Chlorotetracycline Research Articles

Zeolite imidazolate framework-8 as sorbent for on-line solid-phase extraction coupled with high-performance liquid chromatography for the determination of tetracyclines in water and milk samples

Zeolite imidazolate framework-8 (ZIF-8) was used as the novel sorbent for on-line solid-phase extraction coupled with high-performance liquid chromatography (HPLC) for the determination of oxytetracycline (OTC), tetracycline (TC) and chlorotetracycline (CTC) in water and milk samples. 390mg of ZIF-8 was packed into a stainless steel column (3cm long×4.6mm i.d.) which was mounted on the HPLC injector valve to replace the sample loop. On-line solid-phase extraction of OTC, TC and CTC was achieved by loading sample solution at a flow rate of 3.0mLmin−1 for 10min with the aid of a flow-injection system. The extracted analytes were subsequently eluted into a C18 analytical column (25cm long×4.6mm i.d.) for HPLC separation under isocratic condition with a mobile phase (10% MeOH–20% ACN–70% 0.02molL−1 oxalic acid solution) at a flow rate of 1.0mLmin−1. Under optimized conditions, the developed method gave the enhancement factors of 35–61, the linearity range of 5–1000μgL−1, the detection limits of 1.5–8.0μgL−1, quantification limits of 5.0–26.7μgL−1, uncertainties of 0.9–1.1μgL−1, and the sample throughput of 4 samples h−1. The recoveries of OTC, TC and CTC at 50μgL−1 in water and milk samples ranged from 70.3% to 107.4%.

Determination of Chlorotetracycline and Doxycycline in Medicated Feedingstuffs by Liquid Chromatography

Abstract High performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and optimised for the determination of tetracyclines (TCs) in medicated feedingstuffs. The extraction of the analyte from feedingstuffs was performed with Na2EDTA-McIlvaine buffer (pH 2.5 and pH 4). The extracts were cleaned up by solid phase extraction using octadecyl cartridges (C18). The samples were dried up and redissolved in the mixture of oxalic acid and methanol. Separation was performed on reserved phase column (Phenomenex C18, 250 x 4.6 mm, 5 μm) by multistep gradient elution, which provided better chromatographic separation. The analysis was performed at a wavelength of 390 nm. Validation study of the method revealed that all obtained calibration curves showed good linearity R= 0.9985 for doxycycline (DC) and R= 0.9981 for chlorotetracycline (CTC) over the range of 25-2,000 mg/kg. The analytical procedure was successfully adapted for quantitative determination of DC and CTC in medicated feedingstuff samples. Validation included determination of specificity, linearity, and repeatability. Mean recovery for spiked samples was 93.1% for CTC and 93.2% for DC. The results of validation of the analytical procedure proved that presented method is efficient, precise and useful for quantification of DC and CTC in medicated feedingstuffs.

Improved nonaqueous capillary electrophoresis for tetracyclines at subparts per billion level

A NACE method with laser-induced fluorescence detection was modified for sensitive detection of 4 tetracyclines (TCs) in biological samples and feeds. The changes in injection mode, injection times, id of capillary, excitation wavelength, and the use of surfactant and sample stacking technique all contributed to improved LODs of TCs to sub-ng/mL level. With the optimized conditions, the instrumental LODs could reach 1.33 ng/mL for chlorotetracycline (CTC) and 13.3 ng/mL for TC, oxytetracycline (OTC), and doxycycline (DC), an improvement of 10-100-fold over past studies. A simple SPE procedure was further developed for the extraction and concentration of TCs in plasma, urine, feed, and milk. Taken together, the instrumental LOD and feasible SPE concentration factors the overall LODs for CTC could reach 65 pg/mL in feed and milk and 260 pg/mL in plasma and urine. Detection limits for TC, OTC, and DC at sub-ng/mL level were also achieved. The modified CE-LIF method was found to be less complicated and more sensitive than the best current methods using UV or LIF detection, and has been applied successfully to assess oral absorption of DC in swine and chickens and to confirm suspected TC-positive bovine serum samples.

Strong adsorption of chlorotetracycline on magnetite nanoparticles

In this work, environmentally friendly magnetite nanoparticles (Fe 3O 4 MNPs) were used to adsorb chlorotetracycline (CTC) from aqueous media. Fe 3O 4 MNPs exhibit ultrahigh adsorption ability to this widely used antibiotic. The adsorption behavior of CTC on Fe 3O 4 MNPs fitted the pseudo-second-order kinetics model, and the adsorption equilibrium was achieved within 10 h. The maximum Langmuir adsorption capacity of CTC on Fe 3O 4 (476 mg g −1) was obtained at pH 6.5. Thermodynamic parameters calculated from the adsorption data at different temperature showed that the adsorption reaction was endothermic and spontaneous. Low concentration of NaCl and foreign divalent cations hardly affected the adsorption. Negative effect of coexisting humic acid (HA) on CTC adsorption was also observed when the concentration of HA was lower than 20 mg L −1. But high concentration of HA (>20 mg L −1) increased the CTC adsorption on Fe 3O 4 MNPs. The matrix effect of several environmental water samples on CTC adsorption was not evident. Fe 3O 4 MNPs were regenerated by treatment with H 2O 2 or calcination at 400 °C in N 2 atmosphere after separation from water solution by an external magnet. This research provided a high efficient and reusable adsorbent to remove CTC selectively from aqueous media.

Degradation of chlorotetracycline and bacterial disinfection in livestock wastewater by ozone-based advanced oxidation

The simultaneous removal of antibiotics and antibiotic-resistant bacteria by ozone-based advanced oxidation technology (AOT) was investigated for the treatment of anaerobic digestion effluent of piggery wastewater. Chlorotetracycline (CTC) and CTC-resistant bacteria were selected as targets since CTC has been the most used antibiotics in livestock industries in Korea. The performance of four different combinations of oxidation agents was investigated, showing the order of removal rate as O 3 < O 3/H 2O 2 < O 3/UV < O 3/UV/H 2O 2 for wastewater. Applications of O 3/UV/H 2O 2 and O 3/UV degraded almost all CTC within 15 and 20 min, respectively. Only 30% of CTC was degraded even in 40 min by O 3 alone and O 3/H 2O 2 degraded 65% of CTC in 40 min. The combination of ozone/H 2O 2 did not contribute significantly to CTC degradation, but the influence of UV was substantial when coupled with ozone. CTC was partially degraded during oxidation although main HPLC peak was completely vanished. The resulting intermediates of partially oxidized CTC possessed no more antibiotic capability, suggesting that CTC transformed into nontoxic organic constituents. For disinfection, ozone alone was not effective for disinfection; less than 2-log reduction was achieved in 1 h. UV was more superior in reducing bacterial population, showing more than 4-log reduction in 20 min and 5-log reduction in 40 min. UV was also effective in reducing both total and CTC-resistant bacterial population, and the contribution ratio of UV to ozone was about 3:1 in logarithmic scale. The application of O 3/UV together further enhanced the disinfection performance compared to UV alone.

Transformation kinetics and pathways of tetracycline antibiotics with manganese oxide

Tetracycline antibiotics including tetracycline (TTC), oxytetracycline (OTC) and chlorotetracycline (CTC) undergo rapid transformation to yield various products in the presence of MnO 2 at mild conditions (pH 4–9 and 22 °C). Reaction rates follow the trend of CTC > TTC > OTC, and are affected by pH and complexation of TCs with Mg 2+ or Ca 2+. Experimental results of TTC indicate that MnO 2 promotes isomerization at the C ring to form iso-TTC and oxidizes the phenolic-diketone and tricarbonylamide groups, leading to insertion of up to 2 O most likely at the C9 and C2 positions. In contrast, reactions of OTC with MnO 2 generate little iso-OTC, but occur mainly at the A ring’s dimethylamine group to yield N-demethylated products. CTC yields the most complicated products upon reactions with MnO 2, encompassing transformation patterns observed with both TTC and OTC. The identified product structures suggest lower antibacterial activity than that of the parent tetracyclines.

Spectrophotometric study of the interaction between chlorotetracycline and bovine serum albumin using Eosin Y as site marker with the aid of chemometrics

Interaction of chlorotetracycline (CTC) with bovine serum albumin (BSA) was investigated under simulated physiological conditions by spectroscopy with the aid of multivariate curve resolution-alternating least squares (MCR-ALS). Eosin Y was selected as an alternative site I marker on the BSA to study the above molecular interaction. The binding of Eosin Y and CTC to BSA showed that CTC was displaced from CTC–BSA complex by Eosin Y, and Eosin Y–BSA complex was formed. However, the recorded fluorescence spectra of Eosin Y and Eosin Y–BSA overlapped and MCR-ALS was applied to resolve the two-way fluorescence spectra. From the resolved equilibrium concentration profiles, it was observed that Eosin Y competed with CTC in the binding process with BSA; it was also shown that the binding site of CTC on BSA was site I, and this was further confirmed by the fluorescence polarization method. Compared with some common site I markers for BSA, the fluorescence and UV–vis spectral shapes of the Eosin Y–BSA complex were quite different from that of Eosin Y, and this feature facilitated the investigation of the small molecule–BSA interaction.

Adsorption and transformation of tetracycline antibiotics with aluminum oxide

Tetracycline antibiotics (TCs) including tetracycline (TTC), chlorotetracycline (CTC) and oxytetracycline (OTC) adsorb strongly to aluminum oxide (Al 2O 3), and the surface interaction promotes structural transformation of TCs. The latter phenomenon was not widely recognized previously. Typically, rapid adsorption of TCs to Al 2O 3 occurs in the first 3 h ([TC] = 40 μM, [Al 2O 3] = 1.78 g L −1, pH = 5, and T = 22 °C), followed by continuous first-order decay of the parent compound ( k obs = 15 ± 1.0, 18 ± 1.0 and 6.2 ± 0.9 × 10 −3 h −1 for TTC, CTC and OTC, respectively) and product formation. The transformation reaction rate of TCs strongly correlates with adsorption to Al 2O 3 surfaces. Both adsorption and transformation occur at the highest rate at around neutral pH conditions. Product evaluation indicates that Al 2O 3 promotes dehydration of TTC to yield anhydrotetracycline (AHTTC), epimerization of TTC, and formation of Al-TTC complexes. Al 2O 3 promotes predominantly the transformation of CTC to iso-CTC. The surface-bound Al(+III) acts as a Lewis acid site to promote the above transformation of TCs. Formation of AHTTC is of special concern because of its higher cytotoxicity. Results of this study indicate that aluminum oxide will likely affect the fate of TC antibiotics in the aquatic environment via both adsorption and transformation.

Study of robustness based on n-way models in the spectrofluorimetric determination of tetracyclines in milk when quenching exists

An important step in the validation of an analytical procedure is the study of its robustness. In the case of spectrofluorimetric determinations, quenching introduces specific problems which are approached in this paper for the particular case of tetracyclines determination in milk. Quenching can be detected with excitation emission matrices (EEM) signals and a three-way Parallel Factor (PARAFAC) decomposition and modelled by means of a four-way PARAFAC decomposition which reproduces the physical model of this effect. The robustness of the method is evaluated by including changes in seven experimental variables: trichloroacetic acid (TCA) volume solution used in the precipitation of milk proteins, revolutions per minute, time and temperature in the centrifugation step, pH and emission-excitation slit width in the fluorimetric analyte determination and the analyst. The robustness analysis is carried out by means of a Plackett-Burman experimental design as it is suggested by European Decision 2002/657/EC (European Decision (EC) No. 2002/657/EC of 12 August 2002, implementing Council Directive 96/23/EC, concerning the performance of analytical methods and the interpretation of results, Off. J.L 221, 17/8/2002, 8). The analyte concentration will be taken as response in the Plackett-Burman experimental design instead of the signal as it is habitual in these cases. Therefore, a three-way Partial Least Squares (3-PLS) calibration models with EEM signal is needed. When an analogous study is carried out for tetracycline (TC) in the absence of chlorotetracycline (CTC) as interferent, univariate calibration is employed, being able to conclude that in the robustness analysis, different factors are significantly active when quenching exists.

Electrospun poly(D,L‐lactide) fibers for drug delivery: The influence of cosolvent and the mechanism of drug release

AbstractControlled‐drug release from electrospun fibers has found important biomedical applications in wound healing, transdermal delivery, and tissue engineering. In this study, poly(D,L‐lactide) (PDLLA) was electrospun into ultrafine fibers and loaded with tetracycline (TC) or chlorotetracycline (CTC) as model drugs. The influence of a cosolvent (methanol) at various concentrations was studied regarding physical properties, morphology, and in vitro release profiles of the drugs from the PDLLA nanofibers. Swelling tests in a physiological buffer solution were performed to determine the extent and rate of swelling of the fiber mats. The results showed that for both drugs electrospun fiber diameters decreased with increasing amounts of cosolvent, whereas water contact angles and drug‐loading efficiency increased. However, similar in chemical structure, the two drugs exhibited considerably different release mechanisms. The results indicated that the concentration of methanol changed the release profiles mainly based on the morphology of the resultant nanofibers and the polymer/drug/solvent interaction during the electrospinning and drug release process. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Effect of chondroitin sulfate C on sperm capacitation and fertilization parameters in vitro in pigs

The objective of this study was to determine the effects of chondroitin sulfate C (CS-C) on sperm capacitation and fertilization parameters in vitro in pigs. Frozen–thawed ejaculated pig sperm (semen S-484) were incubated with fertilization medium containing CS-C (0–2 mg/ml) for 1 h and the capacitation rate with chlorotetracycline (CTC) assay was examined, which showed that CS-C increased the rate of incapacitation F pattern spermatozoa converted to capacitation B pattern sperm cell in concentration-dependent manner and mostly increased capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in 1 mg/ml concentration. When sperm was incubated for 1, 2 and 4 h in fertilization medium containing 1 mg/ml CS-C, it showed that the capacitated B pattern sperm cell was significantly ( p < 0.01) increased and the AR pattern sperm cell was significantly decreased at each time point in the presence than in the absence of CS-C. For identifying the validity of CS-C in sperm capacitation, sperm–oocyte was inseminated in fertilization medium containing CS-C (0–2 mg/ml) and the rate of fertilized oocytes was examined, which showed that the penetration rates significantly ( p < 0.05) increased from 0.5 to 1.0 mg/ml concentrations (87.4–96.3%) compared with control (74.9%). For identifying the universality of CS-C in sperm capacitation, four different semens (boar S-484, S-454, D-815 and D-748) were incubated in fertilization medium containing CS-C (1 mg/ml) for 2 h, respectively, which showed that CS-C increased the rate of capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in each semen. And it showed that CS-C yielded a higher promote effect (93.9%, 83.9%, 60.7% and 44.9%, respectively) on sperm penetration compared to unaddition control (63.4%, 22.0%, 3.3% and 3.3%, respectively). Sperm–oocyte binding analysis showed that CS-C increased the number of sperm bound to oocyte compared unaddition control in each semen. These results suggested that CS-C is the efficient factor on sperm capacitation in pigs, CS-C may promote sperm from the incapacitated to capacitated state and sequentially prevent sperm from spontaneous acrosome reaction, and thus facilitate the sperm–zona binding and sperm penetration to oocyte.

Effect of Monosaccharide L-fucose and Polysaccharide Fucoidan on Sperm ??L-fucosidase Activity and Relation to Sperm-oocyte Interaction in Pig

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that α-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm α-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm α-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm α-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of α-L-fucose on sperm binding and penetrating into ZP caused sperm α-L-fucosidase inhibited by α-L-fucose.

Screening of Tetracycline Residues in Fish Muscles by CCD Camera-Based Solid-Surface Fluorescence

A methodology for the screening of tetracyclines (TCs), including tetracycline (TC), oxytetracycline (OTC), and chlorotetracycline (CTC), in different fish muscle matrices has been proposed. This method was based on in situ fluorescent derivation of TCs, transferring weakly fluorescing TCs to highly fluorescent species, on alkaline-activated solid silica gel G plates (SGGPs). By coupling solid-surface fluorescence (SSF) with charge-coupled device (CCD) camera imaging, a CCD camera-based SSF (CCD-SSF) methodology has been developed. Calibration curve, repeatability, selectivity, limit of detection (LOD), and limit of quantification (LOQ) have been explored for evaluating the performance of the method itself. Linear calibration curves were obtained over a range of 0.20-1.0 ng/spot for all three TCs. The LODs, defined as 3sigma, for TC, OTC, and CTC were 0.14, 0.15, and 0.16 ng/spot, respectively. The trueness of method was validated by HPLC, and no significant difference between CCD-SSF and HPLC was found, on a basis of 95% confidence level. By spiked recovery studies, a linear calibration curve ranging from 20 to 300 microg/kg of TC in fish muscle samples with a correlation coefficient (R 2) equal to 0.994 was obtained. The total average recovery for TC in fish muscle samples from six different fish matrices, fortified with TC at 50, 100, and 200 microg/kg levels, was 75.7% with average relative standard deviations (RSDs) ranging from 2.0 to 7.7%. RSDs ranged from 2.5 to 5.8% and from 5.2 to 7.6% for in-day and interday repeatability, respectively. The detection and quantification limits in fish muscle matrices were 16 and 53 microg/kg of TCs, respectively. The newly developed CCD-SSF method has been applied to the screening of the TC residues in fish muscle samples. The method has been demonstrated to bear some advantages, such as its simplicity, high throughput, low cost, use of fewer pollutants, and reasonable sensitivity.

Impact of sulfadiazine and chlorotetracycline on soil bacterial community structure and respiratory activity

Veterinary medicines enter agricultural soils by the use of animal excrements as fertilizers. To study their impact on soil bacterial communities, microcosms containing orthic luvisol soil were spiked with the antimicrobial agents sulfadiazine (SDZ) and chlorotetracycline (CTC) at three different concentrations (1, 10, 50 mg kg −1 soil) and incubated for 48 days at 20 °C. The impact on the microbial respiratory activity was measured continuously in a respirometer (Sapromat). Changes in bacterial community structure were visualized by means of PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA derived from soil samples after 1, 7, 11 and 48 days. Additionally, growth inhibitory effects of SDZ and CTC on bacteria previously isolated from the same soil were tested in agar diffusion tests. In microcosms with soil and antibiotics only, no effects could be observed, either on respiratory activity or on bacterial population structure. Therefore, further incubations were conducted in the presence of an additional assimilable carbon source (5 g glucose kg −1 soil). In the presence of glucose, SDZ affected soil respiration as well as the bacterial community structure: Additional bands appeared and some bands already visible at the beginning of incubations increased in intensity. A clear relationship between SDZ concentrations and changes in DGGE patterns became visible. During 48 days of incubation, changes in DGGE patterns were minimal in microcosms with 50 mg SDZ kg −1soil indicating an inhibition of strains, which were capable of growing on glucose in the presence of lower SDZ concentrations. Only a few soil bacterial isolates (5 out of 47 strains tested) were weakly inhibited by SDZ in agar diffusion disk tests. Contrastingly, CTC inhibited growth of 12 soil bacterial isolates significantly in disk tests, but no effects on soil respiration and bacterial community structure could be observed. In the presence of the soil matrix the growth inhibitory potential of CTC decreased due to adsorption or complexation. This was confirmed in growth inhibition experiments with soil suspensions and time-dependent sampling.

Study on the interaction between protein and Eu(III)–chlorotetracycline complex and the determination of protein using the fluorimetric method

Chlorotetracycline (CTC) can react with europium ions Eu3+, and the complex emits the intrinsic fluorescence of Eu3+. The intensity is greatly enhanced by proteins and this forms the basis of a new fluorimetric method for determination of protein. Further research indicates that under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins, in the range 2.0 x 10(-7)-1.0 x 10(-5) g/mL for bovine serum albumin (BSA) (linear equation, I(f) = 34.35933 + 11.54467 x 10(6)C)(r = 0.99895) and 8.0 x 10(-7)-1.0 x 10(-5) g/mL for human serum albumin (HSA) (linear equation, I(f) = 76.58881 + 5.3569 x 10(6)C) (r = 0.99283). Detection limits (S/N = 3) were 8.9 x 10(-9) g/mL for BSA and 3.3 x 10(-8) g/mL for HSA. In an assay for BSA in calf serum, this method gave a value close to that determined by the UV spectrophotometric method.

Light-induced cytosolic calcium transients in green plant cells. I. Methodological aspects of chlorotetracycline usage in algae and higher-plant cells

The fluorescent dye chlorotetracycline (CTC) has several disadvantages compared with ratio dyes like Fura-dextran. However, in many plant tissues the derivatives of Fura cannot be loaded. Thus, the pitfalls and possible precautions for the measurement of the light-induced changes in cytosolic free calcium concentration ([Ca2+]c) were investigated in algae and higher plants. Eremosphaeraviridis de Bary and the flowing cytosol in whorl cells of Characorallina Klein ex Willd. were used as examples for possible pressure injection of Fura-dextran or bis-carboxyethyl-carboxy-fluorescein (BCECF) dextran, illustrating the better calibration in absolute terms provided by these dyes. However, here it is shown that CTC works better than Fura-dextran for monitoring the light-induced changes in [Ca2+]c in the ectoplasm close to the plasma membrane in Chara. Protoplasts of Solanumnigrum L. and whole intact leaves of Viciafaba L. and Nicotianatabacum L. were used as examples of cells that were too fragile for pressure injection of Fura-dextran. The sensitivity of CTC to pH may cause artefacts when light-induced changes in [Ca2+]c in intact leaves are to be measured. If some precautions are met, this problem and others (requirement of constant temperature, sensitivity to other ions, effect on plasma-membrane Ca2+ permeability) can be circumvented, thus making CTC a suitable dye for monitoring light-induced changes in [Ca2+]c in a broad spectrum of different plant cells, tissues and species.

Apoplastic and membrane‐associated Ca2+ in leaves and roots as affected by boron deficiency

A combination of fluorescein‐isothiocyanate (FITC), coumarin‐benzothiazol (BTC), and chlorotetracycline (CTC) fluorescence was used to simultaneously monitor apoplastic pH, apoplastic free Ca2+, and plasma membrane‐bound Ca2+. As early boron deficiency reactions supposedly include alterations of plasma membrane‐bound transport processes besides rapid effects on cell wall physical properties, the corresponding changes were followed in leaves and roots of Vicia faba L. cv. Troy.Boron deficiency did not alter the apoplastic pH, but it reduced plasma membrane‐bound Ca2+ in roots at 4 h and leaves at 3 days after starting the deficiency treatment. The decrease in plasma membrane‐bound Ca2+ coincided with an increase in apoplastic free Ca2+ and K+, and occurred before the first visible symptoms were noticed.It is proposed that less Ca2+ is bound to the plasma membrane due to a reduction of specific Ca2+‐binding sites (borate esters with vic‐diols or polyhydroxy‐carboxylates) before plasma membrane integrity deteriorates.

Effects of extracellular potassium concentrations on acrosome reaction and polyspermy during in vitro fertilization and subsequent development in vitro in the pig

Potassium (K +) concentration in the mammalian oviduct and uterus is particularly interesting due to its unusually high concentration (12 to 25 mM) compared with that in the blood stream (3 to 6 mM). In this study we examined the effects of various K + concentrations in the fertilization medium on polyspermy and subsequent in vitro development of porcine oocytes. In the absence of K + in the fertilization medium, sperm penetration was not observed. The incidence of polyspermy was significantly higher in the fertilization medium that contained 6 or 12 mM K + as compared with 3 mM K +. The mean number of sperm penetrated in oocytes in medium with 6 and 12 mM K + was higher than in medium with 3 mM K +. The addition of 3, 6 or 12 mM K + to the fertilization medium did not significantly affect the proportion of zygotes that developed to the blastocyst stage (14.1, 12.4 and 15.0%, respectively). Chlorotetracycline (CTC) analysis was used to determine the capacitation and acrosome reaction of spermatozoa incubated for 3 h with various concentrations of K +. The number of acrosome reacted spermatozoa decreased with increasing K + concentration. These results suggest that extracellular K + in the fertilization medium affects sperm acrosome reaction which may be related to the sperm penetration.

Relative quantification of membrane-associated calcium in red spruce mesophyll cells

We describe a method for localizing and comparing relative amounts of plasma membrane-associated calcium ions (mCa) in complex tissues and verify the procedure for mesophyll cells of red spruce (Picea rubens Sarg.) needles. This technique incorporates epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image processing and analysis. Using an appropriate standardization for image brightness, the procedure allows relative quantitative comparison of CTC-fluorescence in the plasma membrane-cell wall region that corresponds to relative amounts of mCa. The technique effectively discerned mCa differences among red spruce needle sections exposed to treatments designed to alter mCa levels in vitro. Estimates of mCa for nine red spruce seedlings, were highly repeatable over a 6 week period in late summer. This repeatability verifies that the described methods produce reliable and reproducible estimates of foliar mCa in woody plant foliage. By incorporating image analysis, this technique allows for relative quantitative estimates of mCa specific to the physiologically-important and labile pool of Ca associated with the plasma membrane-wall complex. Such measurements have not previously been reported for woody plant tissues and thus may provide new insights into the relative roles and responsiveness of mCa vs total foliar Ca pools.

Rate of Na +/Ca 2+ exchange across the plasma membrane of synaptosomes measured using the fluorescence of chlorotetracycline. Implications to calcium homeostasis in synaptic terminals

It is shown that the fluorescence of chlorotetracycline (CTC) can be used to be used to continuously monitor Ca 2+ fluxes mediated by the Na +/Ca 2+-exchanger of the plasma membrane of synaptosomes. The kinetics of Ca 2+ uptake can be followed from the kinetics of the increase of CTC fluorescence with external Ca 2+ concentrations in the micromolar range. Since the fluorescence of CTC is not sensitive to Ca 2+ concentration below 20 μM this avoids any significant contribution of Ca 2+ flux through Ca 2+ channels to CTC fluorescence. By replacing KC1 by choline chloride in the buffer to avoid plasma membrane depolarization it is shown that the amplitude of the CTC fluorescence change is dependent upon the Na +-gradient preimposed across the plasma membrane, and the rate constant of the kinetic process is dependent upon the Ca 2+ concentration. The rate constant of the Ca 2+ influx measured with depolarized and non-depolarized synaptic plasma membrane vesicles at 37°C and pH 7.4 were 0.55 ± 0.10 and 0.25 ± 0.02 min −1, respectively. The overall rate of Na +/Ca 2+ exchange calculated under conditions close to physiological Na + and Ca 2+ gradients and membrane resting potential ranged from 15 to 25% of the activity of the plasma membrane Ca 2+ pump under these experimental conditions. The results also point out that membrane depolarization increases approx. 2-fold the rate of Na +/Ca 2+ exchange in synaptic plasma membrane vesicles.