Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling

Determination of the physico-chemical attributes of plant cell walls, such as lignin content and composition, is of paramount importance in germplasm screening and for evaluating the results of plant breeding and genetic engineering. There are escalating needs for analyses to be robust, reproducible, accurate, and efficient. We have recently modified an established protocol for discrimination of lignin monomers, thioacidolysis, with the goal of increasing sample throughput while maintaining accuracy and reducing equipment load and consumption of reagents. Numerous methodological changes related to volume scaling, selection of the processing vessel, and sample handling were addressed. The revised protocol permitted rapid processing of some 50 or more samples per person per day. A direct comparison between methods using hybrid poplar ( Populus alba  ×  tremula ) wood samples, resulted in quantities of p -hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin monomers that were equivalent to those derived from the original protocol. The revised methodology was then applied to quickly generate phenotypic trait data from 267 hybrid poplar trees (including wild type and eight C4H::F5H transgenic lines), for the development of a near-infrared-based model for predicting the proportion of lignin monomers across a broad phenotypic range of S:G. The resulting partial least squares regression model performed well under full cross-validation, giving strong, linear relationships between actual and predicted monomer proportions, and very high predictive accuracy for the predominant G and S monomers. This research brings considerable refinement to the thioacidolysis procedure, and establishes a method for rapidly and accurately quantifying cell-wall lignin composition that could effectively be employed in routine phenotypic screening platforms.     

Rats grouping and the circadian and ultradian synchronization of their carbon dioxide emission by a light-dark 12:12 h alternation.

Carbon dioxide emission (VCO2) was continuously recorded during 19 consecutive days in 25 Sprague Dawley young male rats placed in the same "respiratory chamber", grouped by 5 (G) and then separated (S). All rats were in controlled environmental conditions (20 degrees C temperature, humidity, ventilation, food and water ad libitum) and submitted to a light (100 lux)-dark alternation (LD 12:12). The curves obtained with the respiratory chamber CO2 concentration sampled every 20 minutes were analyzed for circadian periods, amplitudes, phases, ultradian peak oscillation intervals and amplitudes, and VCO2 time variations at L-->D and D-->L light transitions. Analysis of variance and t test show circadian amplitudes significantly (P < 0.001) higher (by 40.9%) than in S; moreover, ultradian peak amplitudes were higher in G than in S (by 78.0% in L and 105.8% in D). The circadian and ultradian (tau > 40 min) period intervals were not significantly different in G and in S. Circadian phase differences between L-->D and D-->L were significantly greater in S (by 50.3 min) but not in G. Light transitions did not significantly modify ultradian phases in G and in S. This data shows a better LD 12:12 synchronization in G than in S, resulting mostly from an increased respiratory amplitude modulation due to interindividual interactions.     

Alkaline treatment of muscle microsomes releases amphiphilic and hydrophilic forms of acetylcholinesterase.

To obtain information about the mode of attachment of amphiphilic monomers of acetylcholinesterase (AChE) in sarcoplasmic reticulum (SR) of skeletal muscle, attempts were made to release the enzyme by alkaline hydroxylamine. About half of the activity measured in microsomes preincubated with 0.5% (w/v) Triton X-100 is detached by incubation of SR with bicarbonate buffer (pH 10.5). Addition of 1 M hydroxylamine to the alkaline buffer did not improve enzyme solubilization. Molecular forms of 16S (A12), 10.5S (G4) and 4.0S (G1) are separated by sedimentation analyses of Triton X-100 or bicarbonate-solubilized AChE. Monomeric AChE, released under alkaline conditions (G1A), displays amphiphilic properties. G1A, but not G4 and A12, forms are retained in a phenyl-Sepharose column and this allows its separation from hydrophilic forms. Isolated monomers extracted with Triton X-100 (G1D) or alkaline buffer showed identical kinetic behaviour. The two forms reacted with lectins in a similar manner. However, thermal inactivation experiments revealed that about 90 and 40% of the activity in the G1D and G1A forms were lost by heating at 50 degrees C, following the same rate constant (k = 0.130 min-1). Addition of Triton X-100 to the G1A form leads to an increase of its thermal sensitivity, the enzyme being fully inactivated very rapidly (k = 0.230 min-1). The results suggest that the hydrophobic moiety of the enzyme might be exposed or hidden depending on the environmental hydrophobicity. Changes in the composition of the solvent will determine the final conformational state of the protein.     

Mass spectrometric detection of nodularin and desmethylnodularin in mussels and flounders

A rapid and simple method for the determination of morphine (M), normorphine (NM), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma by high-performance liquid chromatographic separation with mass spectrometric detection (HPLC-MS) has been developed. Samples (40 microl) were cleaned-up by protein precipitation with two volumes (80 microl) of acetonitrile and reconstituted in formic acid 0.1% in water. Naloxone was used as internal standard. Analytes were separated on a phenyl-hexyl column using a step-gradient (1 ml/min) of acetonitrile and formic acid in water. Acetonitrile was added post-column (0.3 ml/min). Quantification of morphine and its metabolites was achieved with an Agilent 1100 series HPLC-MS system equipped with electrospray interface set to selected ion-monitoring (SIM) mode. Calibration curves covered a wide range of concentrations (2.44-10,000 nM) and were best fitted with a weighed quadratic equation. The limits of quantification achieved with this method were 2.44 nM for M and 4.88 nM for NM, M3G and M6G. The method proved accurate (85-98%), precise (C.V.<10%) and was successfully applied to a wide range of in vitro and in vivo pharmacokinetic studies in rodents.     

Simultaneous determination of flavones and phenolic acids in the leaves of Ricinus communis Linn. by capillary electrophoresis with amperometric detection

Capillary electrophoresis (CE) with amperometric detection (AD) has been developed for the separation and determination of disaccharide glycoside rutin, gentistic acid, quercetin, and gallic acid in the leaves of Ricinus communis Linn. for the first time. The effects of the acidity and the concentration of the running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions for the determination of the four analytes. The detection electrode was a 300microm diameter carbon disc electrode at a detection potential of +0.90V (versus saturated calomel electrode (SCE)). The four analytes could be well separated within 10min in a 40cm length fused silica capillary at a separation voltage of 15kV in a 50mM borate buffer (pH 9.0). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N=3) ranging from 0.8 to 2.9microM for all the analytes. The proposed method has been successfully applied to monitor flavones and phenolic acids in the real plant samples with satisfactory assay results.     

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--II. Studies with the Doluisio technique

The absorption of oestrone (E1), oestrone glucuronide (E1G) and oestrone sulphate (E1S) from the small intestine of anaesthetized rats has been evaluated using the Doluisio in situ technique. Luminal disappearance of E1 was biphasic, which is consistent with a 3-compartment model; t1/2 alpha (first phase) was less than 5 min and t1/2 beta (second phase) approx 27 min for each concentration of steroid studied (trace identical to 10 nM, 1 microM and 10 microM). In contrast, luminal disappearance of E1G and E1S was monoexponential; t1/2 for E1G was 159, 229 and 299 min (trace identical to 200 nM, 10 microM and 100 microM respectively) and for E1S, 215, 174 and 192 min (trace identical to 10 nM, 10 microM and 100 microM respectively). There was a good correlation between the luminal disappearance data and recovery of steroid in bile. Adsorption of E1S was estimated from the initial rapid fall in luminal content within the first 5 min after drug administration. The study provides further evidence that E1S can be absorbed intact. Since saccharolactone only caused a reduction in E1G absorption of 32% we also conclude that part of the administered E1G was absorbed intact.     

Polysome translational state during the cell cycle.

HeLa cells were synchronized with a double thymidine block. Ribosomal subunits, monomers and polyribosomes have been quantitatively analysed at hourly intervals, during interphase, and every 15 min, during mitosis. This analysis was performed on linear 7-47% sucrose gradients. From the beginning of G1 up to the end of S phase, a certain equilibrium among ribosomal subunits, monomers and polyribosomes is maintained, while from the time of entering G2 to M the translation machinery appears to be mobilized in the sense of polysome formation. Under these conditions, the amount of polysomes per cell during the mitotic cycle is expressed by a bi-phasic pattern showing pre- and post-mitotic peaks with a falling-off during S. The G1 peak, meanwhile, is much lower than the G2 peak. The incorporation of [3H]leucine into nascent polypeptide chains on polysomes, as well as into bulk cell proteins and into nuclear and cytoplasmic proteins considered separately, is also represented by a bi-phasic curve which shows, however, a higher peak in G1 and a lower peak in G2, with two fallings-off during S and M, respectively. Since between the G1 and the G2 amino acid pools there are not strong differences of leucine concentration, the discrepancy between the amount of polysomes and the rate of labelling is discussed on the basis of the differences of polysome shape found at the different stages of the cycle. In young cells, in fact, there is an abundance of small polysomes, while in the old cell large polysomes predominate. It is suggested that, in the old cell, the rate of translation on large polysomes could be relatively lower or that among these heavy aggregates a given number of "frozen" polysomes could be present. The ribosome state is considered as a probable limiting-factor of translation, particularly in mitosis.     

Thermostability of bacteriophage T4 fibritin and its deletion mutants]

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-packed subunits each containing 486 amino acid residues) progressively truncated from the subunit N-end was studied during incubation at 40-90 degrees C in the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resulted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly denatured N-terminal part. Fibritins B and C were more stable and were only partly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. The denaturation of mutants B, C, E, and S1 proceeded in one stage without formation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon folding in vivo. The refolding capability was found for fibritin and its mutants denatured by heating at low temperatures in the presence of 2% SDS.     

Column-switching high-performance liquid chromatographic system with a laser-induced fluorimetric detector for direct,automated assay of salivary cortisol

In order to measure human stress, an easy and rapid, fully automated method for the determination of cortisol in saliva has been developed, using column-switching high-performance liquid chromatography with laser-induced fluorescence detection, which involves post-column labeling with sulfuric acid. The developed system requiers only 0.1 ml of saliva, and a simple pretreatment consisting of dilution and filtration is sufficient. The column-switching system consisted of a Polymer-Coated Mixed-Functional silica (PCMF) column for deproteinization, and a CN column for frontal concentration and separation. An ODS column in place of the CN provided a better separation, but required a post-column make-up of water for safe reaction. Detection limit of cortisol was 8 fmol (signal-to-noise ratio = 3), which is adequate for routine determination of normal levels of cortisol (1–20 pmol/ml). The analysis time was about 40 min and reproducibility was excellent with an R.S.D. of less than 5%.     

Simultaneous determination of urinary creatinine and UV-absorbing amino acids using a novel low-capacity cation-exchange chromatography for the screening of inborn errors of metabolism

A simple and versatile low-capacity cation-exchange chromatography system for the simultaneous determination of creatinine and UV-absorbing amino acids was developed. The separation column was packed with a newly developed low-capacity sulfoacylated macro-porous polystyrene-divinylbenzene resin selective for amino-acid cations. Urinary creatinine, creatine, tyrosine, histidine, phenylalanine, and tryptophan were simultaneously separated and determined by an isocratic elution with phosphate/acetonitrile eluent in 25 min. Relative standard deviations (R.S.D.) of the retention times for the analytes were between 0.28 and 1.06%. R.S.D. of peak area responses for the analytes were between 0.75 and 3.51%. The r(2) values for the calibration lines were between 0.9994 and 0.9999. The method could provide the creatinine ratios for the analytes, and was applicable to the screening and/or chemical diagnosis of several inherited disorders of amino-acid metabolism such as phenylketonuria (PKU).     

Regenerative proliferation of mouse epidermal cells following application of a carcinogenic skin irritant (MCA). Micro-flow fluorometric DNA measurements and 3H-TdR incorporation studies of isolated basal cells.

0.025 ml of a 1% solution of the complete skin carcinogen 20-methylcholanthrene (MCA) dissolved in benzene was applied to the back skin of hairless mice. At different time intervals up to 3 days after the carcinogen application groups of animals were injected i.p. with 30 muCi 3H-TdR 30 min before they were killed. Single cell suspensions of epidermal basal cells were prepared by a combined enzymatic and mechanical separation method, and the DNA frequency distribution pattern from each cell suspension was measured by means of micro-flow fluorometry. Smears for autoradiography were made from each cell suspension and the labeling index and mean grain count assessed. After a short initial delay, MCA induced an increase in the labeling index similar to that observed after non-specific cell injury and cell loss. Thereafter, the cells were considerably delayed in their progression through the S phase, with a low exit from S resulting in a transient emptying of the G2 compartment, without indications of any significant delay of the passage through G2 phase. The cells that had been injured by the MCA application in or just before S phase proceeded into the G2 phase and mitosis more than 24 h after the initiation of DNA synthesis. The cell kinetic reaction of epidermis to a single application of MCA is thus very different from that caused by a nonspecific cell damage, e.g. application of the vesicant agent cantharidin or removal of surface cells by cellophane tape stripping.     

Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA.

Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization.     

Enhancing immunoassay detection of antigens with multimeric protein Gs

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.     

魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

Quantitation of cocaine and cocaethylene in canine serum by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic procedure for the determination of cocaine and cocaethylene in canine serum has been developed. The compounds were extracted from 1 ml of alkalinized canine serum with hexane. Chromatographic separation was achieved with a cyanopropyl column (250 × 4.6 mm I.D., 5 μm) using a mobile phase of acetonitrile and phosphate buffer, pH 7.40 (38:62, v/v) flowing at 1 ml/min. Eluate was monitored by a variable-wavelength UV detector set to 230 nm. The extraction procedure yields an average recovery of 99 and 96% for cocaine and cocaethylene, respectively. The between-day coefficients of variation, at 2400 ng/ml, for cocaine and cocaethylene were both 8.6% and the within-day coefficients of variation, at 400 ng/ml, for cocaine and cocaethylene were 7.3 and 8.0%, respectively. A concentration-time profile resulting from administration of 3 mg/kg cocaine and cocaethylene to the dog revealed a similar disposition between cocaine and cocaethylene, with a clearance and volume of distribution at steady-state values of 72.8 and 61.0 ml/min/kg and 2.6 and 2.7 1/kg, respectively.     

One-step rapid determination and purification of puerarin from Radix puerariae by n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith

n-Octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monoliths were prepared for rapid screening, determination and one-step purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata). The modified monolith showed a specific surface area of 17.8 m(2) g(-1), an average pore size of 0.76 microm and a total porosity of 60.8%. Fast separation of R. puerariae crude extract was achieved within 5 min at a flow velocity of 722 cm h(-1) resulting in a puerarin purity of 97%, with a recovery of 85%. This demonstrates the potential of n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith for the rapid analysis and separation of isoflavonoids. Preparative scale sample loading (12 mg in 2 mL) resulted in a purity of 95%, and a recovery of about 69%. HPLC, FTIR, MS and (1)H NMR spectroscopy were used for the characterization and quantification of puerarin in isolated fraction.     

Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.     

Lignin content versus syringyl to guaiacyl ratio amongst poplars

Two oxidation techniques that afford high yields of monomers and dimers were used to more accurately estimate the syringyl to guaiacyl (S:G) ratio of hardwood lignins. Permanganate oxidation of the woodmeal after a CuO pre-hydrolysis step gave poor results and this was attributed to preferential oxidation and degradation of syringyl nuclei by CuO. However, this procedure did provide a good estimate of the percentages of both S and G phenylpropane (C(9)) units that were uncondensed. When the total S and G products from nitrobenzene oxidation (NBO) of the uncondensed fractions were corrected, credible S:G ratios were obtained. These ratios were in good agreement with results from KMnO4 oxidation of dissolved kraft lignin without CuO pre-hydrolysis. The corrected NBO method was used to determine the S:G ratio of 13 poplars, and the values ranged from 1.01 to 1.68. Unlike results from other investigations, an excellent linear correlation (R(2) =0.846) was obtained for a decreasing lignin content (28% to 16.5%) with an increase in the S:G ratio.     

Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of 17OHP in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.