Interfacial photopolymerization of beta-cell clusters: approaches to reduce coating thickness using ionic and lipophilic dyes.

Microencapsulation of insulin-secreting cells is a potential therapy for Type I diabetes. Critical requirements for therapeutic use are the high number of beta-cells to be implanted and a fast insulin diffusion through the encapsulating membrane. The use of thin, conformal coating for beta-cell encapsulation may be a way to reach these goals by decreasing the capsule void volume. This study focuses on the production of very thin membranes by interfacial photopolymerization of beta-cell clusters. Two types of photosensitizing dyes were used: Eosin Y, which stains the cell surface as well as the cytoplasm, and a lipophilic-derivatized eosin that specifically stains the cell membrane. The fraction of encapsulated clusters and membrane thickness were studied as a function of irradiation parameters. In the case of Eosin Y, the fraction of encapsulated clusters is found to depend mainly on an optimal light dose for and above which complete encapsulation is obtained. We found that the membrane thickness decreased with decreasing irradiation time, but does not depend on irradiation intensity. Using Eosin Y, 16 microm thick coatings were obtained, together with a high fraction of encapsulated clusters. The coating thickness was further reduced to 10 microm by using the lipophilic-derivatized eosin photoinitiator. Cell viability and functionality were studied following the encapsulation process using vital staining and measurement of insulin secretion. Cell viability and functionality were preserved following the encapsulation process with Eosin Y and for sufficiently low lipophilic dye concentration. Although it still requires further improvement, the method proposed here provides a promising route to obtain thinner coatings, down to a few microns.     

Intraspecific variability in the response of bloom-forming marine microalgae to changed climate conditions

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO(2) availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24°C, and present day and next century applied atmospheric pCO(2), respectively. In both species, individual strains were affected in different ways by increased temperature and pCO(2). The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO(2) on growth-slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO(2). Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO(2) supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO(2) conditions, with potentially severe consequences for the coastal ecosystem.     

Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and DPPAO

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.     

Using absorbance and fluorescence spectra to discriminate microalgae

The utility of absorbance and fluorescence-emission spectra for discriminating among microalgal phylogenetic groups, selected species, and phycobilin- and non-phycobilin-containing algae was examined using laboratory cultures. A similarity index algorithm, in conjunction with fourth-derivative transformation of absorbance spectra, provided discrimination among the chlorophyll [Chl] a/phycobilin (cyanobacteria), Chl a/Chl c/phycobilin (cryptophytes), Chl a/Chl b (chlorophytes, euglenophytes, prasinophytes), Chl a/Chl c/fucoxanthin (diatoms, chrysophytes, raphidophytes) and Chl a/Chl c/peridinin (dinoflagellates) spectral classes, and often between}among closely related phylogenetic groups within a class. Spectra for phylogenetic groups within the Chl a/Chl c/fucoxanthin, Chl a/Chl c/peridinin, Chl a/phycobilins and Chl a/Chl c/phycobilin classes were most distinguishable from spectra for groups within the Chl a/Chl b spectral class. Chrysophytes/diatoms/raphidophytes and dinoflagellates (groups within the comparable spectral classes, Chl a/Chl c/fucoxanthin and Chl a/Chl c/peridinin, respectively) displayed the greatest similarity between/among groups. Spectra for phylogenetic groups within the Chl a/Chl c classes displayed limited similarity with spectra for groups within the Chl/phycobilin classes. Among the cyanobacteria and chlorophytes surveyed, absorbance spectra of species possessing dissimilar cell morphologies were discriminated, with the greatest range of differentiation occurring among cyanobacteria. Among the cyanobacteria, spectra for selected problematic species were easily discriminated from spectra from each other and from other cyanobacteria. Fluorescence-emission spectra were distinct among spectral classes and the similarity comparisons involving fourth-derivative transformation of spectra discriminated the increasing contribution of distinct cyanobacterial species and between phycobilin- and non-phycobilin-containing species within a hypothetical mixed assemblage. These results were used to elucidate the application for in situ moored instrumentation incorporating such approaches in water quality monitoring programmes, particularly those targeting problematic cyanobacterial blooms.     

Methods to maximise the staining of fungal propagules with fluorescent dyes

The spores and conidia of most fungi have very thick and resistant cell walls that severely impede the staining with fluorescent dyes to allow epifluorescence microscopy to be employed for their direct detection and quantification in natural habitats. In this study, oxidation by sodium hypochlorite and microwave irradiation (MWI) were used to enhance the staining of Aspergillus fumigatus and Penicillium brevicompactum conidia with six fluorescent dyes. Sodium hypochlorite resulted in high percentages of stained conidia (up to 98.8% with 4',6-diamidino-2-phenylindole [DAPI]), but had to be removed prior to staining with consequent heavy conidia losses. By contrast, MWI gave very high percentages, while its enhancement of fluorescence intensity facilitated observation by epifluorescence microscopy.     

Evaluation of delocalized lipophilic cationic dyes as delivery vehicles for photosensitizers to mitochondria

Mitochondria are attractive targets in photodynamic therapy. Two conjugates: TPP–Rh (a porphyrin–rhodamine B conjugate) and TPP–AO (a porphyrin–acridine orange conjugate), each possessing a single delocalized lipophilic cation, were designed and synthesized as photosensitizers. Their ability to target the mitochondria for photodynamic therapy was evaluated. The conjugates were synthesized by conjugating a monohydroxy porphyrin (TPP-OH) to rhodamine B (Rh B) and acridine orange base (AO), respectively, via a saturated hydrocarbon linker. To evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro photodynamic activities were studied in comparison to those of TPP-OH. Although fluorescence energy transfer (FRET) was observed in the conjugates, they were capable of generating singlet oxygen at rates comparable to TPP-OH. Biologically, exciting results were observed with TPP–Rh, which showed a much higher phototoxicity [IC₅₀, 3.95 μM: irradiation of 400–850 nm light (3 mW cm⁻²) for 1 h] than either TPP-OH or Rh B (both, IC₅₀, >20 μM) without significant dark toxicity at 20 μM. This improved photodynamic activity might be due to a greater cellular uptake and preferential localization in mitochondria. The cellular uptake of TPP–Rh was 8 and 14 times greater than TPP-OH and Rh B, respectively. In addition, fluorescence imaging studies suggest that TPP–Rh localized more in mitochondria than TPP-OH. On the other hand, TPP–AO showed some dark toxicity at 10 μM and stained both mitochondria and nucleus. Our study suggests that conjugation of photosensitizers to Rh might provide two benefits, higher cellular uptake and mitochondrial localization, which are two important subjects in photodynamic therapy.     

A convenient buffer system for controlled staining with basic dyes

Summary A new buffer system is described for use in histochemical staining with basic dyes. The buffer is made up by mixing solutions of formic acid and sodium acetate. Tables giving the proportions for closely-spaced pH values in the range 3.0–5.6 are presented. A table for an acid phosphate series down to a pH of 2.0 is also included. The value of these buffer mixtures in histological as well as histochemical staining with basic dyes is stressed.With 1 Figure in the Text     

Modification of the method for staining DNA with basic dyes]

The author studied the mechanisms and the applicability in histochemistry of the sodium bisulfate treatment with subsequent toluidine and methylene blue staining after Felgen's hydrolysis. Bisulfite treatment proved to increase the reaction intensity 11/2-fold; the stain is bound stoichiometrically. Toludidine blue results in a metachromatic and anisotropic staining of the cell nuclei. The method is recommended as a sensitive test for DNA in cytochemical investigations and for the study of dichroism of the DNA-containing structures.     

共生条件下三种荒漠灌木的根系分布特征及其对降水的响应

以全根系挖掘法,对共生于原始盐生荒漠生境中的多枝柽柳[Tamarix ramosissima (Ledeb.)]、梭梭[Haloxylon ammodendron(C. A. Mey.)Bunge]、琵琶柴[Reaumuria soongorica (Pall.) Maxim.]的根系分布特征进行了研究;对降水引发的湿润-干旱周期中植物同化枝水势、蒸腾速率的变化过程进行了跟踪观测,并据此计算3种植物的水分胁迫效应指数和土壤-植物系统导水度,以最终确定3种植物用水策略和其对降水的响应特征.研究结果表明,多枝柽柳的吸收根系分布范围从地下50cm到310cm,单株平均总吸收根表面积为30249.2cm2;梭梭的根系分布范围0～250cm,单株平均总吸收根表面积12847.3 cm2;琵琶柴的根系分布范围0～80cm,单株平均总吸收根表面积361.8 cm2.多枝柽柳为深根植物,主要利用地下水和深层土壤水,在降水引发的湿润-干旱周期中,其植物水分生理参数对降水无响应.琵琶柴为浅根植物,对降水响应极为显著.梭梭的根系分布特征介于多枝柽柳和琵琶柴之间,对地下水和降水都有利用,对降水响应显著.3种荒漠灌木对降水的响应差异显然与其根系分布、水分利用策略密切相关,在未来降水发生变化的情景下,根系分布特征的差异将决定着植物在水分资源竞争中的地位.具有较强根系形态可塑性的物种,如梭梭,将具有明显的竞争优势.     

三种香蘑属野生食用菌菌株的培养条件优化

【背景】供试菌株分别是分离自山西省宁武县管涔山的肉色香蘑Lepista irina、斑褶香蘑L. panaeolus和山西省蒲县五鹿山的紫丁香蘑L. nuda 3种野生食用菌的子实体。【目的】获得3种野生食用菌的最佳培养条件。【方法】以菌丝生长速度为指标研究不同碳源、氮源、碳氮比、pH和培养温度等各因素对菌丝生长的影响,根据Box-Benhnken中心组合试验设计原理,采用3因素3水平的响应面法确定使菌丝体达到最快生长速度的最佳培养碳源、氮源和pH。【结果】肉色香蘑在葡萄糖20.9 g/L、土豆196.47 g/L、pH 6.0、培养温度21 °C的条件下,菌丝日均生长速度达到最大,为1.13 mm/d;斑褶香蘑在甘露醇17.4 g/L、酵母膏8.1 g/L、B族维生素0.1 g/L、K2HPO4 2.5 g/L、MgSO4 2.5 g/L、pH 7.9、培养温度25 °C的条件下,菌丝日均生长速度达到最大,为0.73 mm/d;紫丁香蘑在土豆200 g/L、可溶性淀粉20.5 g/L、KNO3 2.1 g/L、K2HPO4 2.5 g/L、MgSO4 2.5 g/L、B族维生素0.1 g/L、pH 7.0、培养温度25 °C的条件下,菌丝日均生长速度达到最大,为2.38 mm/d。【结论】获得了3种香蘑属菌株的最佳培养条件,为后续优质野生食用菌的引种驯化积累了相关数据和资源。     

Attempts to explain and reduce variability of superovulation

The variability in response to superovulation treatment is a major disadvantage for economical use of embryo transfer and clearly limits use of embryo transfer in animal breeding. Despite considerable efforts, it has not yet been possible to reduce this variability drastically. Less than 40% of the causes for variability has been explained to date. The high proportion of unexplained variability leads to the hypothesis that variability is a special biological function that supports natural selection over the long term. Corresponding data from laboratory animal studies support this hypothesis.     

Long-term staining of live Merkel cells with FM dyes

Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. To find dyes that remain in the cells for a long period of time, we tested many fluorescence dyes and found that FM dyes (such as FM1-43) are useful markers for live Merkel cells. In the rat footpad skin, FM1-43 was shown to stain 95% of live Merkel cells that were already stained with quinacrine. FM4-64 stained 98% of quinacrine-stained Merkel cells. Merkel cells in sinus hair follicles were also stained with FM dyes. The fluorescence intensity of FM dyes was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM-dye-stained cells. To test how long FM dyes remain in live cells, FM-dye-stained Merkel cells in hair follicles were embedded in collagen gel and were cultured in a serum-free medium. FM-dye-stained cells were easily identified even after 7 days of culture. During the culture, Merkel cells changed their shape, moved in the preparation and tended to aggregate on the surface. We conclude that FM dyes are powerful tools for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate FM dyes suggests that vesicles in the cells are likely to have mechanisms of recycling in a manner similar to those in neurons and secretory cells.     

Irradiation of specimens by excitation light before and after staining with pararosaniline feulgen: A new method to reduce non-specific fluorescence in cytofluorometry

Summary Thorough irradiation on specimens with strong green light before or after pararosaniline Feulgen staining destroys specifically the primary-fluorescent substances in the background. By this treatment of pre- or post-irradiation, accuracy of DNA cytofluorometry is markedly improved and the Feulgen specific fluorescence is stabilized. Selecting proper wavelength, this technique will universally be useful in microfluorometry of any fluorochromes for reducing background fluorescence.     

Proteomic capacity of recent fluorescent dyes for protein staining

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby (SR), Deep Purple (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP approximately SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.