Millipore Filtration Research Articles

遊離型低分子Ｐｒｏｔｅｉｎ Ａ高度生産

Protein A reacts with the Fc region of IgG from many mammalian species. Immobilized protein A has been recently used for immunoadsorbent therapy in breast adenocarcinoma patients. We isolated mutant strains from Staphylococcus aureus after mutagenization. These mutants produced high levels of extracellular protein A (102_??_205μg/ml). Protein A from the mutant strains were purified by IgG-affinity chromatography followed by millipore filtration. The molecular weight of the protein A was lower than that of parental protein A by SDS-PAGE.

Effect of albumin on the solubility of cholesterol in bile

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.

Millipore Filtration and Use of RV Medium for Isolation of Salmonella from Preenrichment Broths

A filtration procedure for isolation of Salmonella from preen-richment broth is described. Five ml of broth are passed through a Millipore AP15 pre-filter above a .45μm Millipore filter. The filter is transferred to 25 ml of Rappaport-Vassiliadis broth and incubated at 42°C. Of 76 naturally and 55 artificially contaminated meat and poultry samples, 102 were positive for Salmonella. Of these, 99 were isolated using the filtration technique, 93% of which were obtained following a 6-h incubation period. Isolations from tetrathionate brilliant green and selenite cystine broth were 93 and 84, respectively.

Developmental maturation of calcium transport by rat brush border membrane vesicles.

The developmental aspects of calcium transport across the intestinal brush membrane vesicles was studied utilizing a millipore filtration technique. Calcium transport represented uptake into the intravesicular space as evidenced by osmolality studies, calcium release by the calcium ionophore A23187, and temperature dependency. Calcium transport in both suckling and adolescent rats appears to occur by a saturable mechanism. Calcium uptake was similar in the presence of sodium and potassium gradients, but decreased in the presence of choline gradient. The imposition of negative membrane potential did not enhance calcium uptake compared to voltage clamp conditions indicating an electroneutral process. The initial rate of calcium uptake was linear up to 15 s. Kinetic analysis of calcium uptake at 7 s showed lower Vmax and lower Km in suckling rats compared to adolescent rats. These studies are the first to demonstrate the maturational aspects of calcium entry at the brush border level and are consistent with our previous kinetic studies utilizing whole tissue.

Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. I: Validation of methods.

The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.

Phlorizin increases the permeability of intestinal mucosal membrane to sodium.

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin.

A subpopulation of canine cardiac sarcoplasmic reticulum vesicles has been found to contain a "Ca2+ release channel" which mediates the release of intravesicular Ca2+ stores with rates sufficiently rapid to contribute to excitation-contraction coupling in cardiac muscle. 45Ca2+ release behavior of passively and actively loaded vesicles was determined by Millipore filtration and with the use of a rapid quench apparatus using the two Ca2+ channel inhibitors, Mg2+ and ruthenium red. At pH 7.0 and 5-20 microM external Ca2+, cardiac vesicles released half of their 45Ca2+ stores within 20 ms. Ca2+-induced Ca2+ release was inhibited by raising and lowering external Ca2+ concentration, by the addition of Mg2+, and by decreasing the pH. Calmodulin reduced the Ca2+-induced Ca2+ release rate 3-6-fold in a reaction that did not appear to involve a calmodulin-dependent protein kinase. Under various experimental conditions, ATP or the nonhydrolyzable ATP analog, adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and caffeine stimulated 45Ca2+ release 2-500-fold. Maximal release rates (t1/2 = 10 ms) were observed in media containing 10 microM Ca2+ and 5 mM AMP-PCP or 10 mM caffeine. An increased external Ca2+ concentration (greater than or equal to 1 mM) was required to optimize the 45Ca2+ efflux rate in the presence of 8 mM Mg2+ and 5 mM AMP-PCP. These results suggest that cardiac sarcoplasmic reticulum contains a ligand-gated Ca2+ channel which is activated by Ca2+, adenine nucleotide, and caffeine, and inhibited by Mg2+, H+, and calmodulin.

Improved method for recovery of peritonitis-causing microorganisms from peritoneal dialysate.

The efficacy of recovery of peritonitis-causing microorganisms from peritoneal dialysate fluid by using the Septi-Chek blood culture system (Roche Diagnostics, Div. Hoffmann-La Roche Inc., Nutley, N.J.) was compared with those of other conventional techniques, such as the 20-ml culture and the filtration methods. The recovery of microorganisms by using the Septi-Chek system was found to be as effective as the filtration of 250 ml of dialysate that used a modified Millipore filtration technique (Millipore Corp., Bedford, Mass.). Both methods were found to be superior to the 20-ml culture method. We suggest using the Septi-Chek method as the standard protocol for the culture of dialysate as a relatively inexpensive and labor-saving recovery technique.

The refinement and concentration of agricultural antibiotic A in aqueous solution using membrane processes

This paper looks at the possibilities of refining and concentrating the agricultural antibiotic A (AAA) in aqueous solution using the membrane processes of millipore filtration, ultrafiltration and reverse osmosis. The effects of relevant factors on separation performance are examined and the main factors affecting refinement and concentration are analysed. The most favourable operating parameters are presented, as well as a suitable separation process. Our experiments show that the results of pretreatment are better when using precipitation of oxalic acid and millipore filtration. Cellulose ester membranes can be used to refine and concentrate AAA aqueous solution. When the feed was treated using ultrafiltration with a flux of about 100 ml/cm 2 h for distilled water, the permeate was transparent and limpid; this is a very effective method ofrefinement. When the product was refined by ultrafiltration and then treated using reverse osmosis membranes, with a salt rejection level of 95% or so for tap water, normalization flux was 0.14, and the colour of the permeate was light red-brown according to analysis of the apparent-colour reaction. The concentration of 800–1000 ml feed which had already been refined by ultrafiltration was more than doubled by reverse osmosis at an operating pressure of between 30 and 55 kg/cm 2, lasting for 10 to 13 h at 15 to 50°C, in equipment with an effective film surface area of 80cm 2. The refractivity of the permeate was 1.0–1.2% and the corresponding solute retention levels were over 93% during the whole concentrating cycle. The results offer a basis for the spreading of membrane processes to some areas of biology.

Interaction of Asparagus Root Filtrate with Fusarium oxysporum f.sp. asparagi

Abstract Asparagus (Asparagus officinalis L.) root filtrate (RF) depressed asparagus seedling emergence in a sterile peat–vermiculite medium. In a medium inoculated with Fusarium oxysporum f. sp. asparagi (FO), the effect was magnified. The response to RF dose, regardless of level of FO infestation, was quadratic. Comparisons with a sucrose solution of the same percentage of soluble solids as the RF suggested that reduced emergence may have been due in part to enhancement of FO growth by the energy source added to the medium. However, after germinating seeds in RF until radicle emergence, then rinsing and transferring them to FO-inoculated medium, emergence was reduced relative to controls. Therefore, depression of emergence apparently related both to an autotoxin somehow predisposing young radicles and/or hypocotyls to increased FO infection and to stimulation of FO in the rhizosphere by the soluble solids content of the root exudate. Infection was confirmed to be the only role of FO: sterilization of the spore suspension by Millipore (0.2 µm) filtration eliminated pathogen toxicity.

Micromethod for assaying reverse transcriptase of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus

A micromethod for assaying the reverse transcriptase enzyme of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus in cocultures of clinical specimens for viral isolation was developed and compared with the macromethod in use. Ultracentrifuged, pelleted, and solubilized viral culture supernatants were transferred into either tubes (macromethod) or microtiter plates (micromethod) and incubated with tritiated enzyme substrate. Trichloroacetic acid-precipitated DNA was collected on individual filter papers with a Millipore filtration manifold (macromethod) or on filter sheets using a semiautomated cell harvester (micromethod). Filters were then placed in scintillation fluid and counted on a beta scintillation counter. Results of the micromethod significantly correlated to those of the macromethod, with a linear relationship between the two. The cutoffs for positivity based on the mean + 2 standard deviations for a set of known negative specimens (n = 19) was 4,973 cpm for the micromethod compared with 5,336 for the macromethod. The intrarun and interrun variations were comparable for both methods. There was a 67% increase in the maximal daily number of specimens which could be run (100 versus 60) as well as a reduction in reagent use. In summary, the micromethod utilizing a semiautomated cell harvester is comparable to the existing macromethod in accuracy and is an improvement due to savings in time and reagents.

The effect of changes in the fluid state of rat liver plasma membrane on the transport of taurocholate

The fluidity of basolateral and canalicular rat liver plasma membranes was compared with respect to their response to the membrane perturbants ethanol and calcium. The relation between membrane fluidity and taurocholate transport, a liver plasma membrane function mediated by carrier proteins, was also examined. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as a probe. Uptake of [3H]taurocholate into basolateral rat liver plasma membrane and canalicular rat liver plasma membrane vesicles was measured by a rapid Millipore filtration technique. Anisotropy values were found to be significantly lower for the basolateral rat liver plasma membrane (0.2287 +/- 0.0014) than for the canalicular rat liver plasma membrane (0.2612 +/- 0.0012), indicating that basolateral rat liver plasma membranes are more fluid than canalicular rat liver plasma membranes. Ethanol produced a concentration-dependent effect on lipid fluidity and inhibition of taurocholate uptake, in both membrane preparations. Pretreatment of the membrane with calcium increased the rigidity of both membrane preparations. However, the change in the anisotropy with calcium was only slight in the more rigid canalicular rat liver plasma membrane, while the change in anisotropy was greater and associated with a decrease in taurocholate uptake in the basolateral rat liver plasma membrane. Both the effects of ethanol and calcium were more pronounced in basolateral rat liver plasma membrane than in canalicular rat liver plasma membrane. These results indicate that the fluid state of the hydrophobic bilayer of liver plasma membrane lipids play an important role in regulating bile acid transport in both sinusoidal and canalicular domains.

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na +-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convuluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na +-dependent, but electrically silent transport of propionate is saturable ( K m = 10.9 ± 1.1 mM and V max = 3.6 ± 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of l- and d-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na +-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K + diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na +-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of l- and d-lactate in this region of rabbit kidney proximal tubule.

Purification and characterization of malignancy associated alpha-1 protein from lung tumor of a patient who died of primary carcinoma of the lung.

Malignancy associated ALPHA-1 protein(MAP) was isolated from a kilogram quantity of a lung tumor obtained at an autopsy of a patient who died of primary carcinoma of the lung. Acid extraction, Millipore filtration on a 30,000 molecular weight cut off membrane, treatment of the retentate with 50% saturated ammonium sulfate and gel filtration on Sephadex G-75 yielded a fairly pure material as judged by PAGE and RP-HPLC.

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes.

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.

Endogenous GABA concentration in cortical synaptosomes from young and aged rats

The objective of this study are threefold: (1) to compare the γ-aminobutyric acid (GABA) concentration of a preparation of cortical snaptosomes from young male rats to that of aged rats; (2) to compare the GABA concentration in synaptosomes retained by Millipore filtration for young and aged rats; and (3) to compare the GABA concentration in the synaptosomal preparation to that in synaptosomes retained by Millipore filtration for young and aged rats. The GABA concentration is measured in cortical synaptosomes from Long-Evans rats at 2 months and 30 months of age. Concentration of the synaptosomal preparation declined significantly from 2.97 mM in 2-month animals to 2.68 mM in 30-month animals. In order to compare the results with previous studies of GABA transport, in which Millipore filters are used to separate synaptosomes from incubation medium, the GABA concentration is also measured following placement of synaptosomes on 0.65μ pore size Millipore filters. For both young and aged animals, this population of synaptosomes is found to contain GABA at a concentration more than triple that in the overall population. This is the expected result if the filtration process reduce the non-synaptosomal component of the total preparation, since GABA uptake is known to be a function of the nerve-ending component. Comparison of 2-month and 30-month synaptosomes reveals that the GABA concentration of those synaptosomes retained by the filtration process declined to a greater extent in the aged group than did the content of the overall population (from 9.33mM to 8.37mM). The results are consistent with previous studies of the aging of the GABA transporter in this preparation, in which it was found that the GABA transport capacity declined in the 30-month age group.

Na-H exchange in rat liver basolateral but not canalicular plasma membrane vesicles

Na+-stimulated H+ movement and H+-stimulated Na+ uptake were studied in basolateral (blLPM) and canalicular (cLPM) rat liver membrane vesicles. H+ movement was monitored with the fluorescent amine acridine orange; 22Na uptake was assayed by a rapid Millipore filtration technique. In blLPM, inwardly directed Na+ gradients stimulated H+ efflux and outwardly directed Na+ gradients stimulated proton influx. Outwardly directed proton gradients (pH in 5.9/pH out 7.9) stimulated initial 22Na uptake rates 5- to 10-fold over pH-equilibrated conditions (pH in 7.9/pH out 7.9). Conversely, inwardly directed proton gradients (pH in 7.9/pH out 5.9) inhibited 22Na uptake. pH-dependent 22Na uptake was inhibited by amiloride and harmaline but not by other transport inhibitors, bumetanide, furosemide, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and acetazolamide. Lithium also inhibited H+-stimulated 22Na uptake. Although a component of pH-stimulated 22Na uptake appeared to be dependent on membrane potential, this electrogenic component was amiloride insensitive. Proton gradient-stimulated 22Na uptake in blLPM was saturable, with a Km of 5.4 mM and a Vmax of 14 nmol . min-1 . mg prot-1. In contrast, in cLPM, no Na+ gradient-stimulated proton movement and no pH-dependent Na+ uptake occurred. These findings establish an electroneutral Na-H antiport in blLPM but not cLPM in rat liver. The polarity of this exchanger supports a model of bile formation that is dependent, in part, on canalicular HCO-3 and/or OH- excretion.

Physicochemical Speciation of Trace Elements in River Water by Means of Ultrafiltration

Abstract Ultrafiltration and Millipore filtration techniques have been used in the speciation of trace elements in Tama River water. The size distribution was clarified for 32 elements by means of neutron-activation analysis in order to discuss their chemical or physical forms. Alkali and alkaline-earth elements were primarily found in the dissolved-size fraction (<0.45 μm). Their dissolved species were mostly distributed in the smallest-molecular-weight fraction (<MW 500), although the alkaline-earth elements were also recognized to a significant extent (20–30%) in the middle-molecular-weight fraction (MW 104–500). Transition elements were principally associated with the suspended-size fraction (>0.45 μm) in the upper reaches of the river, but in the lower reaches they were chiefly fractionated as the dissolved species, with the exception of Sc and Fe. The dissolved V and Mn were distributed in the smallest-molecular-weight fraction, while the Co, Ni, and Zn were divided between two smaller-molecular-weight fractions (MW 104–500 and <MW 500). Light rare-earth elements were mostly associated with suspended materials. Heavier rare-earth elements were, on the contrary, mainly found in the dissolved-size fraction, while their dissolved species were concentrated in the middle-molecular-weight fraction. The dissolved species for As, Se, Sb, and W were divided into two smaller-molecular-weight fractions, although As, Se, and W were predominantly found in the middle-molecular-weight fraction and Sb was mainly detected in the smallest-molecular-weight fraction. Aluminium and Ag were mainly associated with suspended materials. Furthermore, the dissolved Al was mostly distributed in the smallest-molecular-weight fraction, while the Ag was concentrated in the largest-molecular-weight fraction (>MW 104).

Water Quality Considerations in Malaysia's First Waterflood

Malaysia's first waterflood, a seawater injection system, was placed in service in the Tapis field in 1982. This paper outlines the design basis and describes the system. Then water quality during and after startup and the formulation of a water quality guide using Millipore membrane filtration tests are discussed. The experience gained and data presented can be applied to current and future waterfloods.

Phosphate transport by jejunal brush border membrane vesicles of the streptozocin-diabetic rat.

We studied the effects of acute diabetes mellitus on jejunal transport of phosphate (32P) by rat brush border membrane vesicles (BBMV) using a Millipore filtration technique. Diabetes was induced by an intravenous (i.v.) injection of 50 mg/kg of streptozocin (STZ). Control and diabetic rats were studied 4 days after the induction of diabetes. In both control and diabetic rats, the presence of a sodium gradient significantly enhanced the uptake of 32P at 20 s and at 1, 2, 5, and 60 min as compared with potassium gradient conditions. Na+-dependent 32P uptake at 20 s and at 1 and 2 min was significantly greater in the diabetic BBMV compared with controls. Na+-independent 32P uptake in both diabetic and control BBMV was similar. To determine whether the enhancement of Na+-dependent 32P uptake in diabetic BBMV is due to an induction of Na+/phosphate cotransporter activity, or a change in Na+ permeability, two additional studies were conducted. Trans-stimulation studies nullifying all electrochemical gradients across the membranes were performed. In diabetic BBMV, 32P uptake at all time points was significantly greater than corresponding values in controls, indicating an increase in the activity of Na+/phosphate cotransporters. 22Na uptake into BBMV at 30 s and at 1, 2, and 60 min was not different between diabetics and controls, indicating that Na+ permeability is not altered in diabetes. Furthermore, kinetic studies using phosphate concentrations between 0.05 and 2.5 mM indicate a significant increase in Vmax capacity of diabetic rats compared with controls without a change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)