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Some properties of electrolyte solutions in nanoconfinement revealed by the measurement of transient filtration potential after pressure switch off.

We have demonstrated that with a composite nanoporous ceramic membrane in a batch membrane cell it is technically feasible to switch off the trans-membrane hydrostatic pressure difference within tens of milliseconds. That enabled us to resolve practically the whole time evolution of transient filtration potential. Measurements of the latter have been complemented by measurements of steady-state salt rejection by the composite membrane and by measurements of the streaming potential and hydraulic permeability of membrane supports available separately. A theory has been developed in terms of network thermodynamics for the electrical response of a bilayer membrane to a pressure perturbation. In combination with the results of salt rejection measurements, from the time transients of filtration potential we could determine the ion transport numbers within the nanoporous layer. Besides that, from the dependence of steady-state salt rejection on the trans-membrane volume flow, we have determined the diffusion permeability of and the salt reflection coefficient in the nanoporous layer. This has enabled us to estimate the contributions of Donnan and non-Donnan mechanisms to the rejection of ions by the nanoporous membrane used in this study. It has been unexpectedly found that the Donnan exclusion played only a secondary role. Our hypothesis is that the non-Donnan exclusion of ions from the nanopores might be caused by changes in water properties in nanoconfinement. Proceeding from the results of steady-state filtration experiments with the membrane and the support, we also concluded that the nanoporous layer was imperfection-free and had a quite narrow pore size distribution, which made it a suitable object for fundamental studies of ion transfer mechanisms in nanopores.

Ceramics↗

Intra-GBM site of the functional filtration barrier for endogenous proteins in rats.

The passage of various endogenous proteins [such as albumin, transferrin, immunoglobulin G (IgG), and immunoglobulin M (IgM)] across GBM was studied in vivo in normal Munich-Wistar rats. Glomeruli were fixed by three different methods: in situ drip-fixation, perfusion- and immersion-fixation; then they were processed for immunogold electron microscopy. The most reproducible results were obtained with in situ drip-fixation. Albumin, transferrin and IgG penetrated into GBM, but IgM did not. Morphometry revealed that density of albumin increased towards the inner 1/5 to 1/3 of GBM (junction of lamina rara interna and lamina densa) and decreased towards the subepithelial region of GBM, whereas density of IgG and transferrin was the highest at the subendothelial site and declined towards the subepithelial side of GBM. These findings suggest that central and/or outer zone of GBM constitute the main filtration barrier for albumin, and that subendothelial zone may contribute also to the charge-selective barrier. It is also suggested that the subendothelial zone acts more effectively as a filtration barrier for IgG and transferrin than for albumin. In the outer zone of GBM, which roughly corresponds to lamina rara externa visualized by conventional electron microscopy, the relative density of IgG and transferrin was higher than that of albumin. Since the pI of albumin was lower than that of IgG and transferrin, this finding suggests that subepithelial zone of GBM also acts as a charge-selective barrier. In conclusion, the main GBM filtration barrier for albumin might be the central and outer zones of GBM, and that for transferrin and IgG might be the entire width of GBM.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

Normal-flow virus filtration: detection and assessment of the endpoint in bio-processing.

The breakthrough of a model virus, bacteriophage PhiX-174, through normal-flow virus filters was studied using both commercial process fluids and model feed streams. The results indicate that (i) PhiX-174 is a reasonable model for a mammalian parvovirus [MMV (murine minute virus)] in virus filtration studies; (ii) PhiX-174 LRV [log(reduction value)] shows a better correlation with percentage flow decline compared with volume processed under a variety of conditions; (iii) although the extent of decline in virus LRV is dependent on the mechanism of filter fouling, the fouling mechanisms operative in a viral validation study are representative of those likely to be found under actual production conditions. The mechanism of LRV decline by many process streams was proposed to be due to selective plugging of small pores. A theoretical model as well as a predictive equation for LRV decline versus flow decay was derived; experimental results from filtration studies using pore-plugging feed stocks were consistent with the equation. As protein solutions may vary in their adsorptive versus plugging behaviour during filtration, an evaluation of the LRV-versus-flow-decay relationship on a biopharmaceutical-product-specific basis may be warranted.

Animals↗

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.

Acetylgalactosamine↗

A theoretical analysis of the use of zonal gel filtration in the detection and purification of protein-ligand complexes.

A simple model of zonal gel filtration is analysed numerically to establish semiquantitatively how the capacity of a protein-ligand complex to survive gel filtration intact depends on several experimental variables and on the kinetic characteristics of the complex. The design of gel-filtration experiments aimed at detecting and partially purifying binding proteins is discussed.

Chromatography, Gel↗

The capillary filtration coefficient for evaluation of capillary fluid permeability in cat calf muscles.

Measuring the capillary filtration coefficient (CFC) from the transvascular fluid filtration following a fixed increase in transcapillary hydrostatic pressure is a common method to estimate capillary hydraulic permeability (conductivity) in an organ. Constant flow pump perfusion with an artificial perfusate and a maximally dilated vascular bed are often used in CFC studies to avoid influence on CFC of variations in vascular tone, blood flow and perfusion pressure. The present study evaluates if capillary hydraulic conductivity can be estimated by the CFC method, when analyzed on a denervated cat skeletal muscle with quite well-preserved local vascular control and perfused with autologous blood. CFC was estimated by increasing venous pressure and by decreasing tissue pressure, and both during autoperfusion and pressure controlled pump perfusion. A constant filtration rate was achieved 3-4 min after the transcapillary pressure elevation, giving a CFC around 0.0090 mL min-1 mmHg-1 100 g. The CFC did not change with arterial pressure or with reduced vascular tone using the tissue pressure method, but decreased slightly with increased arterial pressure and reduced vascular tone using the venous pressure method. CFC variations with arterial pressure were larger during pump perfusion in which myogenic reactivity is depressed, indicating that influence of myogenic tone on CFC is small. We conclude that CFC can be used to evaluate capillary hydraulic conductivity, and also when arterial pressure, vascular tone and blood flow are altered within reasonable physiological limits during the experiment, and the tissue pressure method and autoperfusion is to be preferred.

Animals↗

The bacterial and viral filtration performance of breathing system filters.

The bacterial and viral filtration performance of 12 breathing system filters was determined using test methods specified in the draft European standard for breathing system filters, BS EN 13328-1. All the filters were of two types, either pleated hydrophobic or electrostatic, and these two types differed in their filtration performance. The filtration performance is expressed in terms of the microbial penetration value, defined as the number of microbes passing through the filter per 10 million microbes in the challenge. The geometric mean (95% confidence limits) microbial penetration value was 1.0 (0.5, 3.5) and 2390 (617, 10 000) for the pleated hydrophobic and electrostatic filters, respectively, for the bacterial challenge, and 87 (48, 212) and 32 600 (10 900, 84 900), respectively, for the viral challenge. In general, there was little change in the microbial penetration values following 24 h simulated use. It is concluded that results from the tests specified in the draft standard will allow comparisons to be made between different manufacturers' products enabling an informed choice to be made.

Anesthesia, Inhalation↗

High resolution electroelution of polyacrylamide gels for the purification of single proteins from Mycobacterium tuberculosis culture filtrate.

Culture filtrate from Mycobacterium tuberculosis contains protective molecules which have been used successfully in experimental vaccines against tuberculosis. Despite an increasing number of mycobacterial proteins being characterised, a major effort is still needed to get an overview of the many potentially interesting molecules in culture filtrate. In this study we describe a high throughput method for purification and biological evaluation of protein components in complex protein mixtures. The method presents a new application of the recently developed Mini Whole Gel Eluter and employs this apparatus for the high resolution electroelution of selected molecular mass fractions of protein mixtures previously separated in large polyacrylamide gels. Two novel M. tuberculosis culture filtrate proteins (CspA and TB18.6) were purified by this method, their N-terminal sequences were determined and the open reading frame encoding each of the proteins identified. The immunological recognition of the molecules were evaluated in tuberculosis infected mice and guinea pigs. Both proteins induced DTH responses in guinea pigs and IFN-gamma release from spleen lymphocytes isolated from infected mice.

Amino Acid Sequence↗

Protein synthesis inhibitory activity in culture filtrates from new strains of Streptomyces isolated from Brazilian tropical soils.

AIMS: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. METHODS AND RESULTS: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. CONCLUSIONS: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

3T3 Cells↗

Effects of operating conditions on selectivity of a plasma fractionator in double filtration plasmapheresis.

In a typical double filtration plasmapheresis treatment, plasma fractionation between albumin and some immunoglobulins associated with toxins is limited because none of the currently available plasma fractionators has a strict cutoff property for these proteins. Selectivity of immunoglobulins over albumin depends not only on the cutoff properties of the membrane but on the operating conditions such as the flow rate of the supplied plasma (Q(P)) and retained plasma to be discarded (Q(D)) in the plasma fractionator. We carried out an in vitro study using human plasma harvested by single plasma exchange treatments to assess the selectivity of a plasma fractionator, Evaflux 2A-F (Kawasumi Laboratories, Inc., Tokyo, Japan), under various operating conditions. The results of rate-constant filtration experiments showed that the concentrations in the feed tank and the sieving coefficient (SC) values of every protein were decreased slightly within 2 h after the start of the experiment because of membrane trapping, adsorption, and/or plugging. The time-averaged SC value of albumin increased with flow rate ratio (Q(P)/Q(D)) due to increasing filtration fraction (FF), but relative removal efficiency (mD/mP*) for albumin decreased with Q(P)/ Q(D) due to decreasing Q(D). For immunoglobulins, on the other hand, the SC values were almost unchanged, and the mD/mP* values increased with Q(P)/Q(D) due to an increase in FF. Both increasing Q(P) and decreasing Q(D) are effective means of improving selectivity between these proteins in the plasma fractionator. Membrane fouling is, however, obvious beyond a Q(P)/Q(D) value that is thought to be a critical point. Operation should be conducted below the critical Q(P)/Q(D) value, which depends on the patient's plasma components and the cutoff property of the membrane.

Humans↗

In vitro function of granulocytes isolated from blood of normal volunteers using continuous-flow centrifugation in the IBM-aminco celltrifuge and adhesion-filtration leukapheresis using nylon fiber.

Granulocytes were harvested from each of five healthy male volunteers once by continuous flow centrifugation with the IBM-Aminco Celltrifuge, and once by adhesion filtration leukapheresis with nylon fiber. Granulocyte recovery and purity were significantly better with the filtration leukapheresis system than with continuous flow centrifugation. Measurements of trypan blue dye exclusion and muramidase activity were similar to those in control granulocytes regardless of the method of isolation. Granulocyte-stimulated oxygen consumption was diminished in granulocytes prepared by the adhesion filtration method, but normal in those prepared by continuous flow centrifugation with the IBM-Aminco Celltrifuge.

Blood Transfusion↗

Reduction of human immunodeficiency virus-infected cells from donor blood by leukocyte filtration.

Several filters for leukocyte removal were evaluated in terms of their ability to reduce the cell-associated human immunodeficiency virus (HIV) load in units of blood either inoculated in vitro with lymphocytes from a chronically infected cell line or collected directly from seropositive donors. Filtration of the experimentally inoculated units of blood resulted in a 5.9 log 10 mean reduction (95% confidence interval:7.4-4.5) of tissue culture infectious units (TCIU) as assayed by end-point titration using the coculture assay. Filtration of the units of blood from anti-HIV positive donors lowered the infectivity by over 2 logs, as detected by the coculture and polymerase chain reaction (PCR) techniques. However, residual cell-associated virus was detected in the majority of experiments. Clinical studies are warranted to determine if leukocyte filtration of blood will reduce the risk of transfusion transmitted viral infections.

Acquired Immunodeficiency Syndrome↗

[Analysis of filtration according to implant type after deep sclerectomy in the rabbit].

PURPOSE: The purpose of this study was to compare the aqueous humor outflow using three type of implants after deep sclerectomy. A numeric imaging system with ICG and fluorescein angiography was used. The evolution over time was recorded, while filtration and outflow facility were measured. MATERIAL AND METHODS: Numeric imaging system with ICG and fluorescein angiography were performed before and after deep sclerectomy at regular intervals after surgery. Both resorbable (collagen) and non-resorbable (hema) intrascleral devices have been tested in eight rabbits. The resorption time of the collagen implants has been monitored using UBM technique. The outflow facility was measured with the technique of anterior chamber canulation and constant pressure method. RESULTS: The resorbable collagen implants could be observed with UBM and angiography during the first six weeks post-operatively. The image of the non-resorbable Hema implant remained clearly visible over time. New drainage vessels were observed around the filtering bleb. The filtration significantly increased as the outflow facility improved after surgery, while the intraocular pressure remained stable over time. CONCLUSION: This conjunction of UBM, angiography and outflow measurement allowed us to study and compare three types of implants used as space maintainer after deep sclerectomy, and analyse their effect on the aqueous filtration and outflow facility.

Animals↗

[Effects of horse-chestnut seed extract on transcapillary filtration in chronic venous insufficiency].

The effect of horse-chestnut seed extract (standardized on aescin; Venostasin retard) was assessed in a randomized placebo-controlled crossover double-blind trial of 22 patients with proven chronic venous insufficiency by measuring the capillary filtration coefficient and the intravascular volume of the lower leg by venous-occlusion plethysmography. Three hours after taking two capsules of Venostasin (600 mg; each capsule containing 50 mg aescin) the capillary filtration coefficient had decreased by 22%, whereas after administration of an identical-looking placebo capsule it rose but slightly over three hours. The difference in the effect of Venostasin and placebo is statistically significant (P = 0.006). The intravascular volume was reduced 5% more after Venostasin than the placebo, but this is not statistically significant. It is concluded that Venostasin has an inhibitory effect on oedema formation via a decrease in transcapillary filtration and thus improves oedema-related symptoms in venous diseases of the legs.

Capillary Permeability↗

Induction of transmissible lymphomas in Syrian hamsters by application of DNA from viral hamster papovavirus-induced tumors and by cell-free filtrates from human tumors.

DNA isolated from skin epitheliomas containing papovavirus induced lymphomas within four to eight weeks in 40 to 50 per cent of newborn Syrian hamsters injected. This DNA effect was eliminated by DNase but not by RNase and was not induced by DNA preparations of transplanted epitheliomas or the induced lymphomas. Lymphomas were similarly induced by cellfree filtrates from certain human tumors such as gastric carcinomas and ovarian tumors. Little or no lymphoma effects were observed following injections with filtrates derived from normal human or animal tissues or human blood. The lymphomas induced by DNA and human tumors were transmissible by cell-free filtrates to newborn Syrian hamsters; however, successful serial passage, like the primary lymphomas induced by the DNA preparations, depended upon the use of a newborn hamster from a special breeding colony of hamsters.

Animals↗

Investigation into the pathogenesis of atrophic rhinitis in pigs. II. AR induction and protection after intramuscular injections cell-free filtrates and emulsions containing AR toxin of Pasteurella multocida.

The intramuscular administration of the cell-free culture filtrates of two AR pathogenic Pasteurella multocida (PM) isolates caused Atrophic Rhinitis (AR) in piglets. The culture filtrate of a non-AR-pathogenic PM isolate did not cause AR lesions after intramuscular injection. The severity of the AR lesions appeared to be correlated with the quantity of injected toxin. As the quantity of administered toxin increased, the piglets showed clinical growth retardation, icterus and mortality. Besides lesions typical of AR, severe liver degeneration was observed at necropsy; this liver degeneration was sometimes accompanied by fibrosis. After emulgation of the cell-free culture filtrates in incomplete Freunds adjuvant, AR was caused by administration of the toxin-containing emulsions in piglets of 3 and 6 weeks of age. An experimental water-in-oil emulsion containing the AR toxin of Pasteurella multocida caused AR when intramuscularly injected in 1- to 4-week-old piglets. A PM-AR toxin challenge method is described. After injection of the toxin into piglets born from sows vaccinated against AR, protection was demonstrated. Ten of the 12 piglets (83%) challenged at the age of 3 weeks showed protection. Six animals of the 12 pigs (50%) injected with the toxin at the age of 6 weeks, showed no protection. Differences in protection were noted between and within the litters. After injection with the toxin, all piglets of 3 and 6 weeks of age born from non-vaccinated sows showed deviations typical of AR.

Animals↗

Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes.

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.

Animals↗

The relationship of toxic fractions of a filtrate of Clostridium perfringens type A to the pathogenesis of clostridial myonecrosis.

A crude filtrate from a culture of Clostridium perfringens, type A, was fractionated by a heavy metal-alcohol technique, and some degree of concentration of biologically active factors was achieved. Both the crude material and the fractions obtained were characterized in terms of their alpha toxin, theta toxin, hyaluronidase, and collagenase activities. The filtrate and fractions were tested for their effect on the peripheral circulation of the rat, using the epinephrine threshold technique. The crude material and several fractions caused a sharp increase in epinephrine sensitivity of the capillary bed of the meso-appendix of the rat; fraction R3B1 giving an 86-fold increase in sensitivity. This reaction could be inhibited by specific antitoxic serums but not by normal serum. The "circulation factor" was shown to be heat-labile and appears to be independent of either the alpha or theta toxins. Bilateral nephrectomy greatly reduced, but did not abolish, the effect of the toxin, while the threshold response to epinephrine was not materially changed following bilateral adrenalectomy. The crude filtrate and several fractions were shown to inhibit the phagocytosis of heat-killed B. anthracis to the extent of 40 to 50 per cent. Fraction R3C2 was devoid of all biological properties studied here, except phagocytosis inhibition, suggesting that the factor responsible for this activity is distinct from the "classical" toxins and the "circulation factor." Moreover, a 1:5000 dilution of an antiserum prepared against this fraction would completely neutralize the phagocytosis inhibition factor but failed to inhibit any of the other toxic activities. Since cardiovascular collapse and absence of phagocytosis are two significant clinical findings in gas gangrene, the possible roles of the "circulation" and "phagocytosis inhibition" factors in the pathogenesis of this disease are discussed.

Animals↗