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PubMed2003

Rapid migration in gel filtration of the Cf-4 and Cf-9 resistance proteins is an intrinsic property of Cf proteins and not because of their association with high-molecular-weight proteins.

Gel filtration is frequently used to study the behaviour and composition of protein complexes. In previous studies, gel filtration analysis of solubilised membranes containing the tomato Cf-4 and Cf-9 resistance proteins indicated that these Cf proteins are present in an approximately 400- and 420-kDa protein complex, respectively, which contains only one Cf molecule per complex, does not contain Rho-related proteins, and does not alter in size upon elicitation. Here, we show that inactive Cf-4 and Cf-9 mutant proteins have a similar large apparent size upon gel filtration analysis. The size remains unaltered after pre-treating the samples under harsh conditions, such as boiling with SDS and incubation in 6 m urea. A similar large apparent size was found for Cf-4 and Cf-9 isolated from SDS gel and for Cf-9 expressed by insect cells. Therefore, the large apparent size observed in our studies appears to be an intrinsic property of the Cf proteins, rather than being caused by association with high-molecular-weight protein(s). Taken together, these results suggest that caution should be taken when interpreting data obtained from gel filtration of LRR-containing proteins.

Animals↗
PubMedDate not supplied

Microaggregate blood filtration and the febrile transfusion reaction. A comparative study.

Seventy-four patients with chronic transfusion requirements and histories of repetitive febrile reactions were transfused with 1138 units of microaggregate-filtered red cells. The filtered blood was prepared using either a direct interception or a depth filter. One-half of the units were centrifuged immediately prior to filtration. Microaggregate filtration reduced the overall incidence of febrile transfusion reactions by 77 percent. The centrifugation-filtration protocol reduced the rate of reactions by 98 percent. There were no differences between the ability of the different filters to reduce the reaction rate; however, red cell loss was twice as large with the depth filter as with the direct interception filter. The numerical criterion for "leukocyte-poor blood" was met in all units processed by centrifugation-filtration. Only units processed during the last 2 weeks of shelf-life fulfilled this criterion when centrifugation was omitted from the procedure. The majority of the latter units were clinically tolerated well due to their reduced granulocyte content.

Cell Aggregation↗
PubMed1998

Type II diabetics with macrovascular complications: polymorphonuclear leukocyte (PMN) filtration, PMN membrane fluidity and cytosolic Ca2+ content after activation.

We evaluated polymorphonuclear (PMN) filtration parameters, membrane fluidity and cytosolic Ca2+ content in 21 normal subjects and in 18 type II diabetics with macrovascular complications (MVC). Evaluations were carried out at baseline and after in vitro activation prolonged for 5 and 15 min. PMA (4-phorbol 12-myristate 13-acetate) and fMLP (N-formyl-methionyl-leucyl-phenylalanine) were used as stimulating agents. TMA-DPH (1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene) was used as fluorescent probe for the membrane fluidity tests and Fura 2-AM for the cytosolic Ca2+ content. A significant variation was evident in PMN filtration parameters at 5 and 15 min. No variation was present in PMN membrane fluidity and cytosolic Ca2+ content in normals. In type II diabetics with MVC, we found an increase solely in PMN cytosolic Ca2+ content after PMA activation and an early decrease in PMN membrane fluidity and a late increase in PMN cytosolic Ca2+ content after fMLP activation. After PMA activation alone (at 15 min), PMN filtration distinguishes normals from type II diabetics with MVC. The PMN filtration parameters behave similarly in the two groups, but PMN membrane fluidity and cytosolic Ca2+ content behave differently.

Aged↗
PubMed1970

Unified theory for gel electrophoresis and gel filtration.

Unified theory for gel electrophoresis and gel filtration: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers. This model has been generalized to apply to nonspherical molecules and to several gel types. The model provides equations for inter-relationships between mobility, partition coefficient, gel concentration, and molecular radius; it gives a non-Gaussian distribution of pore sizes as a function of gel concentration. The theory defines conditions for optimal separation and optimal resolution in gel filtration and gel electrophoresis. The difference in resolving power between the two fractionation methods is accounted for by the fact that gel filtration is a form of partition chromatography.

Chromatography, Gel↗
PubMed1997

Induction of rabbit cyclooxygenase 2 in the anterior uvea following glaucoma filtration surgery.

PURPOSE: This study was undertaken to evaluate for the presence of cyclooxygenase 2 (COX2) gene expression in the anterior uvea of rabbits following glaucoma filtration surgery. METHODS: One of the following surgical procedures were performed on the right eye of New Zealand white albino rabbits: (1) paracentesis (2.5 mm limbal incision); (2) iridectomy through a 2.5 mm limbal incision; (3) lamellar scleral flap formation or (4) full glaucoma filtration surgery. The animals were sacrificed within 3 hours of post-surgery, and the anterior uveal tissues were isolated. Polymerase chain reaction-based techniques were employed to assay for the presence of COX2 transcript. RESULTS: A partial coding sequence of the previously unreported rabbit COX2 gene was obtained. COX2 mRNA was detected in the operated eyes of animals that underwent either full filtration surgery or iridectomy through a limbal incision. CONCLUSIONS: In normal rabbit anterior uveal tissue, there appears to be minimal expression of COX2 message. After experimental glaucoma filtration surgery, there is rapid induction of COX2 message.

Amino Acid Sequence↗
PubMed1988

Limitations in the determination of total filtration of x-ray tube assemblies.

A brief introduction is given to various inferential methods for the determination of total filtration of x-ray tube assemblies. The HVL method, which is most commonly used, was investigated in detail. The influence of a number of parameters on the relationship between filtration and HVL was systematically studied. The results clearly show that the HVL method only provides a rough estimate for filtration with errors of typically +/- 30%. As a consequence, quality assurance checks on the acceptance and status test level must consider the limited accuracy of this method. Changing requirements from minimum filtration to minimum HVL permitted at particular kVp does not considerably improve the situation.

Humans↗
PubMed2001

The use of autologous fibrinogen concentrate in treating ocular hypotony after glaucoma filtration surgery.

Antimetabolite therapy with 5-fluouracil (5-FU) or mitomycin-C (MMC) has significantly improved the success rate of glaucoma filtration surgery. However, in some eyes, when filtration is excessive, persistent hypotony may develop. In this study, we describe the experience of using autologous fibrinogen concentrate (AFC) to treat patients with persistent hypotony after glaucoma filtration surgery. Among seven MMC-augmented trabeculectomy patients who developed persistent postoperative hypotony, the effects of AFC intrableb injections were evaluated. Under a microscope, 0.2 ml AFC and bovine thrombin were injected into the blebs of the patients from both sides of the filtering blebs. Postoperative best-corrected visual acuity, anterior chamber status, intraocular pressure (IOP) and fundus examination were followed and compared with those preoperative. On the second day, the mean IOP of seven eyes elevated from preoperative 3.4 +/- 2.1 mmHg to 12.6 +/- 4.2 mmHg, and the anterior chamber became deep without obvious inflammatory response. Within two weeks, macular edema and visual acuity were noted to improve in six eyes (85.7%). In addition, after a mean follow-up of 25 months, the trabeculectomy procedure remained successful in all eyes. AFC appears to be safe and effective in the treatment of ocular hypotony after glaucoma filtration surgery.

Adult↗
PubMed1994

The peripheral microcirculation in atrial fibrillation: preservation of capillary pressure and filtration coefficient.

OBJECTIVE: The aim was to assess whether atrial fibrillation results in disturbances of capillary pressure and capillary filtration coefficient in man. METHODS: Finger nailfold capillary pressure and calf capillary filtration coefficient were measured in subjects in atrial fibrillation and in matched healthy controls in sinus rhythm. Capillary pressure was measured by direct cannulation using an electronic resistance feedback servonulling technique, and capillary filtration coefficient by mercury-in-Silastic strain gauge plethysmography using a technique believed not to invoke the venoarteriolar response. RESULTS: Mean capillary pressure did not differ significantly between subjects in atrial fibrillation and those in sinus rhythm [18.4(SD 5.1) mm Hg in atrial fibrillation v 18.0(2.9) mm Hg in sinus rhythm]. In a subgroup of patients restored to sinus rhythm (n = 7) by dc cardioversion there was no significant alteration in capillary pressure [15.3(4.2) mm Hg v 16.6(2.8) mm Hg]. Capillary filtration coefficient was also similar in subjects in atrial fibrillation to that in healthy controls in sinus rhythm [2.81(0.65) kfu in atrial fibrillation v 2.87(0.69) kfu in sinus rhythm]. CONCLUSIONS: These data would suggest that under resting conditions autoregulatory mechanisms are able to preserve microvascular homeostasis despite the central changes associated with atrial fibrillation.

Atrial Fibrillation↗
PubMed1977

Isolation of ribosomal protein-RNA complexes by nitrocellulose membrane filtration: equilibrium binding studies.

E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions, making it applicable to weak as well as strong protein-RNA associations. We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 X 10(7)M-1.

Binding Sites↗
PubMed1998

Effects of partial liquid ventilation with perfluorocarbons on pressure-flow relationships, vascular compliance, and filtration coefficients of isolated blood-perfused rabbit lungs.

OBJECTIVES: The density of perfluorocarbons is almost twice that of blood. Therefore, we hypothesized that partial liquid ventilation with these fluids markedly affects pulmonary hemodynamics and filtration coefficients. To test these hypotheses we studied pressure-flow relationships, vascular compliances, capillary pressures, and filtration coefficients in normal and perfluorocarbon-ventilated rabbit lungs. DESIGN: Controlled animal study with an ex-vivo isolated lung preparation. SETTING: Research laboratory for experimental anesthesiology at the Heinrich-Heine-University of Düsseldorf. SUBJECTS: Fourteen New Zealand White rabbits. INTERVENTIONS: The lungs were perfused under zone 3 flow conditions with autologous blood at various flow rates (50 to 250 mL/min, closed circuit, roller pump, 37 degrees C) and ventilated with 5% CO2 in air (positive end-expiratory pressure: 2 cm H2O, tidal volume: 10 mL/kg, respiratory rate: 30 breaths/min) without (control group, n=7) and with (n=7) perfluorocarbon administered intratracheally (15 mL/kg). MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial, left atrial, and airway pressures, as well as blood reservoir volume (reflecting changes in pulmonary blood volume) and lung weight, were measured continuously. Inconsistent with our hypothesis, we found no significant differences between both groups in the slopes and intercepts of the pressure-flow relationships. There were no significant differences in capillary pressures determined by double occlusion (6.7+/-1.2 vs. 6.3+/-1.3 cm H2O for control group, p=.53), vascular compliances (0.51+/-0.10 vs. 0.47+/-0.09 mL/cm H2O for control group, p=.38), and filtration coefficients (0.33+/-0.06 vs. 0.37+/-0.07 mL/min/mm Hg/100 g wet weight for control group, p=.80, Mann-Whitney). CONCLUSIONS: Partial liquid ventilation with perfluorocarbons has no relevant effects on pulmonary filtration coefficients and global hemodynamic variables of isolated zone 3 lungs. These findings suggest that right ventricular afterload is not changed with partial liquid ventilation. It is likely, however, that intrapulmonary blood flow is redistributed toward less-dependent regions, although relevant global hemodynamic changes are absent during partial liquid ventilation.

Animals↗
PubMed1996

Prestorage versus bedside white blood cell filtration of red blood cell concentrates: effects on the content of cytokines and soluble tumor necrosis factor receptors.

BACKGROUND: The cytokine network has important implications for the systemic inflammatory and metabolic response in trauma and infection. The objective of this study was to investigate the influence of white blood cell (WBC) filtration on the cytokine content in red blood cell concentrates (RBCs). STUDY DESIGN AND METHODS: Tumor necrosis factor-alpha (TNF), interleukin-1-beta (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and soluble TNF receptors I and II (sTNF-RI, sTNF-RII) were investigated in filtered and nonfiltered RBCs during storage. After 40 days of storage the originally nonfiltered units were filtered. RESULTS: On day 1 prestorage filtered RBCs had lower concentrations of WBCs (p < 0.001), TNF (p < 0.01), sTNF-RI (p < 0.01) and sTNF-RII (p < 0.05) compared to nonfiltered units. IL-1 concentrations increased from day 1 to day 40 (p < 0.05) in nonfiltered RBCs and were higher in nonfiltered units compared to prestorage filtered ones on day 40 (p < 0.05). An increase of IL-8 was found in nonfiltered RBCs as well as prestorage filtered units from day 1 to day 40 (p < 0.05) but the concentrations of IL-8 were higher in nonfiltered units on day 40 compared to prestorage filtered units (p < 0.05). Filtration at the end of the 40-day storage period had no influence on the concentrations of cytokines and soluble TNF receptors. CONCLUSION: The present results suggest that prestorage WBC filtration may be more efficient in reducing the cytokine content of RBCs compared to filtration at the the end of the storage period. The clinical impact of passive transfer of components of the cytokine network via RBCs, e.g., in critically ill patients, is however unclear and needs further investigations.

Adult↗
PubMed1999

The risk profile of glaucoma filtration surgery.

Glaucoma surgery has evolved over the past 30 years from the full-thickness procedure to the guarded filtration procedure. However, many of the risks and complications attendant with the full-thickness procedure, including endophthalmitis, hypotony, cataract progression, and filtration failure, continue to plague the glaucoma surgeon performing the guarded filtration procedure, although at lower incidences. With proper modification of technique, such as with postoperative bleb titration and use of adjunctive antifibrotic therapy based on prognosticators for failure, the success rates of trabeculectomy reoperations can approach those of primary trabeculectomy. Such risk factors for failure include African-American race, higher preoperative intraocular pressures, previously failed filters, younger age, and uveitic and neovascular glaucomas. In this paper, we review a number of studies that analyze the risks, complications, and long-term results of glaucoma filtration surgery and discuss different surgical recommendations based on risk factors for failure as well as for performing concomitant cataract and glaucoma surgery.

Glaucoma↗
PubMed2000

A case of pemphigus vulgaris successfully treated with single filtration plasmapheresis: a correlation of clinical disease activity with serum antibody levels.

We report a patient with pemphigus vulgaris (PV) successfully treated with single filtration plasmapheresis. A 40-year-old man with PV was started on therapy with prednisolone (PSL). Although the dosage of PSL was doubled, and both cyclosporin A (CyA) and pulse therapy were added, the disease was not controlled. After single filtration plasmapheresis began, most of the eroded lesions on the trunk reepithelialized. A switch to double filtration was followed by recurrence. Finally, additional treatments with single filtration plasmapheresis were required to obtain remission. To evaluate the efficacy of the treatment, circulating antibodies were measured by immunofluorescence (IIF) and enzyme-linked immunosorbent assays (ELISAs) using recombinant desmoglein (Dsg) 3. IIF titer and the ELISA scores correlated with the clinical disease activity. It is suggested that ELISA was more sensitive than IIF.

Adult↗
PubMed1977

Studies on the toxin of Aspergillus fumigatus. VII. Purification and some properities of hemolytic toxin (asp-hemolysin) from culture filtrates and mycelia.

A hemolytic toxin has been obtained from mycelia and culture filtrates of Aspergillus fumigatus by the procedures that included precipitation with ammonium sulfate, chromatography of DEAE-Sephadex, affinity chromatography on Concanavalin A-Sepharose and gell filtration on Sephadex G-50, G-100 AND G-150. The purified homolytic toxin was homogeneous on immunological and disk electrophoretic analysis, and the toxin from culture filtrates was identical with that from mycelia by the immunodiffusion technique. The hemolytic toxin was obtained for the first time from fungi and designated as Asp-hemolysin. The molecular weight of Asp-hemolysin was estimated to be appoximately 30,000 by the gel-filtration technique and its isoelectric point was found to be around pH 4.0. This Asp-hemolysin contained large amounts of protein and very small amounts of carbohydrate. The UV absorption spectrum of Asp-hemolysin showed a maximum absorption at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm, E 1% 1CM, was 12.4 and the ratio of absorbance at 280 nm to that at 260 nm was 2.3. The optimum pH for the hemolytic activity of the toxin toward chicken erythrocytes was 5.0 at room temperature and it was active in the pH range of 3.5 to 10.5. The optimum temperature was 21 C and about 50% of the activity was lost by incubation at 50 C for 5 min or 45 C for 23 min. The hemolytic activity was remarkably inhibited by Hg2+, Cu2+, Fe2+, Ag1+, iodine and p-CMB, but enhanced slightly by Zn2+ and Co2+.

Amino Acids↗
PubMed1992

Immunization of mice with mycobacterial culture filtrate proteins.

Culture filtrate proteins were obtained from Mycobacterium tuberculosis cultures after 7 days growth in Proskauer and Beck medium. The protein yield increased substantially to peak about the time the number of viable organisms reached its maximum level (day 8). Examination of the protein concentrate by SDS-PAGE revealed the presence of at least 12 separate protein bands varying from 10 to 90 kD. Mice were injected subcutaneously with 20 micrograms of M. tuberculosis culture filtrate (MTCF) protein suspended in saline or Freund's complete or incomplete adjuvant. The vaccinated mice were subjected to an aerogenic challenge with 10(3) colony-forming unit (CFU) M. tuberculosis Erdman and a significant reduction in the number of viable organisms was observed in the spleens and lungs determined over a 21-day period compared with age-matched normal controls. Mice immunized with the same culture filtrate proteins bound to nitrocellulose particles also showed some resistance to the virulent challenge, suggesting that individual antigens present in the culture filtrate were able to induce a protective T cell-mediated immune response in appropriately immunized mice.

Animals↗
PubMed1984

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate.

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Plasma filtration in Couette flow membrane devices.

This report investigates the effects of transmembrane pressure (ptm), rotation speed, and membrane characteristics on the performance of a rotating membrane device for separating plasma from whole blood. This device consists of 58 cm2 polymeric membrane rotating at high speed (3,000-4,000 r/min) inside a concentric cylinder and produces a Couette type flow in a gap 0.9 mm thick. Blood enters tangentially at the top, exits tangentially at bottom, and plasma is collected by an axial duct. It is found that this device yields maximum filtration velocities (plasma filtration flux per unit area) in the range of 0.5-0.8 cm/min, which are 10-20 times larger than those obtained by using hollow fiber plasma filters. This high performance is not due to the effect of centrifugal forces on red blood cells but rather to the very high shear rates, on the order of 20,000 sec-1, generated in the gap by toroidal flow instabilities (Taylor vortices). When a 0.8 micron pore size Nuclepore (polycarbonate) membrane is used, the filtration velocity presents a narrow peak of 0.6 cm/min at ptm = 10 mm Hg at 3,000 r/min and decreases to 0.35 cm/min at larger ptm. With a 0.5-micron pore nylon membrane the filtration speed increases continuously with ptm and reaches a plateau of 0.5 cm/min.

Cell Separation↗
PubMed2006

In vitro and in vivo evaluation of LEUKOSEP HRC-600-C leukoreduction filtration system for red cells.

BACKGROUND: Documentation of the benefits of leukoreduction has led to the increased use of this technique and the need for development of efficient and effective techniques for its accomplishment. This study investigated the in vitro properties and in vivo autologous radiolabeled recovery of leukoreduced red cells (RBCs) produced through a leukoreduction filtration system for RBCs (LEUKOSEP HRC-600-C, Hemerus Medical). STUDY DESIGN AND METHODS: Normal subjects donated 36 units of RBCs that were leukoreduced on Days 0, 3, or 5 through a "hands-off" technique. Biochemical studies were performed before and after filtration and at the end of 42 days of storage. Units leukoreduced on Days 0 or 5 were held until Day 42 and used for autologous radiolabeled return to determine recovery with 51Cr single-label radiolabeling techniques. RESULTS: Leukoreduction filtration was accomplished in 16.3 +/- 2 minutes on Day 0 at room temperature or 27 to 30 minutes on Days 3 or 5 after refrigeration. Leukoreduction efficiency was 4.6 +/- 0.6 log with a median residual white blood cell (WBC) content of fewer than 3.3 x 10(4) WBCs per unit. RBC recovery was 90 +/- 2 percent. Hemolysis was 0.34 +/- 0.16 percent at the end of 42 days of storage. The in vivo recovery of radiolabeled RBCs 24 hours after autologous return was 80.6 +/- 4.5 percent for RBC units leukoreduced on Days 0 and 5 combined. CONCLUSION: The LEUKOSEP HRC-600-C WBC reduction filtration system produced leukoreduced RBCs efficiently and effectively with acceptable poststorage biochemical measures and posttransfusion recovery after 42 days of storage.

Adult↗