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PubMed2002

Mechanisms of the genotoxicity of crocidolite asbestos in mammalian cells: implication from mutation patterns induced by reactive oxygen species.

Asbestos is an important environmental carcinogen in the United States and remains the primary occupational concern in many developing countries; however, the underlying mechanisms of its genotoxicity are not known. We showed previously that asbestos is a potent gene and chromosomal mutagen in mammalian cells and that it induces mostly multilocus deletions. Furthermore, reactive oxygen species (ROS) are associated with the mutagenic process. To evaluate the contribution of ROS to the mutagenicity of asbestos, we examined their generation, particularly hydrogen peroxide, and compared the types of mutants induced by crocidolite fibers with those generated by H(2)O(2 )in human-hamster hybrid (A(L)) cells. Using confocal scanning microscopy together with the radical probe 5,6 -chloromethy-2,7 -dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), we found that asbestos induces a dose-dependent increase in the level of ROS among fiber-treated A(L) cells, which is suppressed by concurrent treatment with dimethyl sulfoxide. Using N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent) together with horseradish peroxidase, we further demonstrated that there was a dose-dependent induction of H(2)O(2) in crocidolite-treated A(L) cells. The amount of H(2)O(2 )induced by asbestos reached a plateau at a dose of 6 microg/cm(2). Concurrent treatment with catalase (1,000 U/mL) inhibited this induction by 7- to 8-fold. Mutation spectrum analysis showed that the types of CD59(-) mutants induced by crocidolite fibers were similar to those induced by equitoxic doses of H(2)O(2). These results provide direct evidence that the mutagenicity of asbestos is mediated by ROS in mammalian cells.

Animals↗
PubMed1999

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi.

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37 masculineC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Animals↗
PubMed1999

Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore.

Confocal laser scanning microscopy and the potentiometric fluorescence probe tetramethylrhodamine ethyl ester were used to measure changes in membrane electrical potential (DeltaPsi(m)) in individual mitochondria after isolation or in the living cell. Recordings averaged over small mitochondrial populations revealed a gradual decline in DeltaPsi(m) caused by the light-induced generation of free radicals. Depolarization was attenuated by dithiothreitol or acidification. In contrast, individual organelles displayed rapid spontaneous depolarizations caused by openings of the mitochondrial permeability transition pore (MTP). Repetitive openings and closings of the pore gave rise to marked fluctuations in DeltaPsi(m) between the fully charged and completely depolarized state. Rapid spontaneous fluctuations in DeltaPsi(m) were observed in mitochondria isolated from rat heart and in mitochondria in living endothelial cells. The loss of DeltaPsi(m) of mitochondria in the living cell coincided with swelling of the organelle and the breakdown of long mitochondrial filaments. In the individual mitochondrion, oxidative stress initially triggered pore openings of shorter duration, before prolonged openings caused the complete dissipation of DeltaPsi(m) and a measurable efflux of larger solutes. Generalizing this scheme, we suggest that under conditions of prolonged oxidative stress and/or cellular Ca(2+) overload, short openings of MTP might serve as an emergency mechanism allowing the partial dissipation of DeltaPsi(m), the fast release of accumulated Ca(2+) ions and the decreased generation of endogenous oxygen radicals. In contrast, loss of matrix metabolites, swelling and other structural damage of the organelle render prolonged openings of the transition pore deleterious to mitochondria and to the cell.

Animals↗
PubMed2001

Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice.

Purified mouse sperm receptor, a zona pellucida glycoprotein called mZP3, binds to plasma membrane overlying acrosome-intact sperm heads (P.M. Wassarman, 1999, Cell 96, 175-183). Some evidence suggests that mZP3 binds to sp56, a protein reported to be associated peripherally with the plasma membrane of acrosome-intact sperm heads (J.D. Bleil and P.M. Wassarman, 1990, Proc. Natl. Acad. Sci., USA 87, 7215-7219; A. Cheng et al., 1994, J. Cell Biol. 125, 867-878). Here, we report that membrane vesicles prepared from acrosome-intact sperm contain sp56. When these vesicles are incubated with eggs they inhibit binding of sperm to eggs in vitro (ID50 approximately 50-100 microg protein/ml). On the other hand, a monoclonal antibody directed against sp56 relieves the inhibition of binding of sperm to eggs by membrane vesicles. As expected, incubation of intact sperm with the antibody directed against sp56 inhibits binding of the sperm to eggs. Results of immunoprecipitation of sperm extracts incubated with mZP3, by either a polyclonal antibody directed against mZP3 or a monoclonal antibody directed against sp56, suggest that mZP3 is specifically associated with sp56. Results of laser scanning confocal microscopy of fixed sperm probed with antibodies directed against either sp56 or a approximately 155 kDa acrosomal protein, suggest that the two proteins are present in the acrosome, but with different distributions. Furthermore, confocal images of sperm, fixed after exposure to purified mZP3 and probed with antibodies against mZP3 and sp56, reveal overlap between mZP3 and sp56 at the surface of the sperm head. The possible implications of these results are discussed in the context of mammalian fertilization.

Animals↗
PubMed1999

Minimally modified low-density lipoprotein induces monocyte chemotactic protein-1 expression in vivo and a novel model for monocyte adhesion to arterial intima.

OBJECTIVE: To test whether minimally modified low-density lipoprotein (MM-LDL) can stimulate the arterial cells expressing MCP-1 in vivo and thus induce monocyte adherence to endothelium. METHODS: An animal model was constructed to study the function of MM-LDL in vivo. MM-LDL (600 micrograms/ml) was perfused into a segment of the femoral artery of rabbits (both ends of which were occluded with removable ligatures). After exposure to MM-LDL for 2 h, the arterial segments were taken out 6 or 18 h later. The arteries were subjected to in situ hybridization with 35S-labeled antisense monocyte chemotactic protein-1 (MCP-1) RNA probe and processed for scanning electron microscopy. RESULTS: The expression of MCP-1 mRNA of the arterial endothelial cells and smooth muscle cells were increased by 60% and 90% 6 h after perfusion and had a 3.2 folds and 1.6 folds increase 18 h after perfusion respectively as compared with control (arteries perfused with saline). There was also apparent monocyte adherence to the endothelium observed by scanning electron microscopy. CONCLUSION: MM-LDL can stimulate vascular cells in expressing MCP-1 mRNA and enhance monocyte adherence to endothelium in vivo.

Animals↗
PubMed2003

[The influence of surface conditioning on the shear bond strength of La-Porcelain and titanium].

OBJECTIVE: To determine the influence of different surface conditioning methods on bonding strength of low fusing porcelain (La-Porcelain) and titanium. METHODS: The surface of the samples were sandblasted for 2 min with 80-250 microns Al2O3 or coated for two times with Si-couple agent or conditioned by pre-oxidation. The shear bond strength was examined by push-type shear test with a speed of 0.5 mm/min in a universal testing machine. Scanning electron microscopy (SEM) and electron probe micro-analyzer (EPMA) were employed to explore the relationship between bonding strength and microstructures, as well as the element diffusion at the interface between porcelain coating and titanium when heated at 800 degrees C. RESULTS: Bonding strength was not statistically different (P > 0.05) after sandblasting with Al2O3 in particle size ranged from 80 microns to 250 microns. When a Si-couple agent was used, bond of porcelain to titanium was significantly lower (P < 0.05). The shear bond strength of the porcelain to the pre-oxidized titanium surface remained unchanged after heating (P > 0.05). The SEM results revealed integrity of porcelain and titanium. CONCLUSION: La-Porcelain showed a small effect of surface coarseness. Sandblasting the titanium surface with 150-180 microns Al2O3 can be recommended as a method for better bonding between La-Porcelain and titanium. The Si-couple agent coating and pre-oxidation of titanium surface is unnecessary.

Aluminum Oxide↗
PubMed2004

[Studies on the change of intercellular connection and the appearance and function of ectodesmata-like structure in garlic clove during the dormancy development].

The characteristics of intercellular connection have been observed during the dormancy development of garlic by the transmission electron microscope (TEM). The results indicated that the appearances of the different states of intercellular connection were physiological adaptation to the development of garlic clove during storage. Ectodesmata-like existed at the wall boundary between the declining tissue and the living cells in the garlic during germination period. By Confocal Laser Scanning Microscopy (CLSM) combined with fluorescence probe, 457Da Lucifer Yellow (LYCH) which was impermeable to the membrane, could enter the living parenchyma cell through symplastic route. The result proved that ectodesmata-like structure, which may be recognized as the modified plasmodesata, still retained physiological activity during a certain time and function as the channels of the symplastic transport of nutrient.

Garlic↗
PubMed2005

[Study on the interface of Ti-porcelain fused under different surface treatment].

OBJECTIVE: This study was made to observe the topography and ionic diffusion of the elements titanium, silicon, et cetera in the Ti-porcelain interface area which was pretreated with different methods. METHODS: Ti-bond porcelain was fused on commercial pure titanium (CPT) with 8 different pretreatment methods; according to the temperature of the preoxidation, there were 300 degrees C, 400 degrees C, 500 degrees C and 600 degrees C groups, and based on the oxidizing-time in the air, there were 5 min, 15 min, 2 h and 24 h groups. The topography and ionic diffusion of elements in the Ti-porcelain interface area were observed with scanning electron microscopy (SEM) and electron probe microanalysis (EPMA) respectively. RESULTS: There were clefts and exfoliations in the 500 degrees C pre-oxidation group and 600 degrees C pre-oxidation group in SEM images. The distribution of titanium in the interlayer in EPMA images decreased from the titanium surface to porcelain, which was opposite to the distribution of silicon. CONCLUSION: The clefts and exfoliations suggested the position of the fracture and brittleness of alpha-Ti[o] layer. The widths of ionic diffusion in the eight groups were different, but it was difficult to measure and compare them exactly.

Dental Alloys↗
PubMed2005

[Effects of bradykinin on the proliferation, apoptosis and differentiation of human keratinocytes].

OBJECTIVE: To investigate the effects of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocyte (HKC) and the underlying mechanisms. METHODS: HKCs were cultured together with 1 x 10(-4) - 1 x 10(-9) mol/L of BK. With methyl thiotetrazole (MTT) and trypan blue staining it was shown that the BK in dose of 1 x 10(-4) mol/L possessed most powerful inhibitory effect, and the survival rate of HKC was 69.3%. Therefore, BK was employed in the dose of 1 x 10(-4) mol/L in the following studies. When the growth of HKCs reached the logarithmic phase, BK in the concentration of 1 x 10(-4) mol/L was added, and it was categorized as the test group (E). HKCs without BK served as the control group (C). The cell cycle and apoptosis were detected by flow cytometry after being cultured for 24 and 48 hours. The change in intracellular calcium [Ca(2+)](i) was determined by means of laser scanning confocal microscopy with calcium fluorescence probe Fluo-3/AM technique. The expression of HKC differentiation labeling protein keratin10 (K10) and involucrin were detected with Strept Avidin-Biotin Complex (SABC) immunocytochemical assay. RESULTS: The cell ratio in G0/G1 phase in E group increased by 34.57% while in S phase decreased by 58.91% in reference to that in C group. The G1/S phase switching of HKCs was obviously inhibited by BK, and apoptosis was stimulated (apoptotic rate of 15.34% in E group vs 5.60% in C group, P < 0.05). The [Ca(2+)](i) increased transiently in HKCs by 163.0% in E group after 3 minutes of BK activation and decreased thereafter in reference to that in C group. The K10 expression in HKC was down-regulated in E group with positive cell rate of 2.20%, which was lower than that of C group (6.89%, P < 0.05). CONCLUSION: The cell cycle process of HKC could be inhibited by high concentration of BK with increased apoptosis and an increase in [Ca(2+)](i), which might be the mechanism of inhibition of growth of HKC in vitro. Furthermore, the epithelial regeneration and HKC differentiation can also be inhibited by BK.

Apoptosis↗
PubMed1991

[Positive identification of dental porcelain in a case of murder].

Dental prostheses can provide important evidence for use in personal identification. In recent years, the variety of materials used in prostheses has increased with the diversification of dental treatment. The detailed analysis of these materials can provide significant information to assist in personal identification. This paper reports a case when identification was achieved by the comparative analysis of fragment of fused dental porcelain. In April, 1990, a woman aged 24 years was found murdered in her apartment in Hamamatsu City, Shizuoka Prefecture. Near her body a small mass of fused porcelain was recovered. Subsequently, a 48 year old male suspect was arrested. An examination of his teeth revealed a small fragment of dental porcelain adhering to an abutment tooth. This was removed for testing. The suspect denied the charge of murder. A comparative analysis of the porcelain found in the apartment and the porcelain that was removed from the suspect's abutment tooth, was carried out by scanning electron microscopy (SEM) and electron probe X-ray microanalysis (EPMA). The SEM examination demonstrated remarkable similarity in respect of the structure of the porcelain and the nature and distribution of the air bubbles produced during the baking process in both samples. The EPMA showed the elemental composition of each sample to be identical. This case demonstrated that detailed analysis of dental materials may affords a useful aid for personal identification.

Adult↗
PubMed1989

Bonding behavior of a glass-ceramic containing apatite and wollastonite in segmental replacement of the rabbit tibia under load-bearing conditions.

Glass-ceramic implants containing apatite and wollastonite were studied under load-bearing conditions in a segmental replacement model in the tibia of the rabbit. Alumina-ceramic implants were used as a control. A sixteen-millimeter segment of the middle of the shaft of the tibia was resected at a point distal to the junction of the tibia and the fibula. The defect was replaced by a fifteen-millimeter-long hollow, cylindrical implant that was fixed by intramedullary nailing using a Kirschner wire. Two groups of eight rabbits each (one group with a glass-ceramic implant and the other with an alumina implant) were killed twelve weeks after implantation. Two similar groups were killed twenty-five weeks after implantation. The segment of the tibia that contained the implant was excised and tension-tested. The load to failure of glass-ceramic implants containing apatite and wollastonite increased with time. The loads to failure of the glass-ceramic and alumina implants at twelve weeks after implantation were 19.8 +/- 7.06 and zero newtons, respectively. The loads to failure of glass-ceramic and alumina implants at twenty-five weeks after implantation were 126.4 +/- 32.54 and 19.6 +/- 13.92 newtons, respectively. No glass-ceramic implants broke. A calcium-phosphorus layer at the interface of the glass-ceramic and the bone was observed by scanning electron microscopy and electron-probe microanalysis. There was no interposition of soft tissue between the glass-ceramic and the bone, as observed by Giemsa surface staining.

Aluminum Oxide↗
PubMed1982

Changes in Schwann cells and vessels in lead neuropathy.

Transmission electron microscopy (TEM) of peripheral nerve in rats receiving 6% lead carbonate for 4-10 weeks provided evidence of a specific Schwann cell injury, associated with demyelination. Intranuclear inclusions in Schwann cells appeared within 2 weeks of administration of a lead-containing diet. Swelling of Schwann cells and disintegration of their cytoplasm was evident at 4 weeks. Distinctive electron-dense inclusions appeared in both Schwann and endothelial cells during the period of intoxication and were ultrastructurally identical to pathognomonic inclusions of lead poisoning seen in renal tubular epithelial cells. Scanning microscopy (SEM) with electron-probe microanalysis was used to identify the lead-containing deposits. In addition to Schwann cell changes, vessels revealed endothelial cell injury and alteread permeability to macromolecules. Since morphologic changes of Schwann cells precede the development of altered vascular permeability and endoneurial edema, it appears that lead gains access to the endoneurium prior to the development of altered vascular permeability, suggesting that edema and altered endoneurial fluid pressure are epiphenomena that supervene after demyelination occurs. Remyelination, Schwann cell proliferation and formation of onion bulbs are manifestations of persistent toxic injury to myelin-sustaining cells, resulting in chronic demyelination.

Animals↗
PubMed1996

[Confocal laser scanning microscopy for the study of membrane transporter proteins].

Confocal laser scanning microscopy, in combination with fluorescent probes including fluorescence labelled antibodies, enables us to obtain optical sectioning images of fixed and living biological samples. It is a powerful method for studies of membrane transporter proteins in that it provides information on the spatial distribution of not only the transporter proteins themselves, but also the molecules and ions they transport. In the near future, it may also provide information on molecular interaction between and conformation changes of various proteins with the aid of new type of fluorescent probes which utilize the fluorescence resonance energy transfer phenomenon.

Animals↗
PubMed2006

Development of high-throughput, polarization-maintaining, near-field probes.

A novel, chemical-etching technique produces very high throughput, polarization-maintaining probes for near-field, scanning, optical microscopy (NSOM). The process includes coating the tips with aluminum and forming the apertures with a focused ion beam (FIB). The elliptical core fibers used resulted in elliptical apertures for the probes. The throughput of the probes depends on the incident polarization. For polarization parallel to the minor axis, the tip presents an insertion loss of only 20 dB for aperture widths of 55 nm. Probes have a typical polarization extinction of 100 to 1 in the far field. These tips produced NSOM images of gold dots on a GaAs substrate in reflection mode.

Journal Article↗
PubMed2006

Preparation and characterization of anodized titanium surfaces and their effect on osteoblast responses.

In this study, titanium (Ti) surface was modified by anodizing with a mixture of beta-glycerophosphate sodium and calcium (Ca) acetate, and the anodized surfaces were characterized by scanning electron microscopy, X-ray diffraction, and electron probe microanalysis. In vitro osteoblast response to anodized oxide was also evaluated. The anodic oxide produced was observed to have interconnected pores (0.5-2 microm in diameter) and intermediate roughness (0.60-1.00 microm). In addition, anodic oxide was observed to have amorphous and anatase oxide. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Osteoblast differentiation, as indicated by alkaline phosphatase production, was enhanced on anodized surfaces. It was thus concluded from this study that Ca phosphate can be deposited on Ti surfaces by anodization. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of Ca phosphate and higher roughness on anodized Ti surfaces.

Acetates↗
PubMed1999

The NiTi superelastic alloy application to the dentistry field.

The main properties of NiTi alloys are presented, discussed and compared with respect to those of stainless steels. Special regard is given to the use of NiTi alloy as orthodontic wires and endodontic instruments. The superelastic properties of orthodontic wires and endodontic files are measured as a function of the applied stress, of the strain and of the temperature. Both biomedical devices are submitted to surface analysis to control the interface with the biological environment in which they will be immersed.

Biocompatible Materials↗