Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

High-definition mapping of neural activity using voltage-sensitive dyes

The distribution of patterns of activity in different brain structures has been related to the encoding and processing of sensory information. Consequently, it is important to be able to image the distribution of these patterns to understand basic brain functions. The spatial resolution of voltage-sensitive dye (VSD) methods has recently been enhanced considerably by the use of video imaging techniques. The main factor that now hampers the resolution of VSD patterns is the inherent limitation of the optical systems. Unfortunately, the intrinsic characteristics of VSD images impose important limitations that restrict the use of general deconvolution techniques. To overcomes this problem, in this study an image restoration procedure has been implemented that takes into consideration the limiting characteristics of VSD signals. This technique is based on applying a set of imaging processing steps. First, the signal-to-noise (S/N) ratio of the images was improved to avoid an increase in the noise levels during the deconvolution procedures. For this purpose, a new filter technique was implemented that yielded better results than other methods currently used in optical imaging. Second, focal plane images were deconvolved using a modification of the well-known nearest-neighbor deconvolution algorithm. But to reduce the light exposure of the preparation and simplify image acquisition procedures, adjacent image planes were modeled according to the in-focus image planes and the empirical point spread function (PSF) profiles. Third, resulting focal plane responses were processed to reduce the contribution of optical responses that originate in distant image planes. This method was found to be satisfactory under simulated and real experimental conditions. By comparing the restored and unprocessed images, it was clearly demonstrated that this method can effectively remove the out-of-focus artifacts and produce focal plane images of better quality. Evaluations of the tissue optical properties allowed assessment of the maximum practical optical section thickness using this deconvolution technique in the optical system tested. Determination of the three-dimensional PSF permitted the correct application of deconvolution algorithms and the removal of the contaminating light arising from adjacent as well as distant optical planes. The implementation of this deconvolution approach in salamander olfactory bulb allowed the detailed study of the laminar distribution of voltage-sensitive changes across the bulb layer. It is concluded that (1) this deconvolution procedure is well suited to deconvolved low-contrast images and offers important advantages over other alternatives; (2) this method can be properly used only when the tissue optical properties are first determined; (3) high levels of light scattering in the tissue reduce the optical section capabilities of this technique as well as other deconvolution procedures; and (4) use of the highest numerical aperture in the objectives is advisable because this improves not only the light-collecting efficiency to detect poor-contrast images, but also the spatial frequency differences between adjacent image planes. Under this condition it is possible to overcome some of the limitations imposed by the light scattering/birefringence of the tissue.     

Decolourisation of synthetic textile dyes by Phlebia tremellosa

Phlebia tremellosa decolourised eight synthetic textile dyes (200 mg l(-1)) by greater than 96% within 14 days under stationary incubation conditions. High performance liquid chromatography analysis of culture supernatants indicated that Remazol Black B was degraded by the fungus, however, complete mineralisation did not occur as a colourless organic breakdown product accumulated. Laccase activity was detectable in culture supernatants after 5 days when the fungus was grown in the presence of an artificial textile effluent, with activity reaching a maximum of 15 U l(-1) on day 14.     

Dynamics of microbial community for X-3B wastewater decolorization coping with high-salt and metal ions conditions

In this study, decolorization of Reactive Brilliant Red X-3B wastewater by the biological process coping with high salinity and metal ions conditions was investigated, and 16S rDNA based fingerprint technique was used to investigate microbial population dynamics. Results of sequencing batch tests showed that the microbial community could keep efficient with high concentration of dye (1100 mg L⁻¹), salt (150 g L⁻¹ NaCl) and some metal ions such as Mg²⁺, Ca²⁺ (1–10 mmol L⁻¹) and Pb²⁺ (1 mmol L⁻¹). 16S rDNA-based molecular analysis techniques demonstrated that the microbial community shifted during the acclimatization process affected by salt or metal ions. Some stains similar to Bacillus, Sedimentibacter, Pseudomonas, Clostridiales, Streptomyces and some uncultured clones acted for the dynamic succession, supposed as potential decolorization bacteria. This study provided insights on the decolorization capability and the population dynamic shifts during decolorization process of azo dye wastewater coping with salt and metal ions.     

大黄鱼耐低温性状相关微卫星标记的筛选

鱼类的耐低温性状是一种重要的经济性状。为了初步分析大黄鱼的耐低温性状, 文章采用15对荧光微卫星标记, 以SSR-PCR方法对大黄鱼低温耐受组和正常对照组F₁代共40个个体进行了耐低温性状遗传差异分析。结果显示, 标记LYC0002在两组样品中共扩增出5个等位基因(片段大小分别为112 bp、110 bp、108 bp、106 bp和104 bp), 其中LYC00021_{12 bp}等位基因在低温耐受组的出现频率达60%, 而在正常对照组中的频率为零, 表明该等位基因对大黄鱼的温度敏感特性有较明显的偏好性, 可能与某种耐低温基因存在一定的连锁关系。此外, 对LYC0002_{106 bp、}LYC0002_{108 bp}、LYC0002_{110 bp} 和 LYC0002_{112 bp} 4个等位基因分别进行了回收、克隆及测序。序列比对结果显示, LYC0002_{112 bp}等位基因含有10个(CA)重复单元, 而其他3个等位基因依次缺失1个(CA)重复单元, 说明LYC0002在本研究样本中的突变方式为微卫星逐步突变模型(Stepwise mutation model, SMM)。     

麻栎壳斗染料在羊毛染色中的应用

本文研究了提取自麻栎壳斗的植物染料(麻栎染料)的耐酸、碱稳定性,染浴pH值及铝、铁离子等环保型媒染剂对其染毛织物效果的影响,并且探究了其染色动力学.研究表明,麻栎染料在强酸性染浴(pH=3)中对羊毛织物直接性好,染色后毛织物得棕色,也可采用铝离子、铁离子对直接染色后的毛织物进行后媒染,以得到不同色相的毛织物,尤其是铁后...     

Water transfer via ectomycorrhizal fungal hyphae to conifer seedlings

Little is known about water transfer via mycorrhizal hyphae to plants, despite its potential importance in seedling establishment and plant community development, especially in arid environments. Therefore, this process was investigated in the study reported in this paper in laboratory-based tripartite mesocosms containing the shrub Arctostaphylos viscida (manzanita) and young seedlings of sugar pine (Pinus lambertiana) and Douglas-fir (Pseudotsuga menziesii). The objectives were to determine whether water could be transported through mycorrhizal symbionts shared by establishing conifers and A. viscida and to compare the results obtained using two tracers: the stable isotope deuterium and the dye lucifer yellow carbohydrazide. Water containing the tracers was added to the central compartment containing single manzanita shrubs. The fungal hyphae were then collected as well as plant roots from coniferous seedlings in the other two compartments to determine whether water was transferred via fungal hyphae. In addition, the length of the hyphae and degree of mycorrhizal colonisation were determined. Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) analysis was used to identify the fungal species involved in dye (water) transfer. Results of the stable isotope analysis showed that water is transferred via mycorrhizal hyphae, but isotopically labelled water was only detected in Douglas-fir roots, not in sugar pine roots. In contrast, the fluorescent dye was transported via mycorrhizal hyphae to both Douglas-fir and sugar pine seedlings. Only 1 of 15 fungal morphotypes (identified as Atheliaceae) growing in the mesocosms transferred the dye. Differences were detected in the water transfer patterns indicated by the deuterium and fluorescent dye tracers, suggesting that the two labels are transported by different mechanisms in the same hyphae and/or that different fungal taxa transfer them via different routes to host plants. We conclude that both tracers can provide information on resource transfer between fungi and plants, but we cannot be sure that the dye transfer data provide accurate indications of water transfer rates and patterns. The isotopic tracer provides more direct indications of water movement and is therefore more suitable than the dye for studying water relations of plants and their associated mycorrhizal fungi.     

A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.