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N A Swords

Publications and source records attributed to N A Swords.

7 recordsLinked to original sources
PubMed2000

Bone marrow accessory cells regulate human bone precursor cell development.

OBJECTIVE: Much remains to be learned about the intimate relationship between bone marrow and its surrounding tissue: the bone. We hypothesized that bone marrow accessory cell populations might regulate the development of human bone precursor cells. MATERIALS AND METHODS: We used immunologic phenotyping, and isolation methods to fractionate subpopulations of nonadherent, low-density (NALD) human bone marrow cells. These cells were examined for their ability to support the serum-free survival, proliferation, and expression of bone proteins by highly purified populations of human bone precursor cells. Quantitative assessment of the accessory cell populations as well as human bone precursor cells phenotype was performed using multiparameter flow cytometry. Bone protein expression was evaluated by immunocytochemistry, Western analysis, and enzymatic analysis (for alkaline phosphatase activity). RESULTS: Human bone marrow contains a cell population that stimulates the development of purified bone precursor cells. Feeder-layer studies demonstrate that these osteopoietic accessory cells (OACs) do not require cell-cell interaction to promote bone precursor cell development but, rather, produce soluble molecules responsible for their effects. Flow cytometric analyses reveal that bone marrow derived B cells, T cells, macrophages, natural killer cells, and endothelial cells do not produce this stimulatory factor. The (growth) factor cannot be replaced by addition of exogenous cytokines. The isolation of human transforming growth factor beta receptor type II (TGF-betaRII)-positive cells increases OAC-specific activity in bone cell ex vivo expansion cultures. Moreover, isolation of OAC bone marrow cells characterized by high TGF-betaRII expression, relatively low cellular complexity, and small size yields a population that is highly enriched for OACs. CONCLUSION: We conclude that human bone marrow contains a population of OACs that are an obligate requirement for the early phases of bone cell development ex vivo.

Antigen-Presenting Cells↗
PubMed1994

Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex.

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.

Aminocaproic Acid↗
PubMed1993

Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra.

The secondary structure of bacteriorhodopsin is known from electron-diffraction studies, making bacteriorhodopsin a useful test system for analysing environmental influences on membrane proteins using c.d. spectroscopy. The conformational effects of detergent solubilization and incorporation into vesicles of various types were determined by comparison of the calculated secondary structures derived from c.d. spectra with the structure determined from diffraction studies. In addition, two modified forms of the native purple membrane, a shrunken form of the hexagonal lattice and an orthorhombic lattice form, were used to determine the effects of varying membrane fragment size and protein concentration within the membranes. The two different vesicle incorporation procedures yielded bacteriorhodopsin spectra which were nearly identical with each other and very close to the structure calculated from electron-diffraction studies. Solubilization of the native protein in the non-ionic detergent n-octyl glucoside, without subsequent vesicle incorporation, resulted in a significantly altered protein conformation. Organizing the protein in different membrane lattices produced even more apparent deviations from the secondary structure determined by diffraction studies, as a consequence of optical effects caused by the high protein concentrations in the lattices. These studies show the importance of maintaining a 'native' environment, and the influence of particle geometry in interpreting c.d. studies of membrane proteins.

Bacteriorhodopsins↗
PubMed1993

The assembly of the prothrombinase complex on adherent platelets.

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.

Animals↗
PubMed1993

Intact platelet membranes, not platelet-released microvesicles, support the procoagulant activity of adherent platelets.

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Blood Coagulation↗
PubMed1991

Protein-chromophore interactions in bacteriorhodopsin: the effects of a change in surface potential.

The chromophore retinal is bound to bacteriorhodopsin via a protonated Schiff base linkage. The retinal binding site is reported to be buried in the transmembrane portion of the protein, distant from the membrane surfaces. When bound to bacteriorhodopsin, the absorption maximum of retinal is red-shifted from 366 nm to 568 nm producing a purple color. This color persists across a wide pH range. However, when the pH is raised above 12.0, the membranes become pink in color, while at pH values of 3.0 or below, a blue color is produced. The blue color can also be obtained by removing the divalent cations bound to the surface of the protein. In this study, bacteriorhodopsin was examined by circular dichroism and absorption spectroscopy to determine if protein conformational changes were associated with the color shifts. It was found that although the retinal chromophore can be completely removed by bleaching with hydroxylamine with no significant influence on the secondary structure of the protein, a change in the surface charge of bacteriorhodopsin results in measurable conformational change in the protein, which apparently affects the nature of the retinal binding site.

Bacteriorhodopsins↗