Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus

In addition to its extracellular function as a secreted protein, IGF-binding protein (IGFBP)-5 has been postulated to act as a signaling molecule in the nucleus. This study aims to assess the significance of this postulated nuclear localization. By confocal immunofluorescence microscopy, we detected IGFBP-5 in the vesicular compartment of mammary epithelial cells in culture, while no nuclear staining was observed. Immunohistochemistry performed on paraffin sections of the involuting mammary gland revealed IGFBP-5 positive staining of epithelial cells only outside the nucleus. To evaluate the contribution of reuptake of extracellular IGFBP-5, T47D cells were incubated with Alexa Fluor 647-labeled IGFBP-5. The protein was taken up into intracellular vesicles and again was neither detectable in the cytoplasm outside of vesicular structures nor in the nucleus. Quantification of the time and concentration dependence of uptake by immunoblotting revealed that the process was saturable at IGFBP-5 concentrations between 1 and 2 mum and partially reversible with 30% remaining in the cell after a 1-h chase. The observation of nuclear uptake of IGFBP-5 was restricted to artificial conditions such as expression of non-secreted forms of IGFBP-5 or selective permeabilization of the plasma membrane by digitonin.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Differential lysis of plasma membranes and granules of human neutrophils by digitonin

The effect of the detergent digitonin on lysis of granule and plasma membranes of human neutrophils was studied. Either linear or sigmoid dose-response for release of the cytoplasmic marker lactate dehydrogenase and the granule markers lysozyme, beta-glucuronidase, lactoferrin, and myeloperoxidase was noted using digitonin concentrations ranging from 0.001 to 0.1 mM. However, release of the cytosol compartment was far more sensitive to the detergent than the granule compartment, with more release of lactate dehydrogenase than of lysozyme at 0.01-0.08 mM digitonin. Distinction between the two compartments was optimal at 0.025 mM digitonin. By examining in parallel the digitonin-induced release of exogenous fluorescent or luminescent indicators, a granule location was demonstrated for the pH indicator 9-aminoacridine, while the calcium probes aequorin and Quin 2 were released coincident with release of the cytosolic enzyme lactate dehydrogenase. These findings were employed to validate use of the indicators for monitoring of ion translocation in the intact cell. The differential effect of this detergent on subcellular membranes provides a broadly applicable technique for rapid assessment of the subcellular localization of tracer substances. Rapid monitoring may help to avoid problems of redistribution during cell fractionation.     

Cyclosporin a Increases Mitochondrial Calcium Uptake Capacity in Cortical Astrocytes but not Cerebellar Granule Neurons

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca²⁺-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca²⁺ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg²⁺ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca²⁺ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca²⁺]. Permeabilized astrocytes had a fourfold higher Ca²⁺ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca²⁺ uptake capacity was increased twofold by preincubation with 2–5 μM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca²⁺ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.     

Cytochrome c oxidase is not a proton pump.

We conclude that the reduction of O2 to 2 H2O by cytochrome c oxidase of rat liver mitochondria involves the translocation of 4-from cytochrome c at the outer surface of the cristae membrane per O2 reduced and protonated by 4 H+ ions that enter the reaction domain from the inner aqueous phase. This net electron-translocating function of cytochrome c oxidase plugged through the mitochondrial cristae membrane is not linked to a proton-pumping function, such as that proposed by Wikstr?m [7,8].     

Studies on rat liver mitochondria. 6. The effect of contaminating particles in mitochondria stored at 0-4 degrees C

Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.     

Flow cytometric characteristics of subchloroplast particles prepared by the action of various detergents

Agranal thylakoid membranes from leaves of Phaseolus vulgaris L. were fragmented using seven distinct detergents: digitonin, Triton X-100, cetylpyridinium chloride, sodium dodecylsulfate, and Zwittergents 3-12, 3-14, and 3-16, differing in chemical composition and/or electric charges. Subchloroplast particles separated on a Percoll gradient were examined by flow cytometry to determine their size and shape. Vesicle size was also determined by a haematological analyzer, which produced comparable results. Individual green bands consisted of vesicles of fairly wide size distribution. Simple direct proportionality between the particle density and their size was not observed in any case, nevertheless, bigger particles were more abundant in fractions of higher density. Some vesicles had even a larger size than the original thylakoids. This might reflect a specific action of the detergents in low concentrations on agranal membranes, with incorporation of detergent molecules into vesicles. Inner structures of particles of the same size and density were not necessarily identical, but represented several populations, as was apparent from the side scatter analysis. Flow cytometric analysis can thus be used for the investigation of mechanisms of membrane fragmentation by detergents.     

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

毛地黄皂苷诱导的水稻悬浮细胞防卫基因表达的信号传导

用带水稻苯丙氨酸解氨酶PAL 2启动子和GUS读码框嵌合基因的水稻悬浮细胞证明毛地黄皂苷能诱导水稻PAL 2的转录。NAD-PH、NADH、过氧化氢、超氧歧化酶、过氧化氢酶、甘露醇、1,2-羟基苯-3,5-二磺酸(Tiron)、paraquat、精氨酸、还原型和氧化型的谷胱甘肽、N-乙酰-L-半胱氨酸、二硫苏糖醇等都不影响毛地黄皂苷诱导的PAL2转导。只有在超氧歧化酶和过氧化氢酶或超氧歧化酶和Tiron一起时才微弱地抑制这种诱导。毛地黄皂苷能在缺钙培养基中诱导胞外介质碱性化,但它只在含钙离子的条件下才诱导PAL 2转录。钙离子载体A 23187及Stauros-poline不诱导PAL 2基因转录和胞外介质碱性化。钙调素的拮抗剂N-(6-Aminohexy1)-5-chloro-1-naphthalenesulfonamide(W7)和trifluoperazine(TFP)能抑制毛地黄皂苷诱导的PAL 2转录,但一些蛋白激酶的激活剂和抑制剂包括phorbol-12-myristate(TPA)、1-(5-Isoquinoline sulfony1)-2-methylpiperaz-ine(H7)、staurosporine及genestein不干扰它对PAL 2转录的诱导,G蛋白激活剂GTP-γS和抑制剂百日咳毒素也不影响这种诱导。毛地黄皂苷不诱导几丁酶转录。二硫苏糖醇能抑制几丁酶转录。伴刀豆球蛋白及麦胚凝集素能微弱地促进几丁酶基因转录,但对PAL 2表达没有影响。看来毛地黄皂苷诱导的水稻悬浮细胞PAL 2转录与活性氧激活没有简单和直接的关系。但它依赖于胞外介质碱性化和钙-钙调素,它的信号传导途径与诱导几丁酶转录的信号传导途径是不同的。