DAPI: a DNA-Specific Fluorescent Probe

DAPI (4′,6-diamidino-2-phenylin-dole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.     

Leaf litterfall,leaf area index,and radiation transmittance in cypress wetlands and slash pine plantations in north-central Florida

The seasonal dynamics of leaf litterfall and leaf area index (LAI, all-sided basis), light penetration and the vertical distribution of surface area index, and the feasibility of estimating LAI from radiation transmittance were studied from April 1993 to March 1994 in the canopies of three cypress (Taxodium ascendens) wetlands and their surrounding slash pine (Pinus elliottii) uplands in Florida flatwoods. Annual leaf litterfall ranged from 324 to 359 g m^–2 in the wetlands, which was very close to the average for 11 sites throughout Florida of 340±26 g m^–2. The seasonal pattern of the normalized LAI obtained for the dominant tree species in the ecosystems could be used to construct the seasonal dynamics of LAI at the ecosystem scale. The vertical distribution of surface area index in the wetlands was significantly different from that in the surrounding pine uplands. The maximum LAI of cypress wetlands in this area was about 8 m² m^–2, which was higher than the maximum of slash pine plantations of 6 m² m^–2. Cypress leaves were strongly erectophile in space. Results showed that the LAI-2000 canopy analyzer could generally be used to estimate forest LAI, whether the forest canopy was closed or not, if an overall clumping index of 2.00 was applied. However, as LAI decreased, the relative error contained in the radiation-based LAI estimates increased. This indicated that foliage clumping at the stand scale was more important than that at the tree or branch scale.     

拟南芥转录因子HB22与CRY1相互作用研究

通过酵母双杂交的方法,从拟南芥转录因子库中筛选出了6个与CRY1相互作用的转录因子.为了测定其中的HB22与CRY1相互作用的强度,采用了ONPG与CPRG两种方法对其β-半乳糖苷酶活性进行了分析.结果显示在蓝光光强为50μmol/m2s,孵育时间为4 h的情况下,蓝光与暗处理情况下的β-半乳糖苷酶活性比值分别为1.668和2.18.进一步设置蓝光处理时间及光强梯度实验数据显示,在蓝光光强为50μmol/m2s孵育时间为3 h时,二者相互作用强度达到最高.说明HB22与CRY1的相互作用具有蓝光响应.对蓝光处理不同时间的野生型col-4与cry1缺失突变体的材料进行HB22基因的定量PCR分析,发现拟南芥cry1缺失突变体中该基因的表达量比野生型中高,在蓝光处理2 h时,缺失突变体中表达量为野生型中的6倍左右.说明CRY1可能介导蓝光抑制HB22基因表达.     

螺旋藻在光胁迫时的抗逆性研究

螺旋藻对于环境的变化有很强的适应性.以钝顶螺旋藻为实验材料,测定螺旋藻在受到较强光照胁迫时藻体的电导率以及脯氨酸、丙二醛和过氧化氢酶系的含量.在3000 lx光照下,螺旋藻6个藻株的电导率以及脯氨酸、丙二醛和过氧化氢酶系的含量比在1000 lx光照下明显升高,螺旋藻的电导率最高上升了2～6倍;细胞内的脯氨酸含量最多增加5倍,最少增加13%;丙二醛的含量增加40%～100%;过氧化氢酶的含量上升范围在19%～80%,过氧化物酶的含量上升范围在20%～100%.说明螺旋藻在受到光胁迫时自身会启动相关保护机制,产生一定的抗逆性,以适应环境的变化.     

Light-dependent hypersensitive response and resistance signaling against Turnip Crinkle Virus in Arabidopsis

Resistance to Turnip Crinkle Virus (TCV) in Arabidopsis ecotype Dijon (Di)-17 is conferred by the resistance gene HRT and a recessive locus rrt. In Di-17, TCV elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes and high levels of salicylic acid (SA). We have previously shown that HRT-mediated resistance to TCV is dependent on SA-mediated signal transduction and that increased levels of SA confer enhanced resistance to TCV via upregulation of the HRT gene. Here we show that HRT-mediated HR and resistance are dependent on light. A dark treatment immediately following TCV inoculation suppressed HR, resistance and activation of the majority of the TCV-induced genes. However, the absence of light did not affect either TCV-induced elevated levels of free SA or the expression of HRT. Interestingly, in the dark, transgenic plants overexpressing HRT showed susceptibility, but overexpression of HRT coupled with high levels of endogenous SA resulted in pronounced resistance. Consistent with these results is the finding that exogenous application of SA prior to TCV inoculation partially overcame the requirement for light. Light was also required for N gene-mediated HR and resistance to Tobacco Mosaic Virus, suggesting that it is an important factor which may be generally required during defense signaling.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

Resonance light scattering study on the interaction of benproperine phosphate with eriochrome blue black R in the presence of sodium dodecylbenzene sulphonate and its analytical application

The interaction of benproperine phosphate (BPP) with eriochrome blue black R (EBBR) in the presence of sodium dodecylbenzene sulphonate (SDBS) was studied using resonance light scattering (RLS) technology and ultraviolet‐visual (UV‐vis) spectrophotometry. Under optimum conditions, BPP reacts with EBBP and SDBS to form a three‐component complex, which results in strong RLS signal and a new RLS peak. The enhanced RLS intensities are proportional to the concentration of BPP over the range 0.6–28.0 µg/mL, with a detection limit of 0.053 µg/mL. The affecting factors as well as the influence of coexisting substances were investigated. The results indicate that this assay method could be applied to the determination of BPP in pharmaceuticals, serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

An approach to quality management in structural biology: biophysical selection of proteins for successful crystallization

Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519-1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

Simple equations to estimate light interception by isolated trees from canopy structure features: assessment with three-dimensional digitized apple trees

* Simple models of light interception are useful to identify the key structural parameters involved in light capture. We developed such models for isolated trees and tested them with virtual experiments. Light interception was decomposed into the projection of the crown envelope and the crown porosity. The latter was related to tree structure parameters. * Virtual experiments were conducted with three-dimensional (3-D) digitized apple trees grown in Lebanon and Switzerland, with different cultivars and training. The digitized trees allowed actual values of canopy structure (total leaf area, crown volume, foliage inclination angle, variance of leaf area density) and light interception properties (projected leaf area, silhouette to total area ratio, porosity, dispersion parameters) to be computed, and relationships between structure and interception variables to be derived. * The projected envelope area was related to crown volume with a power function of exponent 2/3. Crown porosity was a negative exponential function of mean optical density, that is, the ratio between total leaf area and the projected envelope area. The leaf dispersion parameter was a negative linear function of the relative variance of leaf area density in the crown volume. * The resulting models were expressed as two single equations. After calibration, model outputs were very close to values computed from the 3-D digitized databases.