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Isolation and in vitro fusion of egg and sperm cells in Oryza sativa.

Protocols for isolation of gametes of Oryza sativa were developed and initial results on in vitro fusion of sperm and egg cells are reported. The best yield of viable sperm cells was obtained when pollen grains were cultured in a medium containing of 1.3 mM boric acid, 3.6 mM calcium chloride, 0.74 mM potassium phosphate, and 438 mM sucrose. Embryo sacs were isolated using cell wall degrading enzyme treatments for 2-5 h followed by mechanical manipulation. The maximum yield (38.2%) of egg cell was achieved when 2% cellulase and 0.55% pectinase were used in the medium. However, the optimum concentration of cellulase and pectinase was found to be 1% and 0.85%, respectively. Fluorescein diacetate (FDA) stain was used to determine the viable sperm and egg cells. The optimal procedures (fusion conditions) for gametes fusion occurred in a medium containing calcium chloride at a concentration of 7 mM (pH 7.5) and the best result obtained (55.5%) in terms of fused gametes, is reported.

Cellulase↗

Raman spectroscopic analysis of supersaturated aqueous solution of MgO.B2O3-32%MgCl2-H2O during acidification and dilution.

Raman spectra of supersaturated aqueous solution of MgO.B2O3-32%MgCl2-H2O during acidification/alkalization and dilution have been studied. The assignments of the recorded Raman shift are given. The main existing forms of polyborate anions and their interaction in borate aqueous solution have been proposed through spectroscopic analysis. The experimental results indicate that the higher concentration of cation are beneficial not only to the dissolution of boric acid but also to the polymerization of polyborate anions. The existing forms and interaction among them also depend on the concentration of boron and the pH value in solution.

Anions↗

Mobilisation and attenuation of boron during coal mine rehabilitation, Wangaloa, New Zealand.

Environmental mobility and fate of boron has been traced from source to discharge waters through the rehabilitated Wangaloa coal mine in southern New Zealand. The boron is derived initially from coal, which has up to 450 mg/kg B. The coal also contains pyrite (2-5 wt.% S), which oxidizes to yield a low-pH environment (typical pH 2-5). Weathering of coal-bearing waste rock liberates B into rainwater that infiltrates into waste rock or evaporates to leave a gypsum crust enriched in B, possibly as boric acid or colemanite as inferred from geochemical modelling. Surface waters dissolve this evaporative material periodically, yielding total B concentrations up to 6 mg/L, at pH<4.5. Some of the available B is taken up by plants that have been established on the waste rock, resulting in foliage B concentrations of up to 230 mg/kg (dry weight). Partial attenuation of dissolved B by adsorption to iron oxyhydroxide occurs as groundwater passes through waste rock, but this is inhibited by adsorption competition with dissolved sulphate (up to 600 mg/L). Groundwater flows from the mine through a pit lake and wetland, with total dissolved B near 1 mg/kg after dilution and limited adsorption attenuation has occurred. Despite the widespread B mobility throughout the rehabilitated mine, there is little evidence of B toxicity in plants. The B concentrations in discharging waters are in the environmentally safe range for most aquatic organisms, being neither deficient in B as a micronutrient, nor boron-toxic.

Boron↗

Discriminative power of an assay for automated in vitro screening of teratogens.

Screening for potential teratogenicity of 20 test compounds was performed using a computerised microscope workstation for determination of cytotoxicity, proliferation and morphology of fibroblastoid murine L929-cells. The test compounds, which were divided into four classes according to teratogenicity were: 5-bromo-2(')-deoxyuridine, 6-aminonicotinamide, acrylamide, boric acid, D-(+)-camphor, dimethadione, dimethyl phthalate, diphenhydramine, hydroxyurea, isobutyl-ethyl-valproic acid, lithium chloride, methyl mercury chloride, methotrexate, methoxyacetic acid, penicillin G, all-trans-retinoic acid, pentyl-4-yn-valproic acid, saccharin, salicylic acid and valproic acid. All compounds, with the exception of dimethadione inhibited proliferation in a linear dose-dependent manner, and there were statistically significant compound class-dependent differences between the IC(50)-values for the compounds (p<0.0374), the strongest teratogens being the most potent. Furthermore, the average efficacies (maximum relative change) for 10 parameters describing cell morphology exhibited statistically significant compound class-dependent differences (p<0.0001), the class I and II compounds exhibiting significantly lower efficacies than the class III and IV compounds (p<0.01). Thus, test compounds affected both the proliferation and morphology of L-cells in manner demonstrating a general relationship with the teratogenic potency of the compounds. However, the moderate teratogens dimethadione and lithium chloride only had minor effects on the morphology and proliferation of the cells whereas the non-teratogen diphenhydramine had effects on both proliferation and morphology comparable to the strong teratogens.

Animals↗

Effects of single and repeated exposure to biocidal active substances on the barrier function of the skin in vitro.

The dermal route of exposure is important in worker exposure to biocidal products. Many biocidal active substances which are used on a daily basis may decrease the barrier function of the skin to a larger extent than current risk assessment practice addresses, due to possible skin effects of repeated exposure. The influence of repeated and single exposure to representative biocidal active substances on the skin barrier was investigated in vitro. The biocidal active substances selected were alkyldimethylbenzylammonium chloride (ADBAC), boric acid, deltamethrin, dimethyldidecylammonium chloride (DDAC), formaldehyde, permethrin, piperonyl butoxide, sodium bromide, and tebuconazole. Of these nine compounds, only the quaternary ammonium chlorides ADBAC and DDAC had a clear and consistent influence on skin permeability of the marker compounds tritiated water and [(14)C]propoxur. For these compounds, repeated exposure increased skin permeability more than single exposure. At high concentrations the difference between single and repeated exposure was quantitatively significant: repeated exposure to 300 mg/L ADBAC increased skin permeability two to threefold in comparison to single exposure. Therefore, single and repeated exposure to specific biocidal products may significantly increase skin permeability, especially when used undiluted.

Adult↗

New technetium 99m-labeled brain perfusion imaging agents.

In developing new brain perfusion imaging agents, three types of neutral and lipid-soluble technetium 99m complexes have been reported: 99mTc-ethylene cysteinate dimer, 99mTc-hexamethyl propylene amine oxime, and 99mTc-boric acid adduct of technetium oxime. Structure-activity relationships of these complexes on brain uptake and retention have been investigated. The new 99mTc-brain perfusion imaging agents are useful in detecting various cerebral vascular abnormalities. The exact trapping mechanisms and their relationship with potential clinical applications still remain to be elucidated. However, initial clinical studies with 99mTc agents do show parallel clinical efficacy as those reported for the iodine 123-labeled brain perfusion agents, isopropyliodoamphetamine and N,N,N'-trimethyl-N'-[2-hydroxy-3-methyl-5-iodobenzyl]-1,3-propanediamine .

Animals↗

Reduced need for corneal epithelial debridement during vitreo-retinal surgery using two different viscous surface lubricants.

PURPOSE: Corneal clarity is frequently reduced during vitrectomy and scleral buckling surgery secondary to epithelial edema, requiring epithelial debridement to improve visibility. The presence of toxic preservatives in the corneal lubricant solutions is hypothesized to be a contributing factor to epithelial edema during vitreo-retinal surgery. DESIGN: Interventional study. METHODS: We compared prospectively the corneal clarity and epithelial debridement frequency in 71 patients within a single institution in whom either Goniosol (2.5% methylcellulose, boric acid, edetate disodium, sodium borate, potassium chloride, water, and 0.01% benzalkonium chloride) or GenTeal gel (0.3% hydoxypropyl-methylcellulose, carbopol 980, phosphonic acid, sorbitol, water, and 0.028% sodium perborate) was used as a corneal lubricant during the course of vitrectomy surgery using sutured contact lenses. Corneal clarity was subjectively graded in a scale of I to IV during surgery. Statistical analysis was made between these two groups for parametric and categorical data. RESULTS: All eyes started with a corneal clarity grade of I (best possible). Corneal clarity decreased significantly faster in eyes where Goniosol was used to a median grade of III at 1 hour of surgical time. In eyes where GenTeal gel was used, corneal clarity decreased much slower, to a median grade of I at 1 hour of surgical time. The difference in frequency of epithelial debridement was also statistically significant: 54% and 14% for Goniosol and GenTeal eyes, respectively. Other factors that may cause corneal epithelial edema and affect corneal clarity were not statistically different between the Goniosol and the GenTeal groups, including median operative time, estimated intraocular pressure, pre- and intraoperative topical solutions and medications, and number of prior ocular procedures. CONCLUSIONS: The use of GenTeal gel as a corneal lubricant maintains corneal clarity longer than Goniosol during the course of vitrectomy surgery using a contact lens viewing system, limiting the need for epithelial debridement.

Adolescent↗

Determination of sugar phosphates and nucleotides related to photosynthetic metabolism by high-performance anion-exchange liquid chromatography with fluorometric and ultraviolet detection.

A high-performance anion-exchange liquid chromatography system was constructed to identify sugar phosphates and nucleotides involved in photosynthetic metabolism. First sugar phosphates and nucleotides were separated by a gradient elution with boric acid and sodium phosphate, then they were detected by a fluorescence detector (as fluorescent derivatives with arginine) and UV detector, respectively. Eight authentic sugar phosphates and 11 authentic nucleotides could be analyzed using the system. The applicability of the system to the determination of the corresponding sugar phosphates and nucleotides in extracts from only five soybean leaf discs (8.95 cm2) was shown.

Chromatography, High Pressure Liquid↗

Effect of specimen storage and preservation on toxicological analyses of urine.

1. The stability of nineteen classes of drugs or their metabolites in urine specimens, for the purpose of toxicological screening has been studied for periods of up to thirty-six weeks. 2. The effect of specimen preservation by refrigation and by the addition of boric acid, chloroform, sodium fluoride, mercuric chloride, and buffers, has been assessed. 3. With the exception of flurazepam, glutethimide, and secobarbital, specimens may be retained at room temperature for periods in excess of six weeks without deterioration. 4. Preservation by the addition of sodium fluoride (5 g/l) followed by freezing, thawing, and filtration significantly prevents specimen deterioration, and may be used as the method of choice.

Central Nervous System Agents↗

Determination of amphetamine and methamphetamine in serum via headspace derivatization solid-phase microextraction-gas chromatography-mass spectrometry.

This study evaluates solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) to determine trace levels of amphetamine and methamphetamine in serum. Headspace post-derivatization in a laboratory-made design with heptafluorobutyric anhydride vapor following SPME was compared with that without derivatization SPME. The SPME experimental procedures to extract amphetamine and methamphetamine in serum were optimized with a relatively non-polar poly(dimethylsiloxane) coated fiber at pH 9.5, extraction time for 40 min and desorption at 260 degrees C for 2 min. Experimental results indicate that the concentration of the serum matrix diluted to a quarter of original (1:3) ratio by using one volume of buffer solution of boric acid mixed with sodium hydroxide and two volumes of water improves the extraction efficiency. Headspace derivatization following SPME was performed by using 6 microl 20% (v/v) heptafluorobutyric anhydride ethyl acetate solution at an oil bath temperature of 270 degrees C for 10 s. The precision was below 7% for analysis for without derivatization and below 17% for headspace derivatization. Detection limits were obtained at the ng/l level, one order better obtained in headspace derivatization than those achieved without derivatization. The feasibility of applying the methods to determine amphetamine and methamphetamine in real samples was examined by analyzing serum samples from methamphetamine abused suspects. Concentrations of the amphetamine and methamphetamine ranged from 6.0 microg/l (amphetamine) to 77 microg/l (methamphetamine) in serum.

Amphetamines↗

Simple method for the simultaneous determination of noradrenaline, dopamine and serotonin by stepwise elution from a short column of weak cation-exchange resin.

A simple method for the simultaneous determination of noradrenaline, dopamine and serotonin using a short column of Amberlite CG-50 is described. Noradrenaline and dopamine were eluted from the column with phosphate buffers containing 1.5 and 4.0% boric acid, respectively, and then serotonin was eluted with 1.0 N HCl. Catecholamines were determined by a modification of the ethylenediamine condensation method using potassium ferricyanide as oxidant and isobutanol for extraction of the fluorophores. Serotonin was measured by the acidic o-phthalaldehyde method. The method was applied to the simultaneous determination of noradrenaline, dopamine and serotonin in discrete regions of rat brain.

Animals↗

High-performance chromatographic method for the purification of tissue-type plasminogen activator.

High-performance affinity chromatography was performed on five ligand-bound columns in an attempt to purify tissue-type plasminogen activator (t-PA), which is a glycoprotein with a high affinity for fibrin and also has two Kringle structures and finger-domain in its molecule. The five columns were concanavalin A-5PW, p-aminobenzamidine-5PW, imidinodiacetic acid-5PW, boric acid-5PW and lysine-5PW. All five were able to rapidly separate t-PA from contaminating proteins, with high resolution and recovery.

Animals↗

Retention of ionic and non-ionic catechols in capillary zone electrophoresis with micellar solutions.

The use of micellar solutions in capillary zone electrophoresis has been primarily relegated to separations of non-ionic solutes, while its applicability to cationic species has been unexplored. We have found that the use of sodium dodecyl sulfate micelles in phosphate buffer allows for tremendous gains in selectivity for several cationic and non-ionic catechols over what can be obtained with normal capillary zone electrophoresis. Complexation of catechols with boric acid alters the net charge on the solutes and changes the partitioning behavior to produce adequate selectivity with improved analysis times. Although the mechanisms of solute interaction with the micellar phase for the cationic species are not decisively known, evidence is presented supporting the existence of ion-pairing equilibria simultaneously accompanied by micellar solubilization.

Borates↗

Effect of sample preparation and liquid chromatography column choice on selectivity and precision of plasma catecholamine determination.

Research into the role of the catecholamines has often depended on the reliable determination of plasma catecholamine concentrations, which present a challenge since they are normally in the low pg/ml range. Most methods employ liquid chromatography, with variations in sample preparation, the separation mechanism, and detection. We tested a new approach to sample clean-up using boric acid gel instead of alumina. No advantage was found. We also compared cation-exchange separation with ion-pair chromatography. Several improvements are possible with the former, most notably greater precision, better specificity, and increased throughput.

Aluminum Oxide↗

Conductivity detection for molecular mass estimation of per-O-sulfonated glycosaminoglycans separated by high-performance size-exclusion chromatography.

Chemically per-O-sulfonated polysaccharides, including glycosaminoglycans (GAGs) and hyaluronan oligosaccharides were analyzed using high-performance size-exclusion chromatography (HPSEC) with suppressed conductivity detection. The results were compared to those obtained by gel filtration HPLC using UV detection or fluorescence detection after the post-column reaction with 2-cyanoacetamide in strong alkaline solution. Analysis was performed on a TSKgel G3000SWXL HPSEC column in 5 mM boric acid (pH 7.0 adjusted by 10 mM NaOH). The use of conductivity detection, in the absence of any derivatization and under isocratic conditions gave a limit of detection in the picogram range. Preliminary studies suggest that this approach may be particularly useful in examining sulfonated polysaccharides and oligosaccharides having no UV chromophore, such as those prepared from O-sulfonated fucans and galactans isolated from algae.

Carbohydrate Sequence↗

Rapid and simultaneous analysis of some bioactive components in Eucommia ulmoides by capillary electrophoresis.

A micellar electrokinetic chromatography method was established for the qualitative and quantitative determination of three groups of bioactive components, iridoids, flavonoids and phenolic compounds, in Eucommia ulmoides. Of the eleven bioactive components being studied, ten were successfully separated in 50 mM boric acid buffer at pH 9.5, with 50 mM sodium dodecylsulfate and 4% 1-butanol, at a voltage of 20 kV, temperature of 20 degrees C and injection under high pressure at 138 kPa for 5 s in a fused-silica capillary with peak detection at 214 nm. A high reproducibility and good linearity was obtained. The relative standard deviations of the migration times in eight injections of the standards ranged from 0.64 to 1.88% and those of the corrected peak area ranged from 2.79 to 6.62%. A good linearity, with correlation coefficients in the range of 0.995-1.000, was obtained in the calibration curves of each standard from 1 to 50 ppm. The amount of these bioactive components in the bark and leaves of Eucommia ulmoides were determined.

Calibration↗

Capillary electrophoretic determination of inorganic anions in the drainage and surface water samples.

Capillary electrophoresis was used for separation and quantitation of several inorganic anions in the drainage and surface water samples from the region with extensive use of fertilisers. Baseline separation of 13 small anions including nitrite and nitrate up to the concentrations of 100 mg/l was achieved in less than 5 min. The electrolyte consisted of 3 mM K2CrO4, 30 microM cetyltrimethylammonium bromide and 3 mM boric acid at pH 8. The method yielded precisions of 1.8-7.2% (RSD, n = 10) and detection limits from 4 micrograms/l (Cl-) up to 500 micrograms/l (citrate). The results of the CE method were compared to ion chromatography using water-acetonitrile (86:14) at pH 8.6 adjusted with NaOH as the mobile phase and consistent results were obtained.

Anions↗