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1.
The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement. 相似文献
2.
Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway. 相似文献
3.
Abstract The effect of culture filtrate containing cholera toxin (CT) on rat intestinal permeability was studied using fluorescein isothiocyanate-labelled dextran 3000 (FITC-D3, M r , 3000) as probe molecule. CT was given either perorally, via a gastric tube 90 min before, or locally in conjunction with the permeability measurement in the distal ileum. Compaired to the control animals, either mode of administration resulted in increased permeation of FITC-D3 from the intestine to portal blood. The effect of the local treatment was apparent after 5–10 min and prevailed during the 60-min measurement period. The results indicate that CT not only affects net water transport at the intestinal mucosa but also the passage of larger molecules across the intestinal wall. 相似文献
4.
Megumi Kono Koji O'hara Yoshihiro Arai Hideyuki Fukuda Misaki Asakawa Hideomi Nakahara 《FEMS microbiology letters》1985,28(2):213-217
Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent. 相似文献
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent. 相似文献
5.
In this study, two procedures for the immobilization of β-glucosidase on silica are compared. The first approach comprises a preliminary stabilization of β-glucosidase by coupling with dextran dialdehyde and subsequent immobilization of the obtained β-glucosidase dextran dialdehyde with aminopropylsilica. In the second approach, β-glucosidase is immobilized on silica modified with a dextran-dialdehyde coating. Enzyme immobilized via coupling with dextran dialdehyde and subsequent attachment with aminopropylsilica show a remarkably enhanced thermostability. Enzyme immobilized by the alternative approach demonstrated an inferior thermoresistance. The difference in behavior of the immobilized enzyme obtained via these two methods can be explained considering the number of links between the enzyme and carrier. Enzyme immobilized on dextran dialdehydecoated silica is fixed via a limited number of links. On the other hand, with soluble β-glucosidase-dextran conjugates, the enzyme configuration is already stabilized via a high number of links with the dextran backbone. It is clear from this study that the sequence of reactions in immobilizing enzymes on silica support via a dextran-dialdehyde linker has a significant effect on the final properties. 相似文献
6.
Adsorption of BSA on QAE-dextran: equilibria 总被引:1,自引:0,他引:1
Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc. 相似文献
7.
Fatemeh Sabet Sarvestani Ali-Mohammad Tamaddon Ramin Yaghoobi Bita Geramizadeh Negar Azarpira 《Engineering in Life Science》2023,23(7):2200140
Angiogenesis is a vital step in tissue regeneration. Hence, the current study aimed to prepare oxidized dextran (Odex)/collagen (Col)-hydrogels with laminin (LMN), as an angiogenic extracellular matrix (ECM) component, for promoting human umbilical vein endothelial cell (HUVEC) proliferation and function. Odex/Col scaffolds were constructed at various concentrations and temperatures. Using oscillatory rheometry, scanning electron microscopy (SEM), and cell viability testing, the scaffolds were characterized, and then HUVEC proliferation and function was compared with or without LMN. The gelation time could be modified by altering the Odex/Col mass ratio as well as the temperature. SEM showed that Odex/Col hydrogels had a more regular three-dimensional (3D) porous structure than the Col hydrogels. Moreover, HUVECs grew faster in the Col scaffold (12 mg/mL), whereas the Odex (30 mg/mL)/Col (6 mg/mL) scaffold exhibited the lowest apoptosis index. Furthermore, the expression level of vascular endothelial growth factor (VEGF) mRNA in the group without LMN was higher than that with LMN, and the Odex (30 mg/mL)/Col (6 mg/mL) scaffold without LMN had the highest VEGF protein secretion, allowing the cells to survive and function effectively. Odex/Col scaffolds, with or without LMN, are proposed as a tissue engineering construct to improve HUVEC survival and function for angiogenesis. 相似文献
8.
Min Lu Per- Ake Albertsson G te Johansson Folke Tjerneld 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,680(1-2):65-70
Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na2SO4. 相似文献
9.
Richard L. Schading Raymond I. Carruthers Barbara A. Mullin-Schading 《Biocontrol Science and Technology》1995,5(2):201-208
The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate. 相似文献
10.
A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D 总被引:1,自引:0,他引:1
We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells. 相似文献