Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.     

Application of hemicelluloses precipitated via ethanol treatment of pre-hydrolysis liquor in high-yield pulp

Hemicelluloses in industrially produced pre-hydrolysis liquor (PHL) were precipitated with ethanol. These PHL-derived hemicelluloses (PHL-EH) and a commercial, pure birch wood xylan sample (powder form) (BWX) were bleached using chlorine dioxide (D(0) and D(1)) and hydrogen peroxide (Ep) in the D(0)EpD(1) sequence, and the chemical compositions, molecular weights and charge densities of the treated samples were assessed. When applied to high-yield pulp (HYP) at 50 mg/g, 26 and 20 mg/g of the bleached PHL-EH and BWX, respectively, were adsorbed without significantly affecting paper properties. These results suggest that semi-bleached hemicelluloses could be used to increase the basis weight of paper products. Furthermore, an integrated process was proposed that converts the kraft-based dissolving pulp production process into a biorefinery unit with dissolving pulp, bleached hemicelluloses and lignin as main products.     

Retinal pigmentation, visual acuity and brightness levels.

This study investigates the hypothesis that the degree of retinal pigmentation in the human eye is adaptive as it relates to the maintenance of visual acuity in optically stressful environments, deserts and snowfields. Eighty-four subjects were examined, an estimation of their degree of retinal pigmentation made by ophthalmoscopic examination and their binocular visual acuity tested over ten levels of brightness. The general level of retinal pigmentation did not influence mean visual acuity within the levels of brightness used in this study. The hypothesis was, therefore, rejected, but with the proviso that this study should be extended to even higher levels of brightness than were obtained here. There was no difference in mean pupil size at various levels of illumination between individuals grouped by degree of retinal pigmentation.     

Multi-scale lines and edges in V1 and beyond: Brightness,object categorization and recognition,and consciousness

In this paper we present an improved model for line and edge detection in cortical area V1. This model is based on responses of simple and complex cells, and it is multi-scale with no free parameters. We illustrate the use of the multi-scale line/edge representation in different processes: visual reconstruction or brightness perception, automatic scale selection and object segregation. A two-level object categorization scenario is tested in which pre-categorization is based on coarse scales only and final categorization on coarse plus fine scales. We also present a multi-scale object and face recognition model. Processing schemes are discussed in the framework of a complete cortical architecture. The fact that brightness perception and object recognition may be based on the same symbolic image representation is an indication that the entire (visual) cortex is involved in consciousness.     

Biobleaching of banana fibre pulp using <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C O1 xylanase produced from wheat bran under solid-state cultivation

A cellulase-free xylanase produced by Bacillus subtilis C 01 from wheat bran under solid-state cultivation was tested for its efficacy in biobleaching of raw banana fibre and banana pulp obtained through a mechanical pulping process. Banana pulp samples treated with crude xylanase (450 nkat g⁻¹ pulp) resulted in a 19.6% increase in the brightness as compared to untreated pulp. The presence of chromophores, hydrophobic compounds and an increased reducing sugar (10.79 mg g⁻¹ pulp) quantity in the bleached solution after enzymatic treatment indicated the removal of materials that were absorbed at 237 nm from the banana pulp.     

Hyper production of cellulase-free xylanase by <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> SSBP on bagasse pulp and its application in biobleaching

A cellulase-free xylanase production by Thermomyces lanuginosus SSBP using bagasse pulp was examined under submerged (SmC) and solid-state cultivation (SSC). Higher level of xylanase activity (19,320 ± 37 U g⁻¹ dried carbon source) was obtained in SSC cultures than in SmC (1,772 ± 15 U g⁻¹ dried carbon source) after 120 h with 10% inoculum. The biobleaching efficacy of crude xylanase was tested on bagasse pulp, and the maximum brightness of 46.1 ± 0.06% was observed with 50 U of crude xylanase per gram of pulp, which was 3.8 points higher than the brightness of untreated samples. Reducing sugars (26 ± 0.1 mg g⁻¹) and UV-absorbing lignin-derived compounds in the pulp filtrates were observed as maximum in 50 U of crude xylanase-treated samples. T. lanuginosus SSBP has potential applications due to its high productivity of xylanase and its efficiency in pulp bleaching.     

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells

Background

Dynamin 2 (Dyn2) is a ~ 100 kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state.

Methods

In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells.

Results

Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1 μM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2.

Conclusions and general significance

These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.     

Comparative study on application of T.lanuginosus SSBP xylanase and commercial xylanase on biobleaching of non wood pulps

Biobleaching of three non wood kraft pulps (bagasse, rice straw and wheat straw) by Thermomyces lanuginosus SSBP xylanase and commercial xylanase (cartazyme sandoz), was studied in order to investigate their potential and effect on their various properties (reduction sugars, chlorine dioxide, kappa number, brightness and chromophores). In generally, xylanases released chromophores and reducing sugars and decreased kappa number of pulps. These samples gained over six brightness points over controls. Biobleaching of rice straw pulp with xylanase cartazyme (Sandoz) produced chlorine dioxide savings of up to 25% or 3.5-4 kg chlorine dioxide/ton pulp.     

Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells

Background

Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.

Methods

We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.

Results

Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional.

Conclusions

We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells.

General significance

The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.     

显微摄像条件对细胞形态测试结果的影响

目的:研究显微摄像条件对细胞形态测试结果的影响情况。方法:以苏木精-伊红(HE)染色的胃壁细胞为研究对象,分别在不同光亮度、对比度、饱和度和锐度的成像条件下进行显微摄像,采用图像分析软件(Image-Pro Plus 6.0)测试胃壁细胞的色度学和几何形态学参数,并对测试结果进行分析比较。结果:不同光亮度、对比度、饱和度组的胃壁细胞红、绿、蓝三基色差异均有统计学意义(P<0.05),其中红、绿、蓝基色值在高光亮度和高对比度组最高,同时红基色值在高饱和组最高,而绿、蓝基色值在高饱和组最低(P<0.05);不同锐度的胃壁细胞红、绿、蓝三基色差异没有统计学意义(P>0.05)。不同光亮度、对比度、饱和度和锐度的胃壁细胞的面积、周长、平均直径差异均没有统计学意义(P>0.05)。结论:光亮度、对比度和饱和度对细胞色度学参数影响明显,而对几何形态学参数无明显影响。