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PubMed2000

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells.

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.

Animals↗
PubMed1994

The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones.

Calcium bilirubinate, palmitate, carbonate and phosphate have been identified in the cores of cholesterol and pigment gallstones, suggesting a role for precipitated calcium salts in the early events of gallstone formation. Previous studies that compared the calcium salt contents of cholesterol and pigment stones required destruction of gallstone structure. We have used scanning electron microscopy with windowless energy-dispersive x-ray microanalysis to determine the prevalence of calcium salts in a series of cholesterol (n = 105), black pigment (n = 35) and brown pigment (n = 6) gallstones obtained from 146 consecutive patients undergoing cholecystectomy. These techniques provide specific identification of cholesterol and individual calcium salts as they occur within the core, periphery and shell of gallstones without destroying stone structure. Calcium precipitates more than 0.5 micron in diameter can be detected in a cholesterol background at a detection limit of 0.01% by weight. Calcium salts were detected in the centers of 88% of cholesterol and 100% of black (p < 0.05 vs. cholesterol) and brown pigment stones. Calcium bilirubinate was identified in the cores of 54% of cholesterol and in all pigment stones (p < 0.001 black pigment vs. cholesterol). Calcium palmitate was detected in all brown pigment stones, in 39% of cholesterol stones (p < 0.001 vs. brown pigment) and in 31% of black stones (p < 0.01 vs. brown pigment). Peripheral calcium salts were detected less in cholesterol (19%) than in black or brown stones (100%, p < 0.05). Fourteen percent of cholesterol and black pigment stones were surrounded by shells containing mostly calcium carbonate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

[Analysis of the effects of physico-chemical factors on the stability of lead and mercury levels in biological specimens. I. Biological specimens with the addition of Pb and Hg standards].

For the analysis of the process of precipitation and adsorption in the walls of polyethylene and glass vessels in which biological specimens containing mercury and lead were stored, the techniques of scanning electrone microscopy and X-ray microanalysis were applied. It was observed that the losses of: --lead in stored urine specimens were mainly due to precipitation, --mercury in stored urine specimens occur, among others, through adsorption on vessels' walls. X-ray microanalysis may also facilitate determination of the chemical composition of micro-structural fragments deposited on the surface of vessels.

Adhesiveness↗
PubMed1985

X-ray microanalysis in cryosections of natively frozen Paramecium caudatum with regard to ion distribution in ciliates.

Cells of Paramecium caudatum were shock-frozen without pretreatment for cryoultramicrotomy and freeze-dried for subsequent X-ray microanalysis. Na, Mg, P, S, Cl, K, and Ca were detected in different amounts in several subcellular compartments. In particular, calcium was localized below the cell surface (pellicle). Trichocysts were found to contain significant amounts of Na in their base but not in the tip. Na, Mg, P, S, Cl, K, Ca were found in electron dense deposits within the lumen of the contractile vacuole. A small K concentration was found in the cytoplasm and in the mitochondria. X-ray microanalysis of the element distribution in different subcellular compartments provides information for the understanding of cellular functions such as exocytosis, locomotion, and ion regulation.

Animals↗
PubMed1982

Application of phi (rho z) curves and a windowless detector to the quantitative x-ray microanalysis of frozen-hydrated bulk biological specimens.

Preliminary experiments aimed at establishing the validity of applying phi (rho z) curves to the analysis of elements from carbon to calcium in frozen-hydrated bulk biological specimens are described. Computer generated phi (rho z) curves permit simultaneous absorption and atomic number corrections to be made for quantitative analysis. The results so far obtained, whilst not conclusive, indicate that the method is valid for frozen-hydrated samples. The method allows non-ideal standards of inorganic salts to be used for quantitative light element analysis. Simultaneous oxygen analysis at 15kV with a windowless detector is possible using ice as a standard. With further refinements in spectrum processing, carbon analysis may also be possible using pure carbon as a standard. phi (rho z) curves are used to predict that absorption of x-rays from elements P to Ca will be low and that topographic changes in the specimen surface will have little effect on analyses, particularly with detectors using high take-off angles. Line-scan analyses support this prediction and in addition show that analyses of carbon and oxygen are strongly influenced by topography. The depth resolution of analysis is also predicted by phi (rho z) curves. For elements Na to Ca resolution at 15kV is approximately 2.0 micron and 1.0 micron at 10kV. These predictions are supported by experiments on frozen-hydrated sections. phi (rho z) curve analysis indicates that Na x-rays will be strongly absorbed in a frozen-hydrated specimen. It is shown experimentally that with a windowless detector the practical limit of Na detection in a frozen-hydrated bulk sample is between 10 and 25mM per kg wet wt at 15kV.

Animals↗
PubMed1986

Pt-staining of peroxidatic reaction products at the ultrastructural level.

The use of H2PtCl6 is proposed for the selective visualization of the poly-DAB reaction product created, in aldehyde-fixed tissue, with the cytochemical reaction according to Graham and Karnovsky (1966) or to Hoefsmit (1975). At sites known to contain peroxidatic activity, at the ultrastructural level, an electron-dense reaction product is acquired in otherwise unstained ultrathin sections. The presence of the element platinum in these sites has been demonstrated by X-ray microanalysis, for both the endogenous peroxidase and peroxidase conjugated to antibodies. The absolute platinum concentration has been established in erythrocytes and the granules in eosinophils and monocytes by co-embedded, Pt-containing Chelex ion-exchange beads next to the cells. By the application of the method of integrated morphometrical and chemical analysis (de Bruijn and Zeelen 1984; de Bruijn 1985; de Bruijn and Cleton 1985), both the elemental concentration and the area occupied have been calculated for eosinophil granules. The mean Pt net-intensity values of the cytoplasmic areas, known not to contain the enzyme peroxidase has been measured, and compared to the mean net-intensity Pt values of the granules. It was noted that the cytoplasmic Pt net-intensity values were not zero. The two sets of values are expressed as a mean Pt granule/cytoplasm ratio, this ratio creates a value for the "selectivity" of the reaction. The application of a postfixation reaction with OsO4- containing media, at pH 7.4, in addition to the H2PtCl6 reaction, resulted in a contrasted poly-DAB reaction product at all sites known to contain peroxidatic activity. However, X-ray microanalysis revealed that in addition to platinum, osmium was present.(ABSTRACT TRUNCATED AT 250 WORDS)

3,3'-Diaminobenzidine↗
PubMed2004

Structural and analytical comparison of gallbladder stones collected from a single patient: studies of five cases.

We observed the gross and fine structure of gallbladder stones collected from five adult patients (cases I-V) by optical photography, radiography, scanning electron microscopy, and backscattered electron microscopy, and then measured the components by energy-dispersive X-ray microanalysis and infrared spectroscopy. From the stones, calcium (Ca) phosphate, Ca bilirubinate, and Ca palmitate or fatty acid Ca were identified. The 3 cholesterol stones (case I) and the 2 brown pigment stones (case II) showed macroscopic homogeneity, respectively. In addition, their fine structure and components were also similar to each other. The black pigment stones (case III) showed macroscopic homogeneity, but they were divided into radiopaque (approximately 30 stones) and radiolucent types (approximately 60 stones). The former had Ca phosphate in the center surrounded with Ca bilirubinate, and the latter was dotted with minute deposits of Ca bilirubinate. The 6 cholesterol stones (case IV) were divided into two types in size. The 5 large stones, of macroscopic homogeneity, had a core region of Ca palmitate and clear concentric rings of Ca phosphate, whereas the smaller stone was almost filled with Ca phosphate deposits in the center. From the different distributions of Ca phosphate, the smaller stone may have been formed later than the 5 large stones. Case V contained 4 stones. The 3 large cholesterol stones, of more or less macroscopic homogeneity, had a core region and concentric rings of Ca phosphate, but 1 smaller stone was dotted with minute deposits mainly containing iron (Fe) and/or silicon elements (rare type). Therefore, the stones of cases III, IV, and V showed considerable heterogeneity, respectively. In many stones, the initial precipitation of Ca salts will have become the nidus, and the concentric rings and dotted deposits of Ca salts may have accelerated cholesterol stone growth. In addition, the dotted deposits of Ca bilirubinate in the black pigment stones and the dotted deposits containing Fe in the rare stone may have become also the nidi.

Adult↗
PubMed2004

Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats.

Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.

Animals↗
PubMed1991

Effect of a platinum chemotherapy drug on intracellular elements during the cell cycle, using X-ray microanalysis.

Intracellular elemental concentrations were measured in the cytoplasm, nucleus and nucleolus of cultured Chinese hamster ovary (CHO) cells, using energy dispersive electron probe X-ray microanalysis and transmission electron microscopy. Synchronous CHO cell populations were analyzed at different times during the growth cycle. The elements K, P, Mg and Zn were all more concentrated in the nucleus and nucleolus than in the cytoplasm, while no specific subcellular compartmentalization was evident for the elements Na, Cl, Ca, Fe and S. Significant changes in intracellular elemental concentrations were associated with the progression of cells from G1 phase to S phase of the growth cycle. Most significant were the effects on the monovalent ions, Na, K and Cl. The effect of a second generation platinum chemotherapy agent, cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum IV (iproplatin) on intracellular elements was investigated by analyzing subcellular compartments of drug-treated synchronous CHO cell populations. Changes in intracellular elemental levels occurred, most notably in the nucleus of G1 phase cells, when a general depletion of most essential elements was evident. Attempts to analyze cells for their Pt content proved disappointing, since quantitation for Pt was not possible. However, measurement of Pt peak/background ratio yielded significant Pt peaks on analyzing lysosomes of cells treated with high concentrations of iproplatin. These Pt peaks were associated with high levels of S and Fe.

Animals↗
PubMed1978

Keratopathy following retinal detachment surgery.

Light microscopy, electron microscopy, and histochemical studies were carried out on the corneas of three patients in whom identical keratopathy developed following extensive retinal detachment surgery with silicone-oil injection. These studies revealed the presence of granular deposits in Bowman's layer. Histochemistry and electron-probe analysis showed these deposits to be calcium phosphate. No silicone was detected in the tissue.

Adult↗
PubMed1983

Analysis of surface contaminants on intraocular lenses.

Surface contaminants have been studied on a variety of intraocular lenses by three methods: scanning electron microscopy, energy dispersive x-ray analysis (EDXA), and electron spectroscopy for chemical analysis (ESCA). Many particulate contaminants were observed on both commercially available lenses packaged for implantation and experimental batches of lenses. The nature of these particles could be inferred using the EDXA technique. Thin films of sodium dodecyl sulfate (SDS) that were resistant to removal were found on many of the lenses by ESCA. Cleaning procedures were developed that did not involve SDS and that generated extremely clean lens surfaces.

Animals↗
PubMed1985

Drusen of the optic nerve associated with retinitis pigmentosa.

Globular excrescences of the optic nerve associated with retinitis pigmentosa have been interpreted as drusen or as astrocytic hamartomas. The histopathologic and ultrastructural findings of globular excrescences of the peripapillary region of the optic nerve associated with retinitis pigmentosa were described in a 22-year-old patient who died in a car accident. From our findings we conclude that the globular excrescences of the optic nerve associated with retinitis pigmentosa are definitely drusen and not astrocytic hamartomas.

Adult↗
PubMed1996

Characterization of in vivo wear debris from ceramic-ceramic total hip arthroplasties.

In contrast to the much-studied mechanism of aseptic loosening of the metal-polyethylene joint couple, the mechanism responsible for failure of ceramic-ceramic (CC) total hip arthroplasties (THAs) has not been evaluated. The aim of this study was to conduct a systematic characterization of the in vivo wear debris from 15 cases of CC THAs revised for aseptic loosening. Two methods were used to evaluate the wear debris; a semiquantitative histological analysis of H&E-stained periprosthetic pseudomembranes; and an evaluation of isolated debris particles using SEM, energy-dispersive X-ray analysis, and image analysis. The three main types of particulate debris identified were titanium alloy (TiAlV) and alumina ceramic (Al2O3) of prosthetic origin, and zirconium dioxide (ZrO2) from the contrast agent used in the cement for prosthetic fixation. Alumina debris was present in the smallest proportion (12%) and was consistent with the low wear rate of the CC joint couple. Zirconium dioxide debris was present in the greatest proportion (76%) and was an unexpected finding. The ZrO2 debris represented microstructural grains of the original ZrO2 particles added as contrast agent to the cement. The presence of a histiocytic foreign body reaction to ZrO2 debris on histologic sections leads us to believe that these particles play an important role in aseptic loosening of the CC THAs evaluated in this study.

Alloys↗
PubMed1998

Chemical modification of titanium surfaces for covalent attachment of biological molecules.

The surface of implantable biomaterials is in direct contact with the host tissue and plays a critical role in determining biocompatibility. In order to improve the integration of implants, it is desirable to control interfacial reactions such that nonspecific adsorption of proteins is minimized and tissue-healing phenomena can be controlled. In this regard, our goal has been do develop a method to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive organic molecules. Titanium first was chemically treated with a mixture of sulfuric acid and hydrogen peroxide to eliminate surface contaminants and to produce a consistent and reproducible titanium oxide surface layer. An intermediary aminoalkylsilane spacer molecule was then covalently linked to the oxide layer, followed by the covalent binding of either alkaline phosphatase or albumin to the free terminal NH2 groups using glutaraldehyde as a coupling agent. Surface analyses following coating procedures consisted of X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Enzymatic activity of coupled alkaline phosphatase was assayed colorimetrically, and surface coverage by bound albumin was evaluated by SEM visualization of colloidal gold immunolabeling. Our results indicate that the linkage of the aminoalkylsilane to the oxidized surface is stable and that bound proteins such alkaline phosphatase and albumin retain their enzymatic activity and antigenicity, respectively. The density of immunolabeling for albumin suggests that the binding and surface coverage obtained is in excess of what would be expected for inducing biological activity. In conclusion, this method offers the possibility of covalently linking selected molecules with known biological activity to oxidized titanium surfaces in order to guide and promote the tissue healing that occurs during implant integration in bone and soft tissues.

Biological Factors↗
PubMed1998

XPS and SEM detection of surface changes on 64 ureteral stents after human usage.

Ureteral stents are commonly implanted to assist the postsurgery flow of urine from the kidney to the bladder. We hypothesized that different surface compositions of stent material could result in different conditioning film depositions and potentially altered receptivity to bacterial biofilms. Using XPS, three types of ureteral stents recovered from 64 patients were found to have adsorbed conditioning films that altered the surface composition of the devices. Elements associated with encrustation (calcium, magnesium and phosphorus) were found on 69% of the silicone latex stents, 44% of the low surface energy (LSe) devices, and 38% of the carbon-rich stents. No statistical difference was found in relation to patient gender, stent type, duration of implantation, and encrustation deposition. The composition of the film suggested that the nature of the underlying material did not significantly alter the elements that adsorbed. Thus, devices may take on a similar surface coat within days, and perhaps hours, of exposure to the host. With respect to dense encrustations, fewer appeared on the LSe devices. SEM confirmed the presence and nature of the film crystals and showed bacterial biofilms adherent to devices and encrustations in three patients who had received prophylactic trimethoprim compared to one on ciprofloxacin. In conclusion, although encrustation deposition and biofilm formation on ureteral stents is not unique to Cook devices, the human model and surface science test systems described here are invaluable to evaluate biomaterials used in patients. Unless biomaterials undergo rigorous analysis in vivo, including true assessment of the outcome of prophylactic antibiotic usage, clinicians will be unable to accurately select the best device and management strategy for a given patient.

Adult↗
PubMed1983

Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid.

A simple, rapid screening method using alizarin red S stain and ordinary light microscopy to detect microcrystalline or noncrystalline calcium phosphate salts was used on wet drop preparations of synovial fluids. This proved to be helpful in detecting apatite crystal clumps and small calcium pyrophosphate dihydrate (CPPD) crystals missed by polarized light. The staining was positive in 100% of synovial fluids from patients later proven to have apatite and/or CPPD deposition diseases. Apatite and CPPD crystals were commonly found together in the same fluids. In addition, some synovial fluids from patients with osteoarthritis, renal failure dialysis, rheumatoid arthritis, and gout also exhibited positive staining. The correlation of positive alizarin red S staining with radiologic evidence of osteoarthritis suggests that apatite crystals might be related to articular cartilage degeneration in different rheumatic diseases.

Anthraquinones↗
PubMed2002

Surface modification of poly (D,L-lactic acid) with chitosan and its effects on the culture of osteoblasts in vitro.

Chitosan is a good biodegradable natural polymer, widely used in biomedical fields. In this study, chitosan was used to modify the surface of poly (D,L-lactic acid) (PDLLA) in order to enhance its cell affinity. The properties of a modified PDLLA surface and control were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA), which indicated the changes in surface energy and chemical structure. Scanning electron microscopy (SEM) observation displayed differences in surface morphology between the chitosan-modified film and the control. These data reflected that PDLLA films could be modified with chitosan and in turn may affect the biocompatibility of the modified films. Therefore, adhesion and growth of osteoblasts on modified PDLLA films as well as control were studied. Cell morphologies on the films were examined by SEM and cell viability was evaluated using an MTT assay; the differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity. The ALP activity of modified PDLLA films was significantly higher than that found on the control (p < 0.01). The proliferation of osteoblasts on modified films was also found to be higher than that on the control (p < 0.05), suggesting that chitosan could be used to modify PDLLA and then enhance its cell biocompatibility.

Alkaline Phosphatase↗