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At least 649 records · Page 36Linked to original sources
PubMed1998

Comparison between ELISA and gel-filtration assay for the quantitation of airway mucins.

In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [3H]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0-2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [3H]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP-stimulated culture spent media were added to inhibit the immunoreactivity with mAbRT03. The contents of mucin in the sample were calculated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.

Adenosine Triphosphate↗
PubMed1999

Regional pleural filtration and absorption measured by fluorescent tracers in rabbits.

In prone anesthetized rabbits, we used Evans blue-dyed albumin (EBA) to study regional pleural filtration and FITC dextran to study regional pleural absorption. Evans blue was injected intravenously, and the animals were ventilated for 6 h at either of two levels of ventilation. Postmortem the right rib cage was frozen and thawed before study. EBA fluorescence emitted from the rib cage surface was measured along the cranial-caudal axis near the mid chest with fluorescence videomicroscopy. Fluorescent light intensity increased from the third to the eighth rib in a cyclic fashion, with peaks at the ribs and troughs at the intercostal spaces. This increase was greater at the higher ventilation. Fluorescent images of cross sections of a frozen rib cage verified a cranial-caudal gradient in filtration. Fluorescent images of FITC dextran absorbed from the pleural space into the rib cage surface indicated major areas of absorption at the ventral, caudal, and cranial regions adjacent to the lung margins and areas of absorption scattered in the intercostal spaces. Simultaneous measurements of EBA filtration and FITC absorption showed sites of maximal filtration that were different from sites of maximal absorption. Pleural uptake of fluorescent microspheres (2-microm diameter) located lymphatic stomata distributed randomly within clusters in the intercostal spaces and channels of lymphatic lacunae parallel to blood vessels. Diaphragmatic uptake of microspheres was confined mainly to the ventral surface of the central tendon. Visceral pleural absorption was minimal.

Absorption↗
PubMed2002

Purification of lysozyme by multistage affinity filtration.

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins.

Journal Article↗
PubMed2004

Development of a dialyzer with enhanced internal filtration to increase the clearance of low molecular weight proteins.

Accumulated low molecular weight proteins in hemodialysis patients require a high-flux dialyzer. There have been several methods proposed for enhancing internal filtration, including narrowing the inside diameter of the hollow fibers, lengthening the fibers, and increasing the fiber density ratio. We tried to enhance the internal filtration by increasing the pressure drop in the dialysate compartment through increasing the fiber density ratio. If the fiber density ratio is too high, however, an irregular dialysate path may result, thus decreasing dialysis performance. Therefore, we took note of the shape of the inner housing and added a short taper structure, which improved the dialysate path dramatically. Consequently, we developed an internal filtration-enhanced dialyzer (APS-Prototype) to improve dialysis performance. The internal filtration rate in water (measured by Doppler ultrasound) was 13.2 l/session for the APS-Prototype and 5.3 l/session for the APS-15E. The amount of alpha1-microglobulin (alpha1-MG) in bovine plasma was 0.34 g for the APS-Prototype and 0.11 g for the APS-15E. In addition, the amount of alpha1-MG in vivo was 29.0% +/- 5.8% for the APS-Prototype, significantly higher than that for the APS-15E (13.6% +/- 1.9%). The desirable loss of albumin is 2-4 g in hemodiafiltration, and it was 3.92 +/- 1.03 g for the APS-Prototype. The prototype showed excellent solute removal performance with no clinical or engineering problems.

Adult↗
PubMed1986

Reopening filtration fistulas with the neodymium-YAG laser.

We used the mode-locked neodymium-YAG laser to reopen filtration fistulas in nine eyes in which the fistula had become occluded internally by a variably pigmented membrane, vitreous, or, in one case, capsular lens material. In five eyes filtration was successfully reestablished, reducing the intraocular pressure to less than 21 mm Hg (range, 10 to 20 mm Hg). The remaining four eyes had fistulas thought to be open internally after laser treatment; however, filtration was not established because of external subconjunctival scarring of the bleb. As with needling or argon laser fistular reopening, successful filtration followed Nd-YAG laser fistular surgery only when the fistula was occluded by an identifiable membrane and when the eye already had had a well-established bleb.

Cicatrix↗
PubMed1983

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes.

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.

Azotobacter↗
PubMed1984

A rapid-filtration technique for membrane fragments or immobilized enzymes: measurements of substrate binding or ion fluxes with a few-millisecond time resolution.

The construction and use of a filtration system with milliseconds time resolution is described here. This apparatus allows measurements of substrate binding to immobilized enzyme or ion fluxes through membrane vesicles to be performed over a very large time scale, from 10 ms to seconds. The main advantage of this system compared to the widely used quench-flow technique is that it does not require the use of an inhibitor. Following adsorption of the enzyme in an adequately chosen filter, the reaction is allowed to proceed within the filter during a forced filtration of a buffer containing the reactive substrate (or of a washing solution in the case of efflux measurements). The design allows the duration of filtration and buffer flux to be finely and reproducibly controlled. This paper illustrates the use of this rapid-filtration system for time-resolved measurements of calcium binding and transport by sarcoplasmic reticulum Ca-ATPase and of the initial phase of ADP transport by the ADP/ATP carrier of intact mitochondria.

Animals↗
PubMed1984

Polyethylene glycol interferes with protein molecular weight determinations by gel filtration.

Gel filtration studies on Sephadex G-75 demonstrate markedly increased elution volumes for proteins chromatographed with polyethylene glycol. As little as 5% (w/v) polyethylene glycol in the applied protein sample can reduce apparent molecular weight estimates by gel filtration as much as 55%. Furthermore, gel filtration columns equilibrated with polyethylene glycol are not size-separating columns. Consequently, caution must be exercised when performing and interpreting gel filtration studies of proteins previously treated or precipitated with polyethylene glycol.

Chromatography, Gel↗
PubMed1985

Alterations of myoelectric activity associated with Campylobacter jejuni and its cell-free filtrate in the small intestine of rabbits.

We evaluated the effects of a culture of Campylobacter jejuni and its cell-free filtrate on myoelectric activity of isolated ileal segments in New Zealand White rabbits. Hematoxylin and eosin staining and scanning electron microscopy were used to assess the association between histologic changes and alterations in intestinal myoelectric activity. A culture of C. jejuni was shown to cause a significant increase in repetitive bursts of action potentials (RBAPs) (6.9 +/- 1.2 RBAP/h; p less than 0.001) compared with controls (0.3 +/- 0.1). Cell-free filtrates of C. jejuni cultures were also observed to induce RBAPs (5.0 +/- 0.9 RBAP/h; p less than 0.001). The fraction within the filtrate that induces alterations in motility was not destroyed by heating to 100 degrees C for 15 min (6.3 +/- 1.2 RBAP/h). Although no gross histologic changes were noted by hematoxylin and eosin staining of intestine exposed to a culture of C. jejuni for 8 h, blunting of villi with a cellular infiltrate was noted in rabbits exposed for 24 h. Scanning electron microscopy disclosed patchy villous tip damage in 3 of 5 animals exposed to cell-free filtrates for 8 h. These studies suggest C. jejuni is pathogenic and produces a heat-stable substance that alters intestinal myoelectric activity in rabbits.

Action Potentials↗
PubMed1991

Comparison of the separation of bovine heparin by strong anion exchange and by gel filtration chromatography.

Heparin has been fractionated by strong anion exchange chromatography followed by elution of the pools on a gel filtration column. This resulted in the expected inverse relationship in the elution order for the pools run by the two methods. Also chromatography of heparin was performed in the reverse order: gel filtration first, followed by anion exchange of the pools. For this order of separation four of the five gel filtration pools of different molecular weights eluted at a similar LiCl concentration. The specific activities of different pools of heparin were evaluated using a colorimetric microwell kinetics assay using antithrombin and thrombin. For the pools separated by ion exchange first, there was an exponential increase of specific activity with increasing molecular weight for all pools. For the pools isolated by gel filtration first, the specific activities became level after an initial increase in relation to molecular weight. Thus, unique pools of heparin species are being isolated by different modes of chromatography.

Animals↗
PubMed1981

Large scale isolation of human erythrocyte membranes by high volume molecular filtration.

A molecular filtration procedure for preparing large quantities of human erythrocyte ghost membranes is presented. Hemolysate ghost membranes are rapidly cycled in the retentate channel of the filtration apparatus, while hemoglobin is removed as it passes through Pellicon filters into the filtrate channel. Several-liter quantities of washed packed erythrocytes can be processed in a few hours with this system, and the filtration procedure does not appear to alter intact erythrocyte or ghost membranes. Intact erythrocytes in isotonic solution can be circulated through the retentate channel for 16 h with only 3% hemolysis and with preservation of their original morphology in scanning electron microscopy. Ghost membranes isolated by the procedure are virtually identical in morphology, polypeptide composition and acetylcholinesterase content to membranes isolated by conventional centrifugation techniques.

Cell Fractionation↗
PubMed1993

Antimicrobial activity of neutralized extracellular culture filtrates of lactic acid bacteria isolated from a cultured Indian milk product ('dahi').

Neutralized extracellular culture filtrate obtained from isolates of Lactobacillus acidophilus, Lactobacillus delbruecki ssp. bulgaricus, Lactobacillus salivarius and Lactococcus lactis ssp. lactis from 'dahi' showed weak to moderate inhibition of Staphylococcus aureus, Bacillus cereus, Escherichia coli, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus laterosporus, Bacillus subtilis and Pseudomonas aeruginosa when tested by the diffusion agar well assay method. The effective minimum quantity of lactic culture filtrates required to obtain complete inhibition of an inoculum of 10(3) cfu/ml of the bacteria tested was between 20 and 26% (vol/vol), as determined by the agar incorporation method. Neutralized extracellular culture filtrate of these lactic cultures added at a level of 10% in sterile, 10% reconstituted non-fat dry milk was able to either suppress or retard growth of selected bacterial cultures when incubated at 37 degrees C for 24 h. This study indicated the antimicrobial activity of dahi and the potential of using neutralized extracellular culture filtrate of lactic acid bacteria in the biopreservation of foods.

Animals↗
PubMed1992

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae.

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.

Ammonium Sulfate↗
PubMedDate not supplied

Capillary filtration coefficient in type II (non-insulin-dependent) diabetes.

Changes in microvascular permeability may be important in the pathogenesis of diabetic microangiopathy. In order to assess microvascular fluid permeability, the capillary filtration coefficient was determined in the forearm of 24 normotensive type II diabetic patients with minimal evidence of microangiopathy and satisfactory glycemic control, and 24 age- and sex-matched control subjects, using a sensitive strain gauge plethysmographic system. The median capillary filtration coefficient was not significantly different in the type II diabetic patients and control subjects [5.3 (3.2 - 9.1) x 10(-3) mL.min-1.100 g tissue-1.mm Hg-1 versus 5.4 (3.5 - 8.0) x 10(-3) mL.min-1.100 g tissue-1.mm Hg-1, p = 0.98)]. There were no correlations between capillary filtration coefficient and age, blood pressure, body mass index, duration of diabetes, glycemic control, or the presence of microvascular complications. These findings contrast with type I diabetes, where capillary filtration coefficient is elevated at an early stage in the disease, and lend support to the theory that there are differences in early microvascular functional abnormalities between type I and type II diabetes.

Adult↗
PubMed2004

A rapid filtration apparatus for harvesting cells under controlled conditions for use in genome-wide temporal profiling studies.

Gene expression can respond rapidly to changes in environmental conditions. To effectively monitor these responses, we built a filtration apparatus that allows for the rapid harvesting and processing of moderate volumes of yeast cells under controlled atmospheric conditions (e.g., anaerobic conditions). Harvesting by filtration offers several advantages over that by centrifugation, especially when rapid, repeated sampling of dilute cultures is required. A number of different filter membranes, including cellulose acetate, mixed esters of cellulose, regenerated cellulose, polycarbonate, and polyvinylidene fluoride, were assayed for harvest efficiency and the quality of RNA obtained by hot-phenol extraction from cells directly adhering to the membranes. To determine the suitability of the RNA for microarray analyses, we quantified both cDNA yield from reverse transcription and the indirect coupling of Cyan dyes. In general, filtration times, cell yields, and RNA quality were similar among the filters examined, although some media components (e.g., antifoam) can cause fouling of smaller-pore-sized filters. Thus, choice of a membrane will depend on the particular medium, ease of filter handling, or on other experimental considerations. We routinely use this filtration apparatus with Osmonics 1.2 microm cellulose acetate filters for isolating RNA for genome-wide temporal profiling analyses.

Cell Culture Techniques↗
PubMed2004

Efficacy of glaucoma filtration surgery in pseudophakic patients with or without conjunctival scarring.

PURPOSE: To analyze the intraocular pressure (IOP), glaucoma medication requirements, and visual acuity after glaucoma filtration surgery in pseudophakic eyes. SETTING: Private practice, Boston, Massachusetts, USA. METHODS: The results of glaucoma filtration surgery in 47 eyes of 40 pseudophakic patients with a minimum follow-up of 1 year were retrospectively reviewed. Eyes with previously surgically manipulated conjunctiva in the area of filtration were compared to eyes without previous manipulation. RESULTS: Preoperatively, the mean IOP was 25 mm Hg +/- 7.3 (SD); mean number of glaucoma medications, 3.3 +/- 1.0; and mean logMAR visual acuity, 0.41 +/- 0.38. At the final follow-up visit (mean 36.5 +/- 31.5 months), the respective means were 13.6 +/- 6.6 mm Hg (P<.001), 0.9 +/- 1.4 (P<.001), and 0.46 +/- 0.43 (P=.53). The mean postoperative IOP, glaucoma medication requirements, and visual acuity at the final follow-up in eyes with virgin conjunctiva and eyes with previously manipulated conjunctiva were, respectively, as follows: IOP, 12.5 +/- 3.2 mm Hg and 14.1 +/- 7.6 mm Hg (P=.52); medications, 1.1 +/- 1.3 and 0.9 +/- 1.4 (P=.66); and logMAR acuity, 0.6 +/- 0.52 and 0.4 +/- 0.39 (P=.23). CONCLUSIONS: Glaucoma filtration surgery in pseudophakic eyes significantly improved IOP and reduced glaucoma medication requirements while maintaining stability of vision. There were no statistically significant differences in final IOP, glaucoma medication requirements, or visual acuity between eyes with virgin conjunctiva and eyes with previously surgically manipulated conjunctiva.

Aged↗
PubMed2004

Characterization of dopamine-depleting activity of Nocardia asteroides strain GUH-2 culture filtrate on PC12 cells.

Experimental infection of BALB/c mice with the Gram-positive bacterium Nocardia asteroides (strain GUH-2) results in life-long movement abnormalities including head shaking and spinning when held by the tail. The head shaking is temporarily inhibited by treatment with dopamine's precursor levodopa, suggesting that abnormalities in dopaminergic neurotransmission may be involved in these movement abnormalities. Cell-free filtrates from N. asteroides cultures induce > 70% dopamine depletion in rat pheochromocytoma PC12 cells, suggesting that Nocardia's effects on dopamine neurons may result in part from secreted factors. The nature of this dopamine-depleting activity was examined in the present study. Dopamine-depleting activity in N. asteroides culture filtrate was resistant to heat (100 degrees C x 30 min), proteases, and chloroform extraction, and was present in a low molecular mass (< 3 kDa) fraction. It was partially inhibited by decreasing (to 4.0) or increasing (to 10.0) the filtrate pH. GUH-2 filtrate increased cellular lactate dehydrogenase release by only 2%, and induced apoptotic morphology in only 11% of PC12 cells, suggesting that dopamine-depleting activity was not due to either cell injury or induction of apoptosis. These results suggest that a protease-resistant, low molecular mass substance secreted by N. asteroides may be responsible for its dopamine-depleting effects.

Animals↗
PubMed1999

Effects of passive tilting on capillary filtration in the lower leg in idiopathic dilated cardiomyopathy and after heart transplantation for the same condition.

Abnormal reflex control of the peripheral microvasculature during orthostasis in congestive heart failure (CHF) and after heart transplantation (HT) may cause failure of microvascular homeostasis and peripheral edema. We explored the effect of passive head-up tilt on lower leg capillary filtration measured by strain-gauge plethysmography in 24 patients with CHF, in 20 patients after HT (12 patients with preserved native right atrium, 8 patients without native right atrium), and in 18 controls. We hypothesized that an impaired peripheral microvascular reflex during orthostasis in CHF and HT might allow increased arterial hydrostatic pressure to increase pressure at the capillary level. To identify an impact of changes in arterial hydrostatic pressure, capillary fluid filtration was expressed per mm Hg arterial hydrostatic pressure (capillary filtration coefficient(arterial pressure) [CFC(AP)]) and was measured (1) during elevated venous pressure alone (50 mm Hg venous stasis in supine position), and (2) during elevated hydrostatic pressure at both the venous and arterial side of the vascular tree (head-up tilt with a vertical distance from the right atrium to the strain-gauge of 68 cm of water [50 mmHg]). Elevated venous pressure alone resulted in the highest CFC(AP) in controls (0.79+/-0.28 ml/min x 100 ml mm Hg x 10(-3)+/-SD) versus those with CHF (0.44+/-0.23, p <0.0001) and those after HT (0.54+/-0.22, p <0.01). However, during head-up tilt, CFC(AP) was similar in all 3 groups, because CFC(AP) decreased in controls (to 0.49+/-0.22, p <0.0001), in contrast to unchanged CFC(AP) in those with CHF (0.43+/-0.24) and in those with HT (0.50+/-0.21). HT patients with complete removal of the native right atrium had higher CFC(AP) (0.62+/-0.17) during head-up tilt than patients with preserved native right atrium (0.36+/-0.16, p <0.005). In conclusion, patients with CHF and those after HT have increased capillary filtration to a lesser degree than controls during elevated venous pressure alone. However, during orthostasis this apparent edema-protective mechanism vanishes, probably because of compromised microvascular reflex control. During daily upright activities, this may be one important factor in the edema pathogenesis. The phenomenon is particularly distinct in HT patients without preserved native right atrium.

Adult↗