Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

萝卜硫素通过Nrf2信号通路对大鼠草酸钙肾结石形成的作用和机制研究

摘要 目的：探究萝卜硫素通过Nrf2信号通路对大鼠草酸钙肾结石形成的作用和机制。方法：选取7~8周龄Wistar健康雄性大鼠30只,随机分为空白对照组、模型组和药物干预组。空白对照组给予正常饮用水,标准饲料;模型组给予1%乙二醇饮用水+2%氯化铵2 mL/d灌胃,标准饲料;药物干预组给予1%乙二醇饮用水+2%氯化铵2 mL/d灌胃+0.2 mg萝卜硫素腹腔注射,标准饲料。灌胃给药均连续28天。于第29天收分别集各组大鼠24 h尿液及肾脏标本,使用全自动生化分析仪检测尿液中K⁺、Na⁺、Cl⁺、Ca²⁺、Mg²⁺、P⁵⁺、Fe²⁺含量,使用ELISA法测定尿液中丙二醛( malondialdehyde,MDA)与草酸水平,使用HE染色分析大鼠肾脏标本病理损伤情况,Von Kossa染色比较各组大鼠钙盐沉积情况,免疫组化法测定谷胱甘肽(glutathione,GSH)、核因子E₂相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)、8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHDG)蛋白表达水平。结果：模型组大鼠尿液P⁵⁺、Ca²⁺、草酸、MDA含量显著高于空白对照组,HE染色示模型组肾小管较空白对照组变性严重,Von Kossa染色结果显示模型组钙盐沉积较空白对照组明显增多,模型组GSH、Nrf2免疫组化评分显著低于空白对照组,8-OHdG显著高于空白对照组,差异均有统计学意义（P＜0.05）;药物干预组大鼠尿液P⁵⁺、Ca²⁺、草酸、MDA含量显著低于模型组,HE染色、Von Kossa染色显示肾小管较模型组损伤减轻,钙盐沉积较模型组减轻,免疫组化显示GSH、Nrf2蛋白表达显著高于模型组,8-OHdG显著低于空白对照组,差异均有统计学意义（P＜0.05）。结论：萝卜硫素对大鼠肾小管具有抗氧化损伤作用,可降低草酸钙晶体的释放,抑制草酸钙结石的形成,该作用可能是通过激活Nrf2蛋白及其下游信号通路实现的。     

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus

Background and Aims

Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host–pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus).

Methods

Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar ‘Charlton’ and one susceptible ‘RQ001-02M2’ by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi).

Key Results

The resistant ‘Charlton’ impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in ‘Charlton’ pathogen invasion was mainly confined to the upper epidermis, in the susceptible ‘RQ001-02M2’, colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible ‘RQ001-02M2’ at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant ‘Charlton’, crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in ‘RQ001-02M2’.

Conclusions

This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.     

铝对外生菌根真菌草酸分泌及磷、钾、铝吸收的影响

试验研究了在铝胁迫条件下,6种(株)外生菌根真菌(ECMF)的生长、草酸分泌,以及磷、钾、铝的吸收状况。结果表明,铝对抗(耐)型菌种Pt715、HrSp、CgSIV的生长无抑制作用,但显著抑制敏感型菌株LbS238N、LbS238A和Lb270的生长,说明ECMF对铝胁迫的生长反应可能是筛选抗(耐)铝的指标之一。在铝胁迫条件下,无论是抗(耐)型还是敏感型菌种(株),都会发生一系列有益于抗(耐)铝的生化反应,包括草酸分泌量、菌丝磷和钾含量增加,H+分泌改变等。在培养液中,草酸电离产生的H+仅占H+总浓度的少数,说明溶液中H+的主要来源不是ECMF所分泌的草酸,而是菌丝细胞为保持吸收阳离子的电荷平衡排出的H+或分泌的其它有机酸。     

Concentration of a potent calcium oxalate monohydrate crystal growth inhibitor in the urine of normal persons and kidney stone patients by ELISA-based assay system employing monoclonal antibodies

Standardized calcium oxalate monohydrate (COM) crystal growth assay system was employed to study the ability of various test samples to influence growth rates of COM crystals. The inhibitory activity (IA) of various samples was expressed in terms of inhibitory units. Urine samples obtained from normal persons and kidney stone patients were found to have IA of 3.18 +/- 0.62 and 1.02 +/- 0.08, respectively. A potent inhibitor having molecular weight between 14.2 and 16.2 kDa was found to be primarily responsible for the differences observed in the urinary IAs between normal persons and kidney stone patients. The potent inhibitor was found to be tightly associated with a chromophore resembling Urobilirubin. An ELISA based assay system, using monoclonal antibodies against the above most potent inhibitor confirmed the difference observed in the urinary IA between the normal persons and kidney stone patients. This assay system has the potential to be routinely used to screen human beings for potential stone formers.     

Disordered calcium crystal handling in antisense CLC-5-treated collecting duct cells

Dent's disease, an X-linked tubulopathy secondary to defects in chloride channel CLC-5, is characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Mechanisms leading to nephrocalcinosis are unknown. Using a murine collecting duct cell line (mIMCD-3), we confirm endogenous expression of mCLC-5. During transfection of antisense CLC-5, we observe a reduction in CLC-5 protein expression that correlates with a reduction in the number of acidic endosomal compartments, as determined by quantitative analysis of confocal microscope images using LysoTracker Red. Using wheat germ agglutinin-lectin as an endocytic marker, an arrest of endocytosis is observed in antisense CLC-5 treated cells. Exposure of the cell surface to calcium oxalate crystals results in crystal agglomeration in a minority of sense CLC-5 transfectants (45%) and all antisense CLC-5 transfectants. We conclude that expression of CLC-5 in mIMCD-3 cells allows acidification of endosomes and endocytosis, and that disruption of CLC-5 expression causes abnormal crystal agglomeration.     

β-N-Oxalyl-L-α,β-Diaminopropionic Acid Protects the Activity of Glycolate Oxidase in Lathyrus sativus Seedlings under High Light

The content of -N-oxalyl-L-,-diaminopropionic acid (ODAP) and the activity of glycolate oxidase (GO) were positively correlated in the leaves of grass pea (Lathyrus sativus L.) seedlings. The activity of GO was kept at a steady level under the high light after treatment with ODAP. Although Na₂S can activate GO, it cannot maintain the activity of GO under the high light. The content of ODAP increased and the activity of GO decreased with increasing oxalate concentration used for seedling treatment. The GO activity was high enough to keep photosynthesis at a steady level under high light. These findings suggested that Lathyrus sativus, using oxalate as a precursor to produce ODAP, protected the GO activity at high irradiance by scavenging the hydroxyl radicals.     

Isokinetic Relationship in the Oxidation of Cuprous Stellacyanin by Cobalt(III) Complexes

Rate parameters have been obtained for the oxidation of cuprous stellacyanin by cobalt(III) ions of the form cis(N)-[CoN₂O₄]^?, including cis(N)-[Co(NTA)(gly)]^?, cis(N)-[Co(IDA)₂]^?, [Co(en)(ox)₂]^?(μ 0.5 M(phosphate), pH 7.0), and Co(EDTA)^?(μ 0.1 M(NaCl), pH 7.2, 0.001 M phosphate). An excellent isokinetic correlation between the activation parameters ΔH^≠ and ΔS^≠ exists for the reactions of aminopolycarboxylatocobalt(III) ions with reduced stellacyanin (β = 300 ± 12 K; correlation coefficient = 0.995). It is concluded that enthalpy-entropy compensation in these reactions may be understood in terms of differing orientations preferred by the various oxidants in forming precursor complexes with the reduced blue protein. While ΔH^≠ and ΔS^≠ values for electron transfer from stellacyanin to cis(N)-[CoN₂O₄]^? ions vary over ranges of 10.7 kcal/mol and 34 cal/mol-deg, respectively, room temperature rate constants are relatively constant (3.6–34.5 M^?1 sec^?1), as expected from Marcus theory for outer sphere electron transfer.