Search PubMed⌕ Search

Biomedical subjects

I M Shapiro

Publications and source records attributed to I M Shapiro.

At least 37 records · Page 2Linked to original sources
PubMed1999

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

1-Carboxyglutamic Acid↗
PubMed1999

Induction of apoptosis in human T-cells by methyl mercury: temporal relationship between mitochondrial dysfunction and loss of reductive reserve.

The objective of our study was to define the mechanism by which MeHgCl induces human T-cell apoptosis. We asked the question: does mercury disrupt the Deltapsim and induce a mitochondrial permeability transition state? Using two fluorescent reagents, JC-1 and DiOC6(3), we demonstrated that MeHgCl exposure resulted in a decrease in the Deltapsim. Since a decline in Deltapsim can disturb the pHi, we employed SNARF-1 to assess pHi; results indicate that mercury treatment reduced the pHi from 7.0 to 6.5. Consistent with these observations, we noted that uncoupled electron transfer reactions generated ROS, while cardiolipin, a mitochondrial phospholipid, was oxidized. In concert with the biochemical changes, there was a decrease in overall dimension of the mitochondria of mercury-treated cells and a loss in cristae architecture. The toxicant also depleted the thiol reserves of the cell and promoted translocation of cytochrome c from the mitochondria to the cytosol. Furthermore, when T cells were thiol-depleted, there was increased susceptibility to MeHgCl-induced apoptosis. Finally, we established a temporal relationship between the decline in Deltapsim, generation of ROS, and depletion of thiol reserves. The earliest detectable event was at the level of the mitochondrion; in the presence of MeHgCl there was a profound reduction in mitochondrial Deltapsim and a decline in GSH levels within 1 h. Subsequently, a further decrease in thiol reserves was linked to the generation of ROS. We propose that the target organelle for MeHgCl is the mitochondrion and that induction of oxidative stress leads to activation of death-signaling pathways.

Adult↗
PubMed1999

Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes.

Epiphyseal chondrocytes end their life cycle through apoptosis. While this event provides a mechanism for the removal of terminally differentiated cells from cartilage, agents that promote this physiological process have not been defined. To address this issue, using a cell culture technique that models events that take place in the growth plate, we asked the following questions: Can agents that promote chondrocyte maturation and cartilage mineralization serve as specific triggers for cell death? Are chondrocytes susceptible to apoptogens at a singular developmental stage? Treatment of embryonic tibial chondrocytes with inorganic phosphate (Pi) induced death in a dose- and time-dependent manner. Within 48 hr, 3 mM Pi increased chondrocyte death by 30%; lower concentrations of Pi induced death after 48 hr. To ascertain if death was due to apoptosis, we evaluated Pi-induced death by a number of different methods and compared the results to those induced by the apoptogen, staurosporine. Analysis of the death process indicated that cartilage cells shared many of the common biological features of the apoptotic process. Thus, there was DNA fragmentation, terminal deoxynucleotidyl transferase (TUNEL) labeling, an increase in cells in the sub-G1 fraction of the cell cycle, and morphological evidence of apoptosis. To explore the specificity of the Pi effect, the experiment was repeated using embryonic sternal cephalic and caudal chondrocytes, cells that are at an earlier developmental stage than the terminally differentiated tibial cells. We noted that these cells remained vital despite a major increase in the medium Pi content. Results of this study suggest that Pi is a stage-specific inducer of apoptosis in maturing chondrocytes and that this role may be linked to chondrocyte maturation and mineralization of the extracellular matrix.

Animals↗
PubMed1999

Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate.

In the companion article, we reported that the local phosphate (Pi) concentration triggers apoptosis in epiphyseal chondrocytes. The goal of the current investigation was to evaluate the apoptotic process in relationship to the energy status of cells in the growth plate. For these studies, we used sections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation-dependent loss of mitochondrial function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4-dinitrophenol as well as the site-specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded that oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections using the mitochondrial probes Rh123 and DiOC6(3) provided unequivocal evidence that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoamide dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl-2 expression in these cells. Although immunohistochemical and Western blot analysis showed that the chick cells contained a low level of the anti-apoptotic protein Bcl-2, reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that transcripts were present in chondrocytes. Based on these observations, we suggest that terminally differentiated chondrocytes undergo a maturation-dependent loss of mitochondrial function. In concert with the low expression of Bcl-2, they become sensitive to signals for programmed cell death. We hypothesize that Pi triggers apoptosis in these energy-compromised cells by promoting a mitochondrial membrane transition, thereby inducing the death process.

Adenosine Triphosphate↗
PubMed1999

Effect of serum proteins on osteoblast adhesion to surface-modified bioactive glass and hydroxyapatite.

Previous studies indicate that modification of the surface of porous bioactive glass promotes osteoblast function. We hypothesize that bone formation on treated bioactive glass is due to the selective adsorption of serum attachment proteins. To test this hypothesis, we examined the profile of proteins adsorbed to treated bioactive glass and compared these proteins with those adsorbed to untreated bioactive glass and porous hydroxyapatite. Porous bioactive glass was treated with Tris-buffered electrolyte solution to generate a calcium phosphate-rich surface layer and then immersed in tissue-culture medium containing 10% serum. Proteins adsorbed to the ceramic surfaces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Porous hydroxyapatite bound a higher amount of total protein than did the other substrates. However, surface-modified porous bioactive glass adsorbed more fibronectin than did hydroxyapatite. The effect of serum-protein adsorption on osteoblast adhesion to bioactive glass and hydroxyapatite was also evaluated. Cell adhesion to porous bioactive glass that was surface-modified and serum-treated was significantly greater than to porous bioactive glass that was either surface-modified or serum-treated. Furthermore, cell adhesion to porous bioactive glass treated to form the dual layer of calcium phosphate and serum protein was significantly higher than adhesion to porous hydroxyapatite with adsorbed serum protein. Results of the study strongly suggest that adsorption of serum fibronectin to the surface of modified porous bioactive glass coated with calcium phosphate may be responsible for enhanced osteoblast adhesion.

Absorption↗
PubMed1999

Discovery: Osf2/Cbfa1, a master gene of bone formation.

This report reviews the current research that has impacted on our understanding of osteogenesis. Recent studies indicate that the transcription factor Osf2 (osteoblast specific transcription factor 2)/Cbfa1 (core binding factor activity 1) serves as a Master Gene regulating osteoblast-specific gene expression. The gene is expressed in cells of the osteoblast lineage only, and this expression is regulated by calciotropic agents. Moreover, when expressed in non-skeletal cells, the cells assume many of the characteristics of an osteoblast. In knockout experiments designed to assess the importance of the gene in osteogenesis, no evidence of bone formation could be observed in animals that are homozygous for the deletion. Studies of the heterozygote indicate that osteoblast function is compromised: there is a severe reduction in the number of bone cells, the tissue is deficient in bone proteins, and the activity of the enzyme alkaline phosphatase is low. It was noted that the heterozygote displays abnormalities that are remarkably similar to those exhibited by cleidocranial dysplastics. Indeed, Osf2 mapped close to a chromosomal locus on chromosome 6p21, long suspected of being involved with the disease. A search conducted for Osf2 mutations in kindreds with cleidocranial dysplasia revealed deletions, insertions, and missense mutations; these mutations are found to segregate with patients who are defined clinically as cleidocranial dysplastic. Aside from providing a new insight into a disease state that has so far avoided molecular analysis, results of the studies emphasize that the loss of a Master Gene drastically alters the development and maintenance of the appendicular skeleton and the craniofacial complex.

Animals↗
PubMed1999

Developmental expression of creatine kinase isoenzymes in chicken growth cartilage.

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.

Animals↗
PubMed1998

Si-Ca-P xerogels and bone morphogenetic protein act synergistically on rat stromal marrow cell differentiation in vitro.

This study describes a novel bioactive xerogel glass as a carrier for bone morphogenetic protein (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro. These cells were seeded onto the surface of xerogel glass disks with BMP either incorporated in the glass, adsorbed to the surface of the glass, or added to the culture media and then compared to cells on glass with no added BMP or to cells on tissue culture plastic (TCP) with and without BMP. Cells were cultured for 6 and 10 days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total protein production. Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass. Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation. These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as a delivery vehicle for BMP.

Alkaline Phosphatase↗
PubMed1998

Low-level methylmercury exposure causes human T-cells to undergo apoptosis: evidence of mitochondrial dysfunction.

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are immunotoxic to the human immune system. The major focus of our study is to demonstrate that methylmercuric chloride (MeHgCl) kills human lymphocytes by inducing apoptosis. T-cells exposed to 0.6-5 microM MeHgCl for 24 h were analyzed by flow cytometry. Methylmercury-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin. Furthermore, T-cells exposed to methylmercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side light scatter. The light scatter and fluorescent changes were consistent with morphological alterations displayed by cells during apoptosis. Cell death was further evaluated by assessing annexin V binding to the plasma membrane. Methylmercury-treated cells exhibited increased annexin V binding indicative of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Using the fluorescent probe DiOC6(3), we noted that methylmercury exposure resulted in a decrease in mitochondrial transmembrane potential (Psim). Since a low Psim is associated with altered mitochondrial function, we also determined if exposure to methylmercury potentiated reactive oxygen species (ROS) generation. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, we evaluated the effect of methylmercury on T-cell GSH content utilizing the fluorescent probe monochlorobimane; in the presence of MeHgCl, there is a marked loss in reduced cell thiols. The results of the study indicate that a key event in the induction of T-cell apoptosis by mercuric compounds is depletion in the thiol reserve which predisposes cells to ROS damage and at the same time activates death signaling pathways.

Apoptosis↗
PubMed1998

Inhibition of poly(ADP-ribose) polymerase rescues human T lymphocytes from methylmercury-induced apoptosis.

The objective of this investigation was to determine the role of poly(ADP-ribose) polymerase (PARP) in methylmercuric chloride (MeHgCl)-induced T-cell apoptosis. Following exposure of human T-cells to 2.5 microM MeHgCl, we observed PARP activation within 45 min. Maximal activation was observed at 90 min after MeHgCl treatment; thereafter, PARP activity declined. The loss in enzyme activity was coincidental with the cleavage of 116-kDa intact PARP protein to an 85-kDa fragment. To address the relationship between PARP activation and induction of apoptosis, we first examined the redox status of T cells treated with MeHgCl. We found that exposure of T cells to low concentrations of this toxicant resulted in decreased levels of reduced pyridine nucleotides and an increase in the relative amounts of oxidized flavoproteins. Thus, the possibility exists that activation of PARP leads to NAD+ depletion and thereby alters mitochondrial redox status. To determine if PARP activation is indeed part of the proapoptotic (destructive) response or a component of the antiapoptotic (protective) response, we employed two inhibitors: 3-aminobenzamide and nicotinamide. Pretreatment of T cells with these inhibitors protected cells from MeHgCl-induced apoptosis; this was seen as a reduction in the uptake of Hoechst 33258 and DNA fragmentation. Moreover, these inhibitors blocked MeHgCl-induced oxidative stress as evidenced by a reduction in reactive oxygen species (ROS) generation. These agents, however, failed to block MeHgCl-dependent decline in mitochondrial transmembrane potential (delta psi m). We conclude that PARP activation leads to proapoptotic events that contribute to MeHgCl-induced cell death.

Apoptosis↗
PubMed1998

Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction.

The major objective of our study was to define the mechanism by which mercuric chloride (HgCl2) induces human T-cell death. Human peripheral blood T-cells were exposed to 0-40 microm HgCl2 and then analyzed for biochemical and molecular features of T-cell apoptosis. HgCl2-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin D. To further evaluate cell death and distinguish between apoptosis and necrosis, translocation of phosphatidylserine to the outer layer of the plasma membrane (annexin V binding), DNA fragmentation (TUNEL assay), and cleavage of poly (ADP-ribose) polymerase (PARP) were assessed. In the presence of 20-40 microm HgCl2, T-cells exhibited increased annexin V binding (28%) and DNA fragmentation (31%). HgCl2-dependent PARP cleavage was also observed by Western blot analysis. Because degradative changes associated with apoptosis are often preceded by disruption of mitochondrial function, HgCl2-treated cells were assessed for disruption of the mitochondrial transmembrane potential (DeltaPsim) and development of the mitochondrial permeability transition state. Using DiOC6(3), we demonstrated that HgCl2 exposure resulted in a decrease in the DeltaPsim. Because a decline in DeltaPsim can disturb the intracellular pH (pHi), we used the fluorescent probe, SNARF-1, to assess intracellular acidification. Treatment of T-cells with HgCl2 resulted in reduced pHi from 7.0 to 6.7. Concomitant with these observations, the fluorescent probe, hydroethidine, was utilized to demonstrate that uncoupled mitochondrial electron transport resulted in increased reactive oxygen species (ROS) generation. Interestingly, in spite of these alterations to mitochondrial function, translocation of cytochrome c to the cytosol was not detected; this correlated with enhanced bcl-2 levels in HgCl2-treated cells. In conclusion, HgCl2 exposure results in oxidative stress and activation of death signaling pathways leading to apoptosis. Collectively, our studies indicate that individual mercurial species are capable of inducing T-cell death by activating specific apoptotic cascades.

Apoptosis↗
PubMed1997

Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve.

The focus of this investigation was to examine the effects of low concentrations of organic mercuric compounds on human monocyte function and to relate these effects to apoptosis. Following exposure of monocytes to 0-5 microM MeHgCl, phagocytic function and capacity to generate a respiratory burst, following PMA activation, were determined. We found that the mercury-treated cells exhibited reduced phagocytic activity. Exposure to the same mercury concentration range, also caused a marked increase in cell death. To ascertain if monocyte death was due to apoptosis, a number of flow cytometric studies were performed. Mercury-treated cells exhibited increased Hoechst 33258 fluorescence, while maintaining their ability to exclude the vital dye 7-aminoactinomycin D. Furthermore, monocytes exhibited changes in light scatter patterns that were consistent with apoptosis; these included decreased forward light scatter and increased side scatter. The percentage of cells undergoing apoptosis was dependent upon the mercury content of the medium, regardless of whether the metal was present as methyl, ethyl or phenyl mercury. Mercury-treated cells also exhibited changes in lipid organization within the plasma membrane as evidenced by increased uptake of the fluorescent probe, merocyanine 540, and by elevated annexin V binding to phosphatidylserine. Using the fluorescent probes DiOC6(3) and rhodamine 123 we noted that within 1 h of exposure to mercury, monocytes exhibited a decrease in mitochondrial transmembrane potential (psi m). Since a decreased psi m is associated with altered mitochondrial function, the hypothesis that mercury potentiated reactive oxygen species (ROS) generation and that these species promoted apoptosis was tested. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, since ROS would also lower monocyte reductive reserve, we also measured GSH levels in mercury-treated cells. Chemical measurement of GSH indicated that there was thiol depletion. We suggest that the low thiol reserve predisposes cells to ROS damage and at the same time activates death-signaling pathways.

Apoptosis↗
PubMed1997

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes.

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.

Animals↗
PubMed1997

Porous bioactive glass and hydroxyapatite ceramic affect bone cell function in vitro along different time lines.

We describe the effects on cell function of treating porous bioactive glass (BG) such that its surface is a composite of carbonated hydroxyapatite and serum protein. The effects on bone cell function of porous hydroxyapatite (HA) ceramic and porous glass treated to become amorphous calcium phosphate only also were studied subsequent to their having adsorbed a serum protein layer. Substrates treated for different durations were seeded with MC3T3-E1 cells and cultured for 3-17 days. Whereas cells seeded on any substrates, BG and HA produced collagen types I and III, bone sialoprotein, and osteopontin, there were significant differences between HA and BG, and among the various surface conditions created on BG. Covering the glass surface with hydroxyapatite and serum protein enhanced expression of high alkaline phosphatase activity, high rates of cell proliferation, and production of mineralized extracellular matrix. The enhancement may be due to the adsorption of a high quantity of fibronectin from the serum onto the reacted bioactive glass surface.

Biocompatible Materials↗
PubMed1997

Induction of apoptosis in human T-cells by organomercuric compounds: a flow cytometric analysis.

Although several lines of investigation demonstrate that many heavy metals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determine if organic mercuric compounds kill human lymphocytes by inducing the cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 microM MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cytometry. Mercury-treated cells exhibited increased Hoechst 33258 and 33342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side scatter. The light scatter and fluorescent changes were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis and necrosis, merocyanine 540 staining and annexin V binding to the plasma membrane as well as DNA fragmentation were assessed. Mercury-treated cells exhibited increased merocyanine 540 fluorescence and annexin V binding along with changes in nuclear morphology consistent with the notion of apoptosis. Conventional agarose gel electrophoresis failed to demonstrate low-molecular-weight DNA bands; however, when probed by flow cytometry using both nick translation and a modified TUNEL assay, patterns consistent with nuclear fragmentation were evident. We noted that the percentage of T-cells undergoing apoptosis was dependent upon the amount of serum present in the medium; as serum concentrations were increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) occurred after 24 hr exposure. Induction of apoptosis was dependent on the mercury concentration and independent of the hydrophobicity of the mercury ligand. Finally, we assessed mercury-dependent apoptosis in activated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apoptosis nor necrosis in these populations. It was concluded that the activation process prevented development of a metabolic state that was required for induction of apoptogenic genes.

Annexin A5↗
PubMed1997

Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix.

The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.

Adsorption↗
PubMed1997

Chondrocytes in the endochondral growth cartilage are not hypoxic.

From an anatomic viewpoint, the blood supply to chondrocytes in the growth cartilage is limited. As a result, it has been suggested that these cells are hypoxic and that this condition regulates chondrocyte maturation and cartilage mineralization. We examined the state of chondrocyte oxygenation in the chick growth plate using a hypoxia-sensing drug, EF5. EF5 is a pentafluorinated derivative of the 2-nitroimidazole, etanidazole, that is metabolically reduced by oxygen-inhibitable nitroreductase(s). Reduced EF5 covalently forms adducts with cellular macromolecules that can be visualized with a highly specific fluorochrome-conjugated monoclonal antibody. When EF5 was injected into chicks and tissues were subsequently examined by immunohistochemical techniques, chondrocytes in articular, proliferating, and hypertrophic cartilage exhibited a low level of fluorescence-detectable binding, suggesting the absence of significant hypoxia. We confirmed that the results were not confounded by tissue-specific factors related to low-chondrocyte nitroreductase activity or problems from drug diffusion into cartilage. Using in vitro systems, we showed that, under conditions of imposed hypoxia, EF5 diffused into the tissue and was bound to chondrocytes. With the use of an in vivo model in which hypoxia was artificially induced by death, chick chondrocytes were found to bind the drug. Although the EF5-binding method is not optimally suited for determining the precise oxygen partial pressure in heterogeneous tissues, such as the growth plate, we concluded that chick chondrocytes are not oxygen deficient in vivo.

Animals↗