Biomedical subjects

C G Simon

Publications and source records attributed to C G Simon.

9 recordsLinked to original sources

PubMed2004

Imaging cells on polymer spherulites.

Polymers are commonly used to construct tissue-engineered medical products and the conditions used to process these polymeric materials can affect their biological performance. For semi-crystalline polymers, thermal processing conditions can induce crystallization of the polymer chains into spherulites, which cause the polymer surface to become roughened. This change in surface topology can influence cell behaviours such as adhesion, morphology and proliferation. Herein, a simple microscope technique is presented, which combines fluorescence microscopy with transmitted white light microscopy using crossed polarizers. This technique allows simultaneous imaging of cells and polymer spherulites, which enables the biomaterials researcher to observe the effects of spherulites on cell alignment and morphology.

Animals↗

PubMed1999

Exchange of C(16)-ceramide between phospholipid vesicles.

Ceramide is considered to be an important signaling molecule in cellular processes such as cell growth, secretion, differentiation, and apoptosis. This implies that the molecule is able to move between cellular membranes. However, the ability of the molecule to undergo such exchange has been largely ignored despite the profound impact that this ability would have on its mechanism of action in signal-transduction cascades. With this in mind, the ability of a long-chain, radioactive ceramide, (14)C-C(16)-ceramide, to exchange between populations of lipid vesicles was evaluated. The rate of exchange of (14)C-C(16)-ceramide between lipid vesicles at lipid concentrations commonly found in cells (10-110 mM) was on the order of days (t(1/2) of 45-109 h). Simultaneous observations revealed negligible exchange of (3)H-cholesteryl oleate, which was included as a nontransferable marker to control for artifacts such as vesicle fusion and aggregation. In addition, all of the ceramide was exchangeable, and the exchange followed monoexponential kinetics, indicating that the ceramide underwent transbilayer movement at a rate faster than or equal to its rate of intervesicle exchange. Two conclusions can be drawn from these observations: (i) the spontaneous transfer of ceramide between cellular membranes is too slow to play a role in rapid, inter-membrane signaling phenomena and can only be a factor in cell functions that take place over days; and (ii) without the aid of an exchange protein, ceramide can only interact with target molecules that are located at the membrane where the ceramide is formed.

Cell Membrane↗

PubMed1999

Sphingolipid metabolism during human platelet activation.

Thrombin activation of human platelets causes release of sphingosine-1-phosphate from platelets and an increase in sphingosine levels. Sphingosine-1-phosphate is also known to potentiate platelet aggregation. Thus, these sphingolipids may serve as second messengers during platelet activation making it possible that another sphingolipid, ceramide, might play a role in platelet function. Platelets are known to contain sphingomyelinase activity and hydrolysis of sphingomyelin by this enzyme yields phosphocholine and ceramide. Since ceramide is thought to exert its effects through regulation of protein kinases and phosphatases, both of which are involved in platelet function, it is possible that ceramide produced during platelet activation could be involved in regulating signal transduction events. To investigate this possibility, potential changes in levels of ceramide and sphingomyelin in resting and thrombin-activated platelets have been evaluated. Thin-layer chromatographic analysis of the total mass or of radiolabeled (14C-palmitate or 14C-serine) pools of ceramide and sphingomyelin did not reveal any significant changes in the concentrations of either of these molecules during platelet activation. In addition, activation of platelets labeled with [14C-choline]-sphingomyelin did not lead to production of 14C-phosphocholine, suggesting that platelet activation did not cause sphingomyelinase to hydrolyze the [14C-choline]-sphingomyelin. Taken together, our results suggest that ceramide does not serve as a second messenger during platelet aggregation.

Blood Platelets↗

PubMed1999

Platelet adhesion to collagen type I, collagen type IV, von Willebrand factor, fibronectin, laminin and fibrinogen: rapid kinetics under shear.

Extracellular matrix proteins in the blood vessel wall fulfill an essential role in haemostasis by promoting platelet adhesion at the site of vessel injury. We have combined a continuous-flow system with affinity chromatography to study platelet adhesion under conditions mimicking arterial flow and have examined the adhesion kinetics of unstimulated platelets to collagens type I and IV, von Willebrand factor (vWf), fibronectin, laminin and to fibrinogen. In the absence of red cells, in ACD-prepared plasma adhesion to collagens type I and IV or vWf was rapid, efficient (>50% in <1 s ) and independent of shear rates from 650 to 3400 s(-1) with kinetics following an inverse exponential decay curve. We introduced a simple mathematical model in which this type of kinetics arises, and which may be more generally applicable to various adhesion processes under flow conditions. The model is characterized by the rate of platelet deposition on the adhesive surface being proportional to the number of platelets in the flow. Adhesion to fibronectin was independent of shear rate, but revealed a lag phase of approximately 1.5 s before significant adhesion began. Laminin and fibrinogen supported efficient adhesion at low shear rates (650-1000 s(-1)), but a lag phase of approximately 1.5 s was seen at high shear rates (1700-3400 s(-1)). Control proteins (albumin and gelatin) supported minimal adhesion. Nonspecific adhesion to poly-L-lysine differed from that to other substrate proteins in that the kinetics were linear. In conclusion, human platelets adhered specifically, rapidly (within seconds) and efficiently to several proteins under flow conditions and the kinetics of adhesion depended on the protein serving as substrate as well as on shear rate.

Blood Platelets↗

PubMed1998

Sphingomyelinase activity in human platelets.

The sphingolipid metabolites, ceramide, sphingosine, and sphingosine-1-phosphate, may be involved in several signalling pathways and may regulate cell functions such as cell growth, secretion, differentiation, and apoptosis. During activation of human platelets by thrombin, sphingosine-1-phosphate is released from platelets and can potentiate their aggregation. Thrombin also causes an increase in platelet sphingosine levels. Since these molecules can be derived from sphingomyelin, we have determined whether platelets possess sphingomyelinase and whether this enzyme is regulated during platelet function. Using radioactive sphingomyelin as substrate, we assayed sphingomyelinase activity over the range of pH 4 to 10 and observed optimal activity at pH 5.0-5.5. Little activity was found at neutral or alkaline pH, and the presence of Mg++, Ca++, Zn++, or EDTA in the reaction mixture had little effect on the pH profile. Activation of platelets by thrombin or ADP had no effect on sphingomyelinase activity, but thrombin caused secretion of the acid-sphingomyelinase activity into the media. Thus, human platelets contain an acid-sphingomyelinase which is secreted during thrombin-induced platelet activation.

Acid-Base Equilibrium↗

PubMed1998

Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation.

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.

Adenine↗

PubMed1997

Platelet adhesion to collagen under flow causes dissociation of a phosphoprotein complex of heat-shock proteins and protein phosphatase 1.

Phosphorylation/dephosphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions. Using 32P-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the alpha2beta1 integrin resulted in a strong dephosphorylation of several protein bands. Neither adhesion to polylysine nor thrombin-induced aggregation caused similar protein dephosphorylation. In addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosphatases. Recent studies indicate that phosphatases may be associated with the heat-shock proteins. Immunoprecipitations with antibodies against either the heat-shock cognate protein 70 (hsc70) or heat-shock protein 90 (hsp90) showed the presence of a phosphoprotein complex in 32P-labeled, resting human platelets. Antibody probing of this complex detected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1C alpha and PP1C delta, as well as the M regulatory subunit of PP1 (PP1M). OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platelets adhering to collagen, hsc70 was completely dephosphorylated and hsp90, PP1 alpha, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platelet adhesion.

Adenosine Triphosphatases↗

PubMed1997

Platelet adhesion to collagen activates a phosphoprotein complex of heat-shock proteins and protein phosphatase 1.

Rapid activation of blood platelets is required for effective haemostasis, with shape change, aggregation, secretion of granule contents and cell adhesion occurring in seconds or even milliseconds. Signal-transduction events, evidenced by changes in protein phosphorylation and calcium levels, also take place in this time domain. We have now shown that platelet adhesion to collagen via the alpha 2 beta 1 integrin under arterial shear forces initiated the rapid dephosphorylation of a 67 kDa protein "band" which contained the 70 kDa constitutive heat-shock protein, hsc70. Immunoprecipitation with hsc70 antibodies revealed a large phosphoprotein complex in resting platelets and adhesion caused dissociation of the complex along with dephosphorylation of hsc70. The complex also contained the hsp90 heat-shock protein, protein phosphatase IC, alpha, delta and M subunits, and some 7-8 unidentified phosphoproteins. The data suggest that heat-shock proteins and protein phosphatases are actively involved in integrin-mediated platelet adhesion.

Biotransformation↗

PubMed1990

In vitro susceptibilities of Bordetella pertussis and Bordetella parapertussis to seven fluoroquinolones.

The in vitro susceptibilities of Bordetella pertussis and Bordetella parapertussis to seven fluoroquinolones were assessed by the agar dilution method. Ciprofloxacin and temafloxacin were the most active compounds (MIC for 90% of isolates tested [MIC90], 0.06 microgram/ml), while enoxacin and pefloxacin were the least active (MIC90, 0.5 microgram/ml). Fleroxacin, lomefloxacin, and ofloxacin showed intermediate activities (MIC90s, 0.125 to 0.25 microgram/ml). These results suggest a possible role for the fluoroquinolones in the treatment of pertussis, at least in adult patients.

4-Quinolones↗