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PubMed1994

Electrophoretically mediated microanalysis of calcium.

Electrophoretically mediated microanalysis (EMMA) was used for the determination of calcium. The faster analyte zone containing the calcium was injected spatially behind a slower zone of o-cresolphthalein complexone in a capillary electrophoresis based system. Upon application of the electric field the calcium zone was electrophoretically mixed with the reagent and product was formed. The bulk electroosmotic flow carried the product to the detector where the absorbance of the resulting complex at 575 nm was measured. Quantitation using an internal standard yielded a linear response with an R.S.D. of 8.1%. An inter-method comparison was performed with the standard bulk method and yielded results that did not significantly differ. The advantages of EMMA with respect to traditional methods were addressed.

Calcium↗
PubMed1995

In vitro and in vivo studies of the effect of the concentration of plasticizer di(2-ethylhexyl) phthalate on the blood compatibility of plasticized poly(vinyl chloride) drain tubes.

Evaluation of the effect of the plasticizer di(2-ethylhexyl) phthalate (DEHP) has been carried out on the properties of blood compatibility of plasticized poly(vinyl chloride) (PVC) through an in vitro registering of the amount of total protein in relation to the amount of DEHP. The influence of DEHP on the properties of blood compatibility has also been evaluated in vivo by tracing through a modified method of Gott (LV Gott and A Feruse, Bull NY Acad Med 1972; 48(2): 482-491) the presence of a thrombus and the patency of tubes made from the material tested, as well as the patency of tubes made from the same material having surfaces coated with paraffin and complexone. For all implanted samples the changes in coagulation time and the amount of haemoglobin after their stay inside a blood vessel in the abdominal cavity of a dog have been registered.

Animals↗
PubMedDate not supplied

Antiretroviral activities of anthraquinones and their inhibitory effects on reverse transcriptase.

Alizarin complexone (AC), alizarin Red S (ARS) and various other anthraquinones were evaluated for their inhibitory effects on Rous-associated virus 2 reverse transcriptase (RAV-2 RT). Some 1,2-dihydroxyanthraquinones were active against this enzyme and AC was the most potent inhibitor among these compounds [50% inhibitory concentration (IC50): 3.8 micrograms/ml]. AC slightly inhibited Rous sarcoma virus RT (RSV RT) and human immunodeficiency virus type 1 RT (HIV-1 RT) (IC50: 100 micrograms/ml and 45 micrograms/ml, respectively). However, AC efficiently inhibited focus formation by Rous sarcoma virus (RSV) and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1). Simultaneous administration of AC with RSV to newborn chickens also delayed tumor induction by RSV.

Animals↗
PubMed1993

Fe(2+)-induced lipid peroxidation kinetics in liposomes: the role of surface Fe2+ concentration in switching the reaction from acceleration to decay.

Kinetics of malonyldialdehyde (MDA) accumulation, Fe2+ oxidation, and chemiluminescence (CL) at different initial iron ([Fe2+]) and liposome ([L]) concentrations were measured in liposome suspension. Above certain critical Fe2+ concentrations ([Fe2+]*) the latent period (LP) of LPO development was observed. The method of [Fe2+]* estimation by the dependence of LP value (tau) on [Fe2+] was elaborated. The increase of [L] resulted in decrease of tau and increase of delta MDA as well as SF CL amplitude. [Fe2+]* value changed from 10 to 50 microM with change of [L] from 1 to 4 mg/ml, so that the ratio [Fe2+]*/[L] was kept constant. This may be explained under the assumption that the major part of Fe2+ is bound by the membranes. At concentrations of Fe2+ higher than the critical one, iron chelators (desferrioxamine, o-phenanthroline, and EDTA) and cations (Eu3+, Ca2+, and Fe3+) decreased tau without any essential influence on the CL "slow flash" amplitude (h). Apparently, the only result of iron complexones and cations on LPO is the decrease of Fe2+ ion concentration on the membrane surface. Thus, [Fe2+]* value and surface concentration of Fe2+ are the main parameters determining both kinetics and efficiency of Fe(2+)-induced LPO in membrane systems.

Calcium↗
PubMed1992

Do different fluorochrome labels give equivalent histomorphometric information?

Substances that bind calcium are given to determine where and how fast bone is forming. Several vital dyes are used (tetracycline, calcein, alizarin, xylenol), but it is not known whether the histomorphometric results they provide are equivalent. This work tests whether different fluorochrome labels give the same results when they are quantitatively measured. Twelve-week-old rats (n = 58) were divided into six groups and given double labels IP of calcein, tetracycline HCl, alizarin complexone, or xylenol using a 1-7-1 scheme. Two other groups received either calcein followed by tetracycline, or tetracycline followed by calcein. Our results show that (a) tetracycline hydrochloride leads to a significant underestimation of mineralizing surface when given as the second label, probably because of its weak fluorescence; (b) there were no differences among any of the non-tetracycline labels for any of the histomorphometric measurements; (c) there was no evidence of osteoblast suppression with any label; and (d) there was no evidence that tetracycline caused increased osteoblast resting periods.

Animals↗
PubMed2004

Effect of hypergravity on carboanhydrase reactivity in inner ear ionocytes of developing cichlid fish.

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish. Otolith growth in terms of mineralization mainly depends on the enzyme carboanhydrase (CA), which is responsible for the provision of the pH-value necessary for calcium carbonate deposition. Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (3 g, hg; 6 h) during development and separated into normally and kinetotically swimming individuals following the transfer to 1 g (i.e., stopping the centrifuge; kinetotically behaving fish performed spinning movements). Subsequently, CA was histochemically demonstrated in inner ear ionocytes (cells involved in the endolymphatic ion exchange) and enzyme reactivity was determined densitometrically. It was found that both the total macular CA-reactivity as well as the difference in reactivities between the left and the right maculae (asymmetry) were significantly lower (1) in experimental animals as compared to the 1 g controls and (2) in normally swimming hg-animals as compared to the kinetotically behaving hg-fish. The results are in complete agreement with earlier studies, according to which hypergravity induces a decrease of otolith growth and the otolithic calcium incorporation (visualized using the calcium-tracer alizarin complexone) of kinetotically swimming hg-fish was higher as compared to normally behaving hyper-g animals. The present study thus strongly supports the concept that a regulatory mechanism, which adjusts otolith size and asymmetry as well as otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

Animals↗
PubMed2004

Swimming behaviour and calcium incorporation into inner ear otoliths of fish after vestibular nerve transection.

Previous investigations on neonate swordtail fish (Xiphophorus helleri) revealed that otolithic calcium incorporation (visualized using the calcium tracer alizarin complexone) and thus otolith growth had ceased after nerve transection, supporting a hypothesis according to which the gravity-dependent otolith growth is regulated neuronally. Subsequent investigations on larval cichlid fish (Oreochromis mossambicus) yielded contrasting results, repeatedly depending on the particular batch of cichlids investigated. Like most neonate swordtails, Type I cichlids revealed a stop of calcium incorporation after unilateral vestibular nerve transection. Their behaviour after transection was normal, and the otolithic calcium incorporation in controls of the same batch was symmetric. In Type II cichlids, however, vestibular nerve transection had no effect on otolithic calcium incorporation. They behaved kinetotically after transection (this kind of kinetosis was qualitatively similar to the swimming behaviour exhibited by larval cichlids during microgravity in the course of parabolic aircraft flights). The otolithic calcium incorporation in control animals was asymmetric. These results show that the effects of vestibular nerve transection as well as the efficacy of the mechanism, which regulates otolith growth/otolithic calcium incorporation, are--depending on the particular batch of animals--genetically predispositioned. In conclusion, the regulation of otolithic calcium incorporation is guided neuronally, in part via the vestibular nerve and, in part, via a further pathway, which remains to be addressed in the course of future investigations.

Animals↗
PubMed2004

Calcium-tracers disclose the site of biomineralization in inner ear otoliths of fish.

Since changing gravity (concerning direction and amplitude) strongly affects inner ear otolith growth and otolithic calcium incorporation in developing fish, it was the aim of the present study to locate the site of mineralization in order to gain cues and insights into the provenance of the otoliths inorganic compounds. Therefore, larval cichlid fish (Oreochromis mossambicus) were incubated in the calcium-tracer alizarin complexone (AC; red fluorescence). After maintenance in aquarium water for various periods (1, 2, 3, 6, 9 and 12 h; 1, 2, 3, 5, 6, 7, 15, 29, 36 and 87 d), the animals were incubated in the calcium-tracer calcein (CAL; green fluorescence). AC thus labeled calcium being incorporated at the beginning of the experiment and would subsequently accompany calcium in the course of a possible dislocation, whereas CAL visualized calcium being deposited right at the end of the test. Subsequently, the otoliths were analyzed using a laser scanning microscope and it was shown that the initial site of calcium incorporation was located directly adjacent to the sensory epithelium and the otolithic membrane. Later, calcium deposits were also found on further regions of the otoliths' surface area, where they had been shifted to in the course of dislocation. This finding strongly indicates that the sensory epithelium plays a prominent role in otolithic biomineralization, which is in full agreement with an own electron microscopical study [ELGRA News 23 (2003) 63].

Animals↗
PubMed2004

Efficacy of an ototoxic aminoglycoside (gentamicin) on the differentiation of the inner ear of cichlid fish.

Previous investigations revealed that the growth of fish inner ear otoliths depends on the amplitude and the direction of gravity, thus suggesting the existence of a (negative) feedback mechanism. In the course of these experiments, it was shown that altered gravity both affected otolith size (and thus the provision of the proteinacious matrix) as well as the incorporation of calcium. It is hitherto unknown, as of whether sensory hair cells are involved either in the regulation of otolith growth or in the provision of otolithic material (such as protein or inorganic components) or even both. The ototoxic aminoglycoside gentamicin (GM) damages hair cells in many vertebrates (and is therefore used for the treatment of Meniere's disease in humans). The present study was thus designed to determine as of whether vestibular sensory cells are needed for otolith growth by applying GM in order to induce a (functionally relevant) loss of these cells. Developing cichlid fish Oreochromis mossambicus were therefore immersed in 120 mg/l GM for 10 or 21 days. At the beginning and at the end of the experimental periods, the fish were incubated in the calcium-tracer alizarin complexone (AC). After the experiment, otoliths were dissected and the area grown during GM-exposure (i.e., the area enclosed by the two AC labellings) was determined planimetrically. The results showed that incubating the animals in a GM-solution had no effect on otolith growth, but the development of otolith asymmetry was affected. Ultrastructural examinations of the sensory hair cells revealed that they had obviously not been affected by GM-treatment (no degenerative morphological features observed). Overall, the present results suggest that hair cells are not affected by GM concerning their possible role in (general) otolith growth, but that these cells indeed might have transitionally been impaired by GM resulting in a decreased capacity of regulating otolith symmetry.

Animals↗
PubMed2005

The bisphosphonate pamidronate on the surface of titanium stimulates bone formation around tibial implants in rats.

Many materials with differing surfaces have been developed for clinical implant therapy in dentistry and orthopedics. We analyzed the quantity of new bone formed in vivo around calcium-immobilized titanium implants with surfaces modified using pamidronate (PAM), a nitrogen-containing bisphosphonate (N-BP), implants of pure titanium, and titanium implants immobilized with calcium ions. New bone formation was visualized using fluorescent labeling (calcein blue and alizarin complexone) with intravenous injection at 1 and 3 weeks after implantation. After 4 weeks, undecalcified sections were prepared, and new bone formation around the implants was examined by morphometry using confocal laser scanning microscopy images. After 1 week, more new bone formed around the PAM-immobilized implant than around the calcium-immobilized and pure titanium implants. This was also seen with the new bone formation after 3 weeks. After 4 weeks, significantly more new bones were formed around the BP-immobilized implant than around the calcium ion-implanted and pure titanium implants. The new N-BP-modified titanium surface stimulates new bone formation around the implant, which might contribute to the success of implant therapy.

Animals↗
PubMed2004

A microbead array chemical sensor using capillary-based sample introduction: toward the development of an "electronic tongue".

The development of a micromachined fluidic structure for the introduction of liquid samples into a chip-based sensor array composed of individually addressable polymeric microbeads is presented. The micromachined structure consists of micromachined storage cavities combined with a covering glass layer that confines the microbeads and fluidic channels. In our sensor array transduction occurs via optical (colorimetric and fluorescence) changes to receptors and indicator molecules that are covalently attached to termination sites on the polymeric microbeads. Spectral data are acquired for each of the individual microbeads using a charged-coupled device (CCD) allowing for the near-real-time analysis of liquid sample. Hence the micromachined fluidic structure must allow for both optical access to the microbeads and fluid flow through the micromachined cavities that serve as the microreactors/analysis chambers. One of the key parts of the structure is a passive fluid introduction system driven only by capillary force. This simple means of fluid introduction realizes a compact device. The capillary flow on the inlet channel has been studied, and the responses of the microbeads (alizarin complexone) to a liquid sample have been characterized. The test results show that this system is useful in a micro-total-analysis-system (mu-TAS) and biomedical applications.

Biomimetics↗
PubMed2005

Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis.

Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 mum thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.

Animals↗
PubMedDate not supplied

Disturbance of electrolyte balance in vitreous of chicks with form-deprivation myopia.

PURPOSE: To investigate the changes in the electrolyte and protein concentrations in the vitreous of 3-week-old chicks with form-deprivation myopia (FDM). METHODS: FDM was induced in 2-day-old male white leghorn chicks by covering the left eye with a translucent plastic goggle and leaving the right eye uncovered to serve as control. After 19 days the animals were euthanized, and the axial dimensions of the eyes were measured with a caliper in an unfixed condition. The liquid vitreous and aqueous humor were removed by paracentesis, and blood was collected from the jugular vein. Sodium, potassium, and chloride concentrations were determined using ion-selective electrodes. Calcium and phosphate concentrations were determined by colorimetric assays using orthocresol phthalein complexone and bacterial xanthine oxidase, respectively. RESULTS: The concentrations of potassium and phosphate were decreased, whereas chloride concentration was increased in the vitreous of the FDM eyes (P < .01). Sodium and calcium concentrations were similar to those in the control eyes. No significant changes in the concentration of electrolytes were observed in the aqueous humor. No significant differences were found in the protein concentrations in the liquid vitreous, gel vitreous, and aqueous humor. CONCLUSIONS: Form-deprivation induced a significant increase of the volume of the liquid vitreous in the eye of the FDM chick. The increased liquid vitreous of the myopic eye was accompanied by an alteration of the electrolyte balance, by a mechanism that has not yet been clarified.

Animals↗
PubMed2002

An improved labelling technique for monitoring microcrack growth in compact bone.

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.

Animals↗
PubMed2001

Determination of trace alkaline earth metals in brines using chelation ion chromatography with an iminodiacetic acid bonded silica column.

The chromatographic behaviour of alkaline earth metals on iminodiacetic acid bonded silica was studied. It was found that the ionic strength of the eluent greatly affected both retention time and selectivity by controlling the extent to which either simple ion exchange or surface complexation was responsible for retention. With a 0.1 M KNO3 eluent, the retention order was Mg(II), Sr(II), Ca(II) and Ba(II), indicating a strong contribution to retention from ion exchange. However, when using a 1.5 M KNO3 eluent, Ba(II) was found to elute first, indicating complexation to be more dominant under these conditions (pH 4.2). The effect of the ionic strength of the sample was also studied and it was found that by matching the eluent cation with that of the sample matrix, efficient separations of alkaline earth metals in 1.0 M NaCl and KCl brines could be obtained without matrix system peaks. Using post-column reaction with o-cresolphthalein complexone, trace levels of Ca(II) and Mg(II) were determined in medicinal NaCl saline solution and laboratory-grade KCl.

Chelating Agents↗
PubMed2000

Somatic growth and otolith growth in juveniles of a small subtropical flatfish, the fringed flounder, Etropus crossotus.

A growth experiment was conducted with juvenile fringed flounder (Etropus crossotus) from North Inlet (South Carolina, USA) to provide information on the growth of a small, shortlived flatfish with a subtropical and tropical distribution. The fringed flounder has a maximum life span of 1.5 y and its long spawning period from March through October complicates the determination of growth rates based on length frequency data. Otoliths of juveniles with a standard length (SL) 23.1-53.0 mm were marked with Alizarin complexone and the fish were held in the laboratory for 66 days at 14, 20, 24 and 29 degrees C while being fed ad libitum. The mean somatic growth increased with temperature from 0.1 mm SL day(-1) at 14 degrees C to 0.4 mm SL day(-1) at both 24 and 29 degrees C. The maximum observed somatic growth rate was 0.7 mm SL day(1) at 29 degrees C. The number of micro-increments formed in otoliths was not significantly different from the expected value, validating formation of one increment per day. The significant relationship between increment width and somatic growth rate can be used to estimate somatic growth rates of individual wild fish based on daily increment information in their otoliths.

Journal Article↗
PubMedDate not supplied

Effects of vestibular nerve transection on the calcium incorporation of fish otoliths.

Previous investigations revealed that the growth of fish inner ear otoliths (otolith size and calcium-incorporation) depends on the amplitude and the direction of gravity, suggesting the existence of a (negative) feedback mechanism. In search for the regulating unit, the vestibular nerve was transacted unilaterally in neonate swordtail fish (Xiphophorus helleri) which were subsequently incubated in the calcium-tracer alizarin-complexone. Calcium incorporation ceased on the transacted head sides, indicating that calcium uptake is neurally regulated. Grant numbers: 50 WB 9533, 50 WB 9997.

Animals↗
PubMed2002

Function-morphological investigations of fish inner ear otoliths as basis for interpretation of human space sickness.

In man, altered gravity may lead to a vestibular dysfunction causing space motion sickness. A hypothesis was developed, according to which asymmetric inner ear statoliths might be the morphological basis of space sickness. The animal model, fish, revealed further information: inner ear "stone" (otolith) growth is dependent on the amplitude and the direction of gravity, regulated by a negative feedback mechanism. The present study was focused on the question, where the regulation centre of adaptive otolith growth may be situated. Therefore, the vestibular nerve was unilaterally transected in neonate swordtail fish (Xiphophorus helleri). As growth marker, the calcium tracer alizarin-complexone was used. It was found that otolith growth had ceased on the operated head sides indicating that the brain is significantly involved in regulating otolith growth. About 2 weeks after nerve transection, otoliths had regained normal growth, probably due to nerve regeneration. Concerning fish, it has now to be tested, if this regeneration is affected by altered gravity, e.g. in a long-term experiment on the International Space Station. Regarding mammals, it has to be proved if asymmetric statoliths are the basis of kinetosis and whether or not the mammalian brain has an effect on statolith growth in the course of compensating altered gravity.

Animals↗