosts into echinocytic form, as induced by incubation in isotonic buffer, resulted in a disruption of skeletal continuity underlying the surface contour of the membrane spicule. The submembrane reticulum extended into the base and the neck of the spiny processes of the crenated ghosts but was absent at the tip of these projections. In addition, membrane vesicles without a submembrane reticulum were detected either attached to the tips of the spicules or released into the supernatant from the echinocytic ghosts. Protein analysis revealed that the released vesicles were enriched in bands 3, 4.1 and 7 and contained very little of the membrane skeletal proteins, spectrin and actin. The data indicate that during echinocyte formation, parts of the lipid bilayer physically separate from the membrane skeleton, leading to a formation of skeleton-poor lipid vesicles.

Actins↗

[Localization of regions of DNA attachment to the nuclear skeleton within chicken alpha-globin genes in functionally active and functionally inactive nuclei].

Short DNA fragments remaining associated with the nuclear skeleton after nuclease treatment and high salt extraction of chicken erythroblasts and erythrocytes nuclei were used as hybridization probes in order to find the positions of DNA attachments to a nuclear skeleton inside the domain of alpha-globin genes. It is found that in erythroblasts nuclei a rather long region including coding sequences is associated with the nuclear skeleton. This region coincides with the region preferentially digested by DNAase I in chromatin. In erythrocytes nuclei, a specific attachment point is found in the region between 4500 and 300 bp upstream to pi-gene. Several attachments in this region are detected also in erythroblast nuclei, but they are not prominent in this case. The results are discussed in terms of the existence of stable (structural) and temporal (functional) attachments of DNA to the nuclear skeleton.

Animals↗

On the thickness of the red cell membrane skeleton: quantitative electron microscopy of maximally narrowed isthmus regions of intact cells.

The thickness of the red cell membrane skeleton was deduced from measurements of the isthmus zones of intact cells that were maximally narrowed by one of two independent methods. The first method involved application of viscous drag to red cells entrapped between spider web fibers. The second method utilized cellular dehydration followed by spectrin denaturation at 49.5 degrees C. Measurements on thin sections showed that the isthmus is narrowed to approximately 120 nm by either method, suggesting that the membrane skeleton occupies a zone beneath the lipid bilayer that is up to 60 nm in thickness. The tertiary and quarternary structure of band 3, a major integral membrane protein that anchors the membrane skeleton to the lipid bilayers may be a critical determinant of the location of the membrane skeleton within the red cell.

Biomechanical Phenomena↗

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes.

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.

Actins↗

Evaluation of renal-skeleton ratio of technetium-99m phosphonate in multiple myeloma.

The kidneys were evaluated on [99mTc]phosphonate bone scans using 35 studies from 23 individuals with multiple myeloma; these images were compared with those from 50 controls. In each case, the kidneys could be visualized and calculation was made of the renal:skeleton ratio. Two myeloma patients showed an elevated renal:skeleton ratio. One was due to reduced vertebral uptake of [99mTc]phosphonate following therapeutic radiation. In the second case, the elevated ratio was related to renal uptake of the tracer (independent of urinary retention), and was consistent with nephrocalcinosis. No significant correlation between the renal:skeleton ratio and the degree of hypercalcemia, proteinuria, or renal impairment was found. We conclude that bone scintigraphy represents a safe, simple means of demonstrating renal presence and activity in multiple myeloma patients. However, calculation of the renal:skeleton ratio is not directly helpful in clarifying the events of calcium metabolism.

Bone and Bones↗

The distribution of pneumatisation in the skeleton of the adult domestic fowl.

The occurrence of pneumatisation in the skull of the adult domestic fowl was investigated in eight birds by gross and histological examination. It was found to occur regularly throughout the neurocranium and in the quadrate and variably in the mandible, but to be absent in the facial skeleton. Close agreement was found between gross and histological examination. The entire skeletons of fifty one adult birds all from the same hatch and kept under the same conditions were examined grossly for evidence of the occurrence and extent of pneumatisation. Previous findings for the skull were confirmed with the additional information that pneumatisation in the pterygoid was variable. Variation in the extent of skull pneumatisation was less than in the postcranial skeleton, where only cervical vertebrae 5-9 were found to be regularly pneumatised. The humerus and coracoid were variably pneumatised, often unilaterally. The os coxae and sternum had a very low incidence of pneumatisation. The three cockerels in the group appeared to be relatively well pneumatised. Some possible factors governing the occurrence and extent of pneumatisation in the skeleton are discussed.

Air↗

Increased cation permeability in mutant mouse red blood cells with defective membrane skeletons.

Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

Anemia, Hemolytic↗

Molecular mechanism of histamine release: the role of intermediate filaments and membrane skeletons.

It has been recognized that cytoskeletons play some important roles in the histamine release from mast cells. We previously reported the role of microfilaments and microtubules in the histamine release from mast cells, and in the present study, the roles of intermediate filaments and membrane skeletons were investigated. When permeabilized mast cells were stimulated with Ca2+, a translocation of protein kinase C from cytosol to the membrane fraction was observed. This lead to the phosphorylation of vimentin, one of the component proteins of the intermediate filaments. Phosphorylation of vimentin induced disruption of intermediate filaments and resulted in an increase in the mobility of granules. This may be favorable for the initiation of degranulation. In the membrane skeletons of rat mast cells, alpha- and beta-fodrin, ankyrin and actin were found. Changes in the distribution of the fodrin network were elicited by antigen-antibody reaction. It is suggested that membrane skeletons may act as a barrier between the plasma membrane and the granule membrane and that the changes in the distribution of membrane skeletons may facilitate the initiation of the fusion of the plasma membrane and granular membrane.

Animals↗

Effect of oil-attached BCG cell-wall skeleton on the induction of pleural fibrosarcomas in mice.

The effect of oil-attached BCG cell-wall skeleton on the induction of pleural fibrosarcomas by 3-methylcholanthrene in mice was examined. The pleural fibrosarcomas were induced by a substernal injection of 3-methylcholanthrene into the thoracic cavity of ddO mice. One week after the injection of 3-methylcholanthrene, 100 mug of oil-attached BCG cell-wall skeleton was injected subcutaneously every week for 10 weeks. In the observation period of 130 days, the incidence of pleural fibrosarcoma was 67% in the control mice and 39% in the mice treated with BCG cell-wall skeleton. The latent period of tumor induction was prolonged in the mice treated with BCG cell-wall skeleton.

Animals↗

Three-dimensional ultrastructure of in situ membrane skeletons in human erythrocytes by quick-freezing and deep-etching method.

The erythrocyte membrane skeleton is a network of some structural proteins, principally composed of spectrin, actin and protein 4.1, which is also attached to the cytoplasmic surface of the lipid membrane. The purpose of the present review is to describe the three dimensional ultrastructure of in situ membrane skeletons in human erythrocytes. The in situ membrane skeletons are densely organized, and the spectrin itself has a property of extension or contraction with reacting the external mechanical forces. The lengths between the intersections are changed according to the mechanical strength, and also vary even in one erythrocyte. To clarify the in situ organization of membrane skeletons, the quick freezing and deep-etching method should be used for fresh unfixed erythrocytes because of their fragility during preparation steps.

Cytoskeletal Proteins↗

Cross-linking of the IgM receptor induces rapid translocation of IgM-associated Ig alpha, Lyn, and Syk tyrosine kinases to the membrane skeleton.

Cross-linking-induced association of membrane IgM (mIgM) with the cytoskeleton is well documented. However, its functional significance during B cell activation is not yet understood. One possible need for mIgM/cytoskeleton interactions may be to recruit the B cell receptor (BCR)-associated signaling molecules to the cytoskeletal matrix for the propagation of downstream signaling. We first verified whether BCR-associated Ig alpha translocates to the cytoskeleton together with mIgM in polyclonal anti-IgM-treated murine B lymphoma cell line, BAL17.7.1. Co-capping experiments and the purification of the membrane skeleton under conditions that preserve IgM-Ig alphabeta) interactions confirmed that Ig alpha translocates to the cytoskeleton as part of the BCR complex. Furthermore, two BCR-associated kinases that are known to play critical roles in anti-IgM-induced B cell signaling, the src family kinase Lyn and the non-src family kinase Syk, accumulate in the membrane skeleton shortly after BCR cross-linking, when most of IgM and Ig alpha accumulate in this fraction. The kinetics of recruitment of the bulk of Ig alpha, Lyn, and Syk into the membrane skeleton appeared to precede the accumulation of their hypertyrosine-phosphorylated forms, suggesting that activation of the BCR-associated signaling molecules occurs in this fraction. These data suggest that cross-linked mIgM translocating to the membrane skeleton serves as a vehicle for active signaling molecules to be recruited to this vicinity. This may promote B cell activation events by providing high affinity interactions between signaling molecules and their substrates supported by the cytoskeletal matrix.

Animals↗