Membrane Filter Assay Research Articles

Changes in the polymorphonuclear leukocyte function of blood samples induced by storage time, temperature and agitation

We have investigated changes in polymorphonuclear leukocyte (PMN) functions of blood samples caused by such typical elements of laboratory handling as storage time, temperature and agitation. The blood of five healthy subjects was stored upright in test tubes at 4, 22 and 37°C over periods of 20 min, one, two, six and 24 h. Controlled agitation was performed on a shaker. The following PMN functional parameters were measured: the white blood cell count (WBC), migration, elastase (EL) release, reactive oxygen species (ROS) production and lipid peroxidation. Migration was determined in a whole-blood membrane filter assay; ROS production by latex-stimulated, luminol-enhanced chemiluminescence (CL) in a whole-blood assay; EL as EL α1-antitrypsin complex; and lipid peroxidation by malondialdehyde (MDA) generation. The reactions after handling were compared with the values measured immediately after blood withdrawal which served as reference values of `genuine' PMN reactivity. The outstanding result was the marked scatter between the individual reactions. Overall, the proportion of migrating PMNs in the blood total decreased, while CL, correlating positively with MDA, increased with the time of storage. EL increased considerably in some of the samples. Agitation raised CL and MDA. The effect of temperature was apparent only after 24 h at 37°C. There was evidence that inhomogeneities in the blood samples were another interfering factor, since resuspension of sedimented blood after storage can be incomplete. In order to obtain reliable results from PMN functional tests, whole-blood assays and processing of blood samples within 20 min after blood withdrawal are recommended.

8-Cyclopentyl-1,3-dipropylxanthine and Other Xanthines Differentially Bind to the Wild-Type and ΔF508 Mutant First Nucleotide Binding Fold (NBF-1) Domains of the Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Certain alkyl xanthines such as CPX (8-cyclopentyl-1,3-dipropylxanthine) stimulate Cl- efflux from cells bearing the delta F508 genotype common to most cases of cystic fibrosis. We have hypothesized that the CFTR molecule itself might be the site for CPX action, perhaps in the region of the first nucleotide binding fold (NBF-1) domain. Therefore, to test this hypothesis directly we have used a rapid membrane filtration assay to measure the kinetics of association and dissociation of [3H]CPX to both recombinant NBF-1 and recombinant NBF-1 bearing the delta F508 mutation. We report that [3H]CPX binds with higher affinity to the delta F508-NBF-1 of CFTR (Kd = 1.0 nM) than to the wild-type NBF-1 of CFTR (Kd = 17.0 nM). These Kd values were calculated from direct measurements of the association and dissociation rate constants. The rate constants for the dissociation reaction of the wild-type NBF-1 and delta F508-NBF-1 of CFTR were not different from each other. However, the corresponding rate constants for the association reaction were k(+1) (NBF-1) = 4.7 +/- 0.9 x 10(4) M(-1) s(-1) and k(+1) (delta F508-NBF-1) = 1.6 +/- 0.3 x 10(5) M(-1) s(-1), respectively. These Kd values were corroborated by equilibrium-binding experiments, which gave very similar values. We have also measured the relative displacement of various xanthines from both wild-type NBF-1 and delta F508-NBF-1, in anticipation that the order of potencies for binding might parallel the action of the different xanthines on CF cells. For wild-type NBF-1, the rank order was DA-CPX > DAX > CPX > caffeine > adenosine >> IBMX > 2-thioCPX. For delta F508-NBF-1, the rank order was DAX > CPX > caffeine > DA-CPX > adenosine >> IBMX > 2-thioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action on CF cells may involve direct interaction with the CFTR molecule itself.

Placental membrane transport

Brush border (microvillous) plasma membranes (BBM) and the basal surfaces (BCM) from the syncytiotrophoblast of human term placenta were prepared by a method of sonication, dialysis and differential centrifugation in specific buffer systems. Such plasma membranes formed closed, osmotically active, right-side-out vesicles in which amino acid transport could be studied unidirectionally by a carefully designed membrane filtration assay under reduced pressure. In such vesicles, L-leucine (1 mM) was found to be transported in a time-dependent manner, peak accumulation being attained at 45 s in both BBM and BCM. The accumulation of L-leucine in the vesicles was dependent on an inward NaC1 gradient, as replacing the Na+ with K+, Li+ and choline, or replacing the C1- with S0(2-4) failed to influence the amino acid movement. Leucine transport in the vesicles also appeared to be dependent on the substrate concentration, indicating saturation at a higher concentration. The transport process showed a k(t) (affinity constant) of 3.85 and 6.67 mM, while the values recorded for the J max (maximum apparent initial velocity) were 270.27 and 384.62 nmol/mg protein-1 per min in the BBM and BCM respectively. Leucine transport was inhibited by a number of amino acids, among which amino isobutyric acid (AIB) produced the maximum inhibition. The k(i) inhibition constant) for different amino acids has also been listed. Lysine showed the least k(i) value, thus showing it to be the most inhibitory compound. These findings are discussed in relation to the mechanism and regulation of transplacental amino acid transfer.

The influence of trauma on changes in neutrophil granulocyte function assessed by an analysis of granulocyte migration.

We examined the effects of trauma on polymorphonuclear leucocyte (PMN) migratory parameters and PMN elastase release, with the aim of tracing an acute inflammatory reaction from its very beginning to the phase of recovery. Fifteen patients who underwent monotrauma surgery, followed by uneventful healing, served as inflammation model. PMN activation was studied by measuring their readiness to migrate (TMI) and their penetration potency (DC) in a whole blood membrane filter device, in which a chemoattractant depot (FMLP) was integrated. Control chambers lacking FMLP provided parameters of the spontaneous migration. In healthy controls (n = 64), the numbers of invading PMNs decreased continuously from the outermost layer towards the interior of the filter device. FMLP did not influence the mobilization rate of PMNs immigrant from the blood into the filter, but those cells that did migrate penetrated deeper (P < 0.05). After trauma, the spontaneous and FMLP-stimulated DC was increased (P < 0.05). Trauma also tended to inhibit PMN migratory activity episodically; depression of the unspecific immune function (low TMI values) was found on the 3rd (P < 0.0001) and 12th (P < 0.01) postsurgical days. There was no correlation between the migratory parameters and the inflammation parameter, PMN elastase release. Preliminary results indicate that analyses of PMN migratory parameters by a whole blood membrane filter assay could provide a valuable adjunct in monitoring trauma-associated immunologic changes.

Downregulation of hepatic beta-adrenergic receptors after trauma and hemorrhagic shock

Although it is well known that sympathoadrenal activity increases under various adverse circulatory conditions, it is not known whether there are any alterations in hepatic plasma membrane beta-adrenergic receptors after trauma-hemorrhage and crystalloid resuscitation. To study this, rats underwent a 5-cm midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximal bleedout (MB) volume was returned in the form of Ringer lactate. The animals were then resuscitated with four times the volume of MB in the form of Ringer lactate over 60 min. Hepatic plasma membranes were isolated using discontinuous Percoll gradient centrifugation. The maximal binding capacity and dissociation constant (i.e., 1/affinity) of [125I]iodopindolol binding to beta-adrenergic receptors were determined using a membrane filtration assay and Scatchard analysis. The results indicate that there was a significant decrease in the maximal binding capacity at the time of MB, which persisted despite crystalloid resuscitation after hemorrhage. However, there were no significant changes in the dissociation constant at any time point during this study. The downregulation of beta-adrenergic receptor binding capacity may be responsible for metabolic abnormalities observed after hemorrhagic shock.

Migratory activity of blood polymorphnuclear leukocytes during juvenile rheumatoid arthritis, demonstrated with a new wholeblood membrane filter assay

Polymorphonuclear leukocyte (PMN) migration is measured in whole blood in a migration chamber consisting of a membrane filter (3-microns pores, 140 microns thick) with an integrated chemoattractant depot (FMLP in solid form) attached to a plastic container. Control chambers lack FMLP (blanks). One test unit requires 300 microliters blood. Numbers and distribution of the PMN immigrants into the filters are determined microscopically. Altogether 26 measurements of PMN migration in five juvenile rheumatoid arthritis (JRA) patients with varying disease activity were compared with the reactions of a healthy control group (N = 32). Correlations were calculated with conventional laboratory parameters (WBC, PLT, BSR, CRP, Hgb, serum Fe) and disease activity. In comparison with healthy controls, PMNs of JRA patients generally show a markedly increased penetration depth into the filters irrespective the presence of the chemoattractant or the disease activity. Increased migratory reactions to FMLP in comparison to blanks were found during high disease activity only. The PMN penetration depth correlates positively with the CRP, and reciprocally with the Hgb blood levels. The migration assay combines fast and simple processing with good preservation of the genuine PMN activation state.

Phagocyte Function in Familial Hypercholesterolaemia: Peripheral Blood Monocytes Exposed to Lipopolysaccharide Show Increased Tumour Necrosis Factor Production

We studied functions of polymorphonuclear leucocytes (PMN) and monocytes from peripheral blood of subjects with familial hypercholesterolaemia (FH). FH monocytes exposed to Escherichia coli lipopolysaccharide (LPS) in 10% human AB serum generated tumour necrosis factor (TNF) significantly more than did control monocytes. After lowering of serum low-density lipoprotein (LDL) levels by drug treatment, FH monocytes exposed to LPS in the absence of exogenous lipoproteins generated significantly more TNF than did control monocytes. These findings suggest that increased TNF production is not affected by hypolipidaemic treatment and may not derive from differences between uptake of exogenous LDL by FH monocytes and control cells. Chemotaxis, chemokinesis, and random migration of both FH PMN and FH monocytes were normal, as determined by agarose assay and membrane filter assay. FH PMN showed increased luminol-enhanced chemiluminescence in response to 2.5 x 10(-8) M, but not to 2.5 x 10(-6) M, N-formyl-methionyl-leucyl-phenylalanine, and not to serum-treated zymosan or to phorbol myristate acetate. Luminol- and lucigenin-enhanced chemiluminescence responses of FH monocytes were normal. In summary, the major aberration found in the present study was that FH monocytes stimulated with LPS show enhanced production of TNF. The possibility that exaggerated TNF production contributes to an early development of atherosclerotic lesions in FH subjects warrants further studies.

A paper membrane filter assay for ciliate chemoattraction

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (∼10 5 cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.

EnterotoxigenicEscherichia coliin the domestic environment of a Malaysian village

The membrane-filter assay, GM1-ELISA, and DNA-DNA hybridization assay, were used to detect enterotoxigenic Escherichia coli (ETEC) in samples of water, weaning food, food preparation surface swabs, fingerprints of mothers, and the fingerprints and stools of children under 5 years of age, in 20 households in a Malaysian village. Weaning food and environmental samples were frequently contaminated by faecal coliforms, including ETEC. The membrane-filter assay detected and enumerated faecal coliforms and LT-ETEC in all types of water and weaning food samples. Highest concentrations of faecal coliforms and LT-ETEC were found in weaning food, followed by well-water, stored water and stored drinking water. The GM1-ELISA detected LT-ETEC in weaning food, food preparation surfaces, fingerprints and stool samples. The DNA-DNA hybridization assay detected a larger proportion of STa2-ETEC than the other toxotypes, either singly or in combination. All the assays in combination detected the presence of ETEC in all types of samples on at least one occasion in each household. It was not possible to classify households as consistently more or less contaminated with ETEC. On individual occasions it was possible to show a significant association of the presence of LT-ETEC between the fingerprints of children and their stools, fingerprints of mothers and children, and weaning food and the stools of the child consuming the food.

The value of assay repetition in epidemiological studies

In a recent study, we compared a new membrane-filter assay for the detection of the heat-labile enterotoxin (LT) of Escherichia coli against established assays. Each of a set of 300 cultures was tested at least twice by the membrane-filter assay. Analyses have been performed to assess whether repeat testing for this assay is justified in epidemiological research. The methods developed have wider applicability.

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization.

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.

MEMBRANE FILTER ASSAY FOR DETECTION OF ENTEROTOXIGENIC ESCHERICHIA COLI IN EPIDEMIOLOGICAL STUDIES

A membrane filter assay is described for detection of heat-labile toxin (LT) production by Escherichia coli. Bacterial colonies were isolated on a membrane filter which was then incubated on an agar medium containing anti-cholera toxin. LT produced by bacterial colonies diffused through the membrane filter, complexed with the antiserum, and formed a zone of precipitation in the agar. Requirements for reagents and materials were simple. The membrane filter assay showed good agreement with both the ganglioside-enzyme-linked immunosorbent assay and the DNA-DNA hybridisation assay and may prove to be an important technique for the study of the epidemiology of enterotoxigenic E coli in developing countries.

LDL receptors in bovine tissues assayed as the heparin-sensitive binding of 125I-labeled LDL in homogenates: relation between liver LDL receptors and serum cholesterol in the fetus and post term

The heparin-sensitive binding of 125I-labeled LDL in homogenates of bovine tissues was determined using a membrane filter assay. The binding fulfilled several criteria which have been established for the binding of LDL to its receptor, namely: (a) saturability, (b) dependence on Ca 2+, (c) sensitivity to proteolytic destruction and (d) heat sensitivity. The adrenal cortex and the active corpus luteum exhibited the highest binding activity of the 22 different tissues assayed. Tissues from the central nervous system had low binding activity. Livers from fetal animals had higher binding than livers from young and adult animals and the binding of 125I-LDL to fetal liver homogenates showed an inverse correlation to the serum cholesterol levels, indicating that the LDL receptors in fetal liver may play a role in the regulation of the serum cholesterol level in the fetus during gestation. After birth, the binding of 125I-LDL to calf liver homogenates decreased to levels found in adult animals and this was paralleled by an increase of total serum cholesterol, suggesting that the rapid rise in serum cholesterol in mammals observed soon after birth may be caused by a decrease of the receptor-mediated catabolism of LDL in the liver.

Biochemical studies of taste sensation: IX. Enhancement of l-[ 3H]glutamate binding to bovine taste papillae by 5′-ribonucleotides

The interaction of the taste substance monosodium L-glutamate with taste receptors has been investigated. Binding of L-[3H]glutamate was measured to preparations of bovine circumvallate (taste) papillae (type I preparation) and to control tongue epithelial preparations (type II preparation) devoid of taste receptors. Binding is operationally defined using a membrane filtration assay. Substantially greater binding occurred to the type I preparation than to the type II preparation, and the binding to the type I preparation showed evidence of saturation. The apparent Kd of L-glutamate was estimated to be in the range of 20--30 mM. The unique taste effect of L-glutamate was considered to depend importantly on its demonstrated synergism in combination with certain 5'-ribonucleotides. A several-fold enhancement of binding of L-[3H]glutamate occurred in the presence of certain 5'-ribonucleotides. 5'-GMP, 5'-IMP and 5'-UMP each increased the binding of L-[3H]glutamate, while 5'-XMP, 5'-AMP and 5'-CMP did not. None of these nucleotides affected the lower level of binding to the type II preparation. Neither the free bases, adenine and guanine, their nucleosides nor their di- or triphosphonucleotides were effective in increasing L-[3H]glutamate binding to the type I preparation. The nucleotide specificity of the glutamate binding enhancement therefore shows a marked similarity with the nucleotide specificity in evoking the synergistic taste effect in humans.

On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is discussed.

Studies of RNA release reaction catalyzed by E. coli transcription termination factor rho using isolated ternary transcription complexes.

Protein factor rho catalyzes site-specific termination of transcription in a reaction requiring hydrolysis of nucleoside triphosphate with eventual release of RNA from RNA polymerase and DNA template. We have characterized the rho-catalyzed RNA release reaction using isolated transcription complexes. Transcription complexes containing T7 D111 DNA, RNA polymerase, and 3H-labeled nascent RNA were formed and isolated by gel filtration on an Agarose 5M column. When the ternary complexes were incubated with rho factor in the presence of ATP, or dATP, significant amounts of nascet RNA were released from the complexes as determined in a membrane filtration assay. Gel electrophoretic analysis of RNA has revealed that rho releases selected species of discrete-sized RNA from among those originally present in the ternary complexes. These results show that rho essentially acts to release RNA from those ternary complexes which have come to pause, and that this reaction proceeds in a discrete step separately from the pausing of RNA synthesis. Under the conditions used, the extent of RNA release widely varied at individual pausing sites and thus the action of rho exhibited certain site-selectivity.

Kinetic studies of the interaction between MS2 phage and F pilus of Escherichia coli.

The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation (kd) of the MS2-phage--F-pilus complex is very slow and follows first-order kinetics with a half-life of 4.2 h at 30 degrees C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half-life of the complex is about 1.0 min. The rate of association is very fast and follows second-order kinetics with the rate constant for association (ka) being 8 x 10(7) M-1 s-1 at 30 degrees C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non-electrostatic respectively. The mechanism of the binding reaction is discussed.

Sulfhydryl groups of pyocin R1. Morphology and activity modified with sulfhydryl reagents.

Electron microscopic observation of pyocin R1 with the negative staining technique demonstrated that pyocin R1 retained its phage tail-like shape of an extended sheath even when it was inactivated by treatment with p-chloromercuribenzoic acid (PCMB) or 4-(p-sulfophenylazo)-2-mercuriphenol (SAMP). Thus it was shown that the contraction and extension of the sheath does not occur reversibly on the modification of sulfhydryl groups accompanying the change of activity. The activity lost under these conditions was restored to the original level by treatment with 2-mercaptoethanol (2-ME). Numbers of sulfhydryl groups in the pyocin R1 particle were determined to be 208 mol and 152 mol per mol (11.8 x 10(6) daltons) by spectrophotometric titration with SAMP and by membrane-filter assay with radioactive PCMB, respectively. Most of these cysteine residues appeared to be localized in the substructure other than the sheath and core. It was also shown that all of these sulfhydryl groups were not necessary for expression of its activity but a part of them were essential for adsorption to the sensitive cells.

Radiolysis of Chromatin Extracted from Cultured Mammalian Cells: Formation of DNA–protein Cross Links

Chromatin extracted from Chinese hamster lung fibroblasts has been examined for the formation of radiation-induced DNA-protein cross links, using a membrane filter assay. The relative efficiencies of the aqueous radical intermediates, OH., eaq- and O2-, were investigated. Cross links were found in gamma-irradiated isolated chromatin and in chromatin irradiated in the cell before isolation. When isolated chromatin was irradiated under conditions in which the chromosomal proteins were dissociated from the DNA, no cross links were detectable. The most efficient radical for the production of cross links in irradiated, isolated chromatin was found to be the hydroxyl radical, whereas, the superoxide radical was essentially ineffective. For chromatin irradiated in the cell before isolation, the greatest effect was seen for cells irradiated in an atmosphere of nitrous oxide, suggesting the hydroxyl radical may be involved in the formation of cross links in intact cells also. The formation of cross links in chromatin irradiated in cells before isolation was considerable less efficient than in irradiated, isolated chromatin.

In Vitro Replication of Cowpea Mosaic Virus RNA III. Template Recognition by Cowpea Mosaic Virus RNA Replicase

Cowpea mosaic virus (CPMV) RNA replicase has been purified about 200-fold from CPMV-infected Vigna unguiculata leaves. Optimal reaction conditions for replicase activity have been established that allow RNA synthesis to proceed for at least 15 h. Using a polymerase assay under conditions optimal for CPMV RNA-directed RNA synthesis, all natural RNA species tested appeared to be able to direct the incorporation of labeled ribonucleotides, whereas synthetic homoribopolymers were either inactive or only slightly active. Using a nitrocellulose membrane filter assay to measure complex formation between the replicase preparation and various RNA species, all natural RNA species tested, except that of the comovirus radish mosaic virus, appeared to be unable to compete with (32)P-labeled CPMV RNA in binding to replicase. We propose that CPMV replicase is actually template specific but does not display this property in a polymerase assay, since labile complexes between heterologous templates and replicase become stabilized by the formation of phosphodiester bonds. From homoribopolymer competition binding experiments we conclude that the polyadenylic acid on the CPMV genome might be a part of the replicase binding site.