kDa Molecular Weight Cut-off Membrane Research Articles

Scaleable purification process for gene therapy retroviral vectors

Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology. A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 microm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold. Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77+/-11%). Up-scaling of the process resulted in high titer vector preparations, 3.2x10(8) infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination. The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies.

Formation of dialyzable iron during in-vitro digestion and extraction of mycoprotein

Mycoprotein is the biomass of the fungus Fusarium venenatum. The ability of mycoprotein to enhance production of dialyzable iron following in-vitro digestion or extraction was studied, with the aim of identifying the components responsible. Digested mycoprotein, extracted mycoprotein and digested chicken breast muscle all produced about ten times as much dialyzable iron as the control. All of the dialyzable iron from mycoprotein was ferric. Essentially all the dialyzable iron passed through a 1 kDa molecular weight cut-off membrane. Iron binding components were extracted from mycoprotein using weak acid but not water. Extraction of mycoprotein produced both dialyzable and non-dialyzable components, which bound ferric iron. Analysis of the digests and the dialyzable fraction of acid extraction showed that they contained siderophores, which contributed to the formation of dialyzable iron.

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak ( Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC 50 value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.

Structural characterization of water-soluble polysaccharides from Opuntia monacantha cladodes in relation to their anti-glycated activities

An aqueous extract of polysaccharides from Opuntia monacantha cladodes (POMC) was preliminarily purified by 5 kDa molecular weight cut-off ultrafiltration membrane to remove impurities with low molecular weight. Then the retentate was fractionated by ethanol solution and chromatographed on a DEAE Sepharose Fast Flow anion-exchange column to yield a major fraction (POMC IV) which was eluted by 0.5 M NaCl. POMC IV was subjected to further purification on a Sephadex G-50 gel filtration column. Two major fractions, POMC V and VI, were collected. By analyses using gel permeation chromatography (GPC), high-performance liquid chromatography (HPLC) and gas chromatography (GC), POMC V, which had a molecular weight of 28.7 kDa, was comprised mainly of rhamnose, arabinose and glucose in the molar ratio of 9.15:1.00:6.84, with 3.07% (w/w) of glucuronic acid, while POMC VI, which had a molecular weight of 10.8 kDa, was comprised mainly of rhamnose, mannose and glucose in the molar ratio of 8.72:1.00:6.19, with 4.68% (w/w) of glucuronic acid. Six distinct-absorbance peaks, at 1742, 1633 and 1417 cm −1 in the infrared (IR) spectra of POMC V, and at 1729, 1596 and 1407 cm −1 in the IR spectra of POMC VI, resulted from the presence of uronic acids. The peaks at 1043 and 890 cm −1 were characteristic of rhamonse and β- d-glucose, respectively. From the profiles of 13C and 1H nuclear magnetic-resonance (NMR) spectra, the main (1 → 2)-α- l-rhamnopyranose units were obviously characterized by six strong signals at 99.24 (C-1), 77.52 (C-2), 70.19 (C-3), 71.33 (C-4), 69.81 (C-5) and 17.45 ppm (C-6). The signal at 175.92 ppm was due to C-6 of β- d-glucuronic acid units. The 1H spectrum signal at 1.20 ppm was assigned to the CH 3 of α- l-rhamnopyranose units. The evaluation of anti-glycation activity suggested that POMC had good potential for inhibiting the formation of advanced glycation endproducts. Time- and dose-dependent effects were also observed for all POMC samples.

Flux enhancement during ultrafiltration of produced water using turbulence promoter

Concentration polarization and membrane fouling remain one of the major hurdles for the implementation of ultrafiltration of produced water. Although many applications for ultrafiltration were already suggested, only few were implemented on an industrial scale. Among those techniques, turbulence promoter can be more simple and effective in overcoming membrane fouling and enhancing membrane flux. As for the result that turbulence promoter increase fluid velocity, wall shear rates and produce secondary flows or instabilities, the influence of turbulence promoter was investigated on permeate flux during produced water ultrafiltration and the potential application of this arrangement for an industrial development. Experimental investigations were performed on 100 KDa molecular weight cut-off PVDF single-channel tubular membrane module using four kinds of turbulence promoters. It is observed that the significant flux enhancement in the range of 83%–164% was achieved while the hydraulic dissipated power per unit volume of permeate decreased from 31%–42%, which indicated that the using of turbulence promoter is more efficient than operation without the turbulence promoter. The effects of transmembrane pressure and cross-flow velocity with and without turbulence promoter were studied as well. Among the four kinds of turbulence promoters, winding inserts with 20.0 mm pitch and 1.0 mm wire diameter showed better performances than the others did.

Temperature-induced increase in cellular chelating potential associated with reduced thermotolerance

Hot springs panic grass, Dichanthelium lanuginosum var sericeum (Schmoll), successfully colonizes geo-thermally heated soils that are subject to chronic temperatures ranging from 40 to 50 °C, occurs at altitudes in excess of 2500 m, and tolerates low soil moisture content for extended periods of time. We utilized superoxide dismutase (SOD; EC 1.15.1.1) as an indicator of oxidative stress response to compare the impacts of temperature on wheat ( Triticum aestivum L.), a cool-season grass, with the temperature responses of three isolates of D. lanuginosum. One of the D. lanuginosum isolates was collected from a non-thermal site and had reduced capacity to adapt to growth at elevated temperature. Wheat SOD activity in crude leaf extracts was significantly reduced by incubation at 45 °C. In contrast, SOD activity in crude leaf extracts from a thermophilic D. lanuginosum isolate increased after incubation at 45 °C. Significant increases in cellular chelating capacity occurred in wheat and the D. lanuginosum isolated from a non-thermal environment after plants were exposed to elevated temperatures. Ultra-filtration of leaf extracts through 10 kDa molecular weight cutoff membranes removed much of the chelating activity and restored apparent SOD activity. The D. lanuginosum isolates with the greatest thermotolerance retained SOD activity when exposed to elevated temperatures and did not show statistically significant increases in cellular chelation potential. Wheat SOD activity increased in response to heat shock. The chelation activity was associated with low molecular weight components (<10 kDa) that did not bind C 18 media. Ultra-filtration removal of these low molecular weight chelating components increased SOD activity and replacement of low molecular weight chelating components with an equivalent amount of EDTA suppressed SOD activity.

Separation and Concentration of Health Compounds by Membrane Filtration

The performance of nano-filtration (NF) for separating phenolic compounds from sugar in apple juice was studied using 1 and 0.25 kDa molecular weight cut-off (MWCO) spiral wound membranes. If these phenolic compounds could be recovered, they could stabilize the juice from haze formation or be added as antioxidants to foods and beverages in order to increase their health properties. Batch experiments were conducted on a pilot scale rig using a diluted clear apple juice concentrate. For the 1 kDa MWCO membrane, the research determined the effect of operating conditions on process efficiency and membrane fouling. The concentration of polyphenolics on the retentate side increased by a factor of up to 4 and the sugar concentration increased by 1.5 times under optimum conditions of lower temperature (30oC), acidic pH (2), lower trans-membrane pressure (5 Bar) and higher initial sugar concentration (20 oBrix). Despite the increase in polyphenolics in the retentate, there was little difference in the phenolic composition between retentate and permeate solutions. As the molecular mass of the rejected phenolics was smaller than the membrane cut-off, this indicated that the rejection was related to the formation of a secondary membrane formed as a result of fouling. A mass balance of polyphenolics in the final retentate and permeate compared with the initial feed solution indicated that up to 4.3 gm of polyphenolics were bound per m2 of membrane. The permeate solutions collected from the 1 kDa MWCO membrane were then filtered using a 0.25 kDa MWCO membrane. Most phenolic compounds were retained by the membrane and the concentration increased by a factor of up to 2. Catechin, rutin, phloridzin and quercetin derivatives were concentrated on the retentate side. However, around 20 - 40% of chlorogenic acid and epicatechin was observed on the permeate side. It is concluded that membrane separation represents a potentially efficient and cost-effective technology to separate the phenolic fraction of fruit juice in a form suitable for use as a functional ingredient.

Enhancing the microdialysis recovery for sampling of Cu and Ni by incorporating humic acid in the perfusion liquid

A strategy to enhance the microdialysis relative recovery for sampling of Cu and Ni ions is presented. Enhanced recovery was achieved by incorporating humic acid, a binding agent, in the microdialysis perfusion liquid during sampling from a Cu and Ni standard solution mixture. All microdialysis sampling experiments were carried out at room temperature under quiescent conditions using a concentric type of microdialysis probe with an adjustable effective dialysis length. For all metal determinations electrothermal atomic absorption spectrometry was employed. Metal recoveries were shown to be dependent on the membrane molecular weight cut-off, perfusion rate, sample solution pH, perfusion liquid composition as well as perfusion liquid pH. Complete recoveries (100%) of Cu and Ni were obtained by microdialysis sampling using a 10 kDa molecular weight cut-off polysulfone membrane at a flow-rate of 2 μl/min employing a 0.05% (w/v) optimal composition of humic acid incorporated in the perfusion liquid. The optimal sampling pH of humic acid was determined to be 6 where most oxygen containing functional groups are dissociated and available for metal binding. These data have important ramifications for sampling and determination of metal ions in small sample solutions (∼10 ml) at very low concentrations in the ppb range.

Vibratory shear enhanced processing membrane filtration applied for the removal of natural organic matter from surface waters

In this study, the performance of a vibratory shear enhanced unit (termed hereafter as VSEP) was evaluated for the removal of humic acids in the presence of inorganic particles (clays) from synthetic aqueous dispersions, simulating contaminated surface waters. For this purpose, several major parameters were examined, such as the specific membrane type, the feed quality (i.e. the concentration of main components in the feed, as well as the pH value) and the vibration amplitude. It was found that the permeate flux was stabilized after certain operation (filtration) time regardless of feed quality for each membrane process. The percentage recovery of liquid volume during the performed semi-batch laboratory-scale experiments exceeded 90%. The increase of pH value from 6 to 9, during microfiltration (MF), resulted in 30% decrease of permeate flux and 20% enhancement of HA rejection, indicating that the form of HA in solutions, as well as their interactions with ions and/or inorganic clay particles may play an important role in the filtration process. Although clay particles were removed almost completely during all the examined filtration processes, HA molecules removal exceeded 99% only during nanofiltration (NF), while for the other processes the removal did not exceed 95%. The permeate flux was affected by the respective feed quality and membranes’ type (pore diameters), but also by the applied vibration amplitude as well, especially during ultrafiltration (UF), using the 10 kDa molecular weight cut-off (MWCO) membrane and NF processes. The permeate flux was found to be proportional to γ ¯ w 0 .21 for UF and to γ ¯ w 0 .31 for NF of humic acids solutions, where γ ¯ w is the mean (induced) shear rate at the membrane surface. Finally, although the operating pressures, especially for MF and UF were relatively high, when compared with other commercially available membrane treatment systems, the examined VSEP unit presented rather stable hydrodynamic behavior, enabling the efficient treatment of surface waters.

Cerebral microdialysis methodology—evaluation of 20 kDa and 100 kDa catheters

Microdialysis monitoring of cerebral metabolism is now performed in several neuro-intensive care units. Conventional microdialysis utilizes CMA70 catheters with 20 kDa molecular weight cut-off membranes enabling the measurement of small molecules such as glucose, lactate, pyruvate and glutamate. The CMA71 100 kDa molecular weight cut-off microdialysis catheter has recently been introduced to allow detection of larger molecules such as cytokines. The objective of this study was to perform in vitro and in vivo testing of the CMA71 microdialysis catheter, comparing its performance with the CMA70. In vitro comparison studies of three of each catheter using reference analyte solutions, demonstrated equivalent recovery for glucose, lactate, pyruvate and glutamate (range 94–97% for CMA70 and 88–103% for CMA71). In vivo comparison involved intracranial placement of paired CMA70 and CMA71 catheters (through the same cranial access device) in six patients with severe traumatic brain injury. Both catheters were perfused with CNS Perfusion Fluid without dextran at 0.3 µl min−1 with hourly sampling and bedside analysis on a CMA600 microdialysis analyser. The two catheters yielded equivalent results for glucose, lactate, pyruvate, glutamate and lactate/pyruvate ratio. CMA71 microdialysis catheters can, therefore, be used for routine clinical monitoring of extracellular substances, as well as for their intended research role of larger molecular weight protein sampling.

Modeling Trichloroethylene Adsorption by Activated Carbon Preloaded with Natural Dissolved Organic Matter Using a Modified IAST Approach

A model was developed, using an approach based on the Ideal Adsorbed Solution Theory (IAST), to predict trichloroethylene (TCE) adsorption by granular activated carbon (GAC) preloaded with natural dissolved organic matter (DOM) isolated from three surface water sources. The IAST model was formulated for a bi-solute system in which TCE and DOM single-solute uptakes were described by the Langmuir-Freundlich and Freundlich isotherms, respectively. The effect of DOM molecular size and polarity (as measured by XAD 8 resin fractionation) on TCE uptake by preloaded GAC was assessed to identify a reactive fraction of natural water DOM for the purpose of modeling competitive adsorption. Consistent with previous work that identified low molecular weight species as the most reactive with regard to preloading effects (i.e., reducing target compound uptake), the low molecular weight components of the polar (hydrophilic) and nonpolar (hydrophobic) DOM fractions, isolated using ultrafiltration (1 kDa molecular weight cutoff membrane), exhibited significant competitive effects. Furthermore, the effects of these fractions on TCE uptake were similar; therefore, theywere considered together to represent a single "reactive fraction" of DOM. On the basis of this finding, isotherms for the <1 kDa low molecular weight DOM fraction of the whole water were measured, and molar concentrations were computed based on an average molecular weight determined using size-exclusion chromatography. The IAST model was modified to incorporate surface area reduction due to pore blockage by DOM and to reflectthe hypothesis thatTCE molecules can access adsorption sites which humic molecules cannot, thus preventing competition on these sites. The model was calibrated with data for TCE uptake by carbon preloaded with the <1 kDa low molecular weight DOM fraction and was verified by predicting TCE uptake by carbon preloaded with whole natural waters for both constant GAC dose (hence constant DOM loading) and variable GAC dose (hence variable DOM loading) TCE isotherms. Preloading by DOM reduced volume in GAC pores having widths smaller than 1.25 nm (likely accessible only to TCE) to a greater extent than total pore volume, suggesting preferential blockage of micropores. Such preferential pore blockage may explain, in part, why increased DOM loading decreases the fraction of the total surface area on which no competition between TCE and DOM occurs.

Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates

The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson’s disease, Alzheimer’s disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500 kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific.

Interbiopolymer complexing between β-lactoglobulin and low- and high-methylated pectin measured by potentiometric titration and ultrafiltration

The interactions between proteins and polysaccharides are of great interest to the food industry. Few studies have been carried out on diluted β-lactoglobulin/pectin systems and none has provided a complete investigation on the nature of the interactions involved. The two main objectives of this study were to determine the conditions for the formation of complexes and to identify the type of interactions involved in β-lg/low-methylated (LM) and β-lg/high-methylated (HM) pectin systems. The pH values leading to the formation of soluble complexes (pH c) were determined using potentiometric titrations. The nature of interactions was determined using sodium chloride, urea and different temperatures. The extent of complex formation was quantified by filtering β-lg/LM- and β-lg/HM-pectin solutions, at pH values between 4.5 and 7.0, through a 100 kDa molecular weight cut-off (MWCO) membrane. The pore size of the membrane was used to retrieve the non-complexed protein in the permeate. These proteins were measured using the bicinchoninic acid protein assay (BCA). The amount of β-lg complexed was greater with LM- than with HM-pectin. At pH 4.5 in deionised water and at a protein/polysaccharide (w/w) ratio of 4:1, about 96% of the β-lg complexed with LM-pectin, whereas only 78% complexed with HM-pectin. The destabilising effect of sodium chloride, urea and temperature has demonstrated that interactions in the systems are mainly caused by electrostatic forces and, to a lesser extent, hydrogen bonding.

High-resolution plasma protein fractionation using ultrafiltration

The effects of pH and ionic strength (salt concentration) on transmission of single protein human serum albumin (HSA) and human immunoglobulins (HIgG) through a 100 kDa molecular weight cut-off (MWCO) polyethersulfone membrane have been studied using a pulsed sample injection technique. Experimental results obtained clearly demonstrate the effectiveness of changing solution pH and ionic strength optimisation for selective transmission of HSA or HIgG. These also demonstrate the usefulness of pulsed injection experimental technique for the optimization of ultrafiltration processes. A pulse input carrier phase ultrafiltration (CPUF) was employed to fractionate a binary protein mixture of HSA and HIgG at low NaCl concentration, showing that it is feasible to separate the binary protein at optimised conditions.

Ultrafiltration of partially hydrolyzed rice bran protein to recover value‐added products

AbstractRice brans were treated with a protease together with a disulfide bond‐breaking agent (Na2SO3) to achieve a 2–4% peptide bond hydrolysis (DH). Ultrafiltration (UF) with 3 kDa molecular weight cut‐off (MWCO) membrane led to substantial loss of feed protein due to permeation. Using 1 kDa MWCO membrane increased protein yields, but it was not effective in purifying the protein hydrolysates despite the increase in membrane area and operating time. The efficiency of this UF process can be improved using a larger MWCO membrane (e.g., 2 kDa MWCO), which may facilitate complete removal of phytate. Based on disparity of molecular sizes, use of phytase may also increase purity of protein retentates and allow the recovery of functional inositol phosphates in permeates. The presence of Na2SO3 during proteolysis to 2% DH of preheated bran (100°C, 10 min) repaired the damage caused by preheat treatment by increasing protein recovery but increased the concentration of small peptides in hydrolysates, i.e., &lt;1 kDa, particularly for highly aggregated proteins. Heat treatment is necessary to stabilize rice bran, but the sulfite treatment may be avoided to increase UF yield and purity of protein retentates and allow higher DH values for hydrolysis of stabilized brans. Accordingly, this UF process can be an efficient method for recovering high‐value components from rice bran, an underutilized rice milling co‐product, for many industrial applications.

Design of a molecular chaperone-assisted protein folding bioreactor.

Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.

Analysis of phospholipase A2 glycosylation patterns from venom of individual bees by capillary electrophoresis/electrospray ionization mass spectrometry using an ion trap mass spectrometer

A method based on tryptic digestion, ultrafiltration and capillary electrophoresis/mass spectrometry (CE/MS) has been developed for the analysis of the glycosylation pattern in the phospholipase A2 (PLA) of individual honeybees. Without reducing the disulfide bonds, PLA was digested with trypsin and filtered with a 3 kDa molecular weight (MW) cut-off membrane. With this procedure, the glycopeptides could be isolated from the nonglycosylated peptides. After tryptic digestion and ultrafiltration, the disulfide bonds were reduced before analysis by CE. To reduce the adsorption, CE separation was performed on successive multiple ionic-polymer (SMIL) polybrene (PB) coated capillary columns. The SMIL-PB columns allowed partial separation of the glycopeptides and eight glycopeptides were identified by on-line coupling of CE with electrospray ionization (ESI) mass spectrometry. The analysis of phospholipase A2 from the venom of individual bees indicated that the variation and relative abundances of different glycopeptides were similar between the younger and the older bees.

Effect of ultrafiltration of yeast extracts on their ability to promote lactic acid bacteria growth.

Five yeast extracts (YE) were fractionated by ultrafiltration (UF) with 1, 3, and 10 kDa molecular weight cutoff membranes, concentrated by freeze-drying, and the resulting powders of yeast extract filtrates (YEF) were evaluated for their growth-promoting properties on nine cultures of lactic acid bacteria (LAB). There was an increase in alpha-amino nitrogen content of the YEF powders as the pore size of the UF membranes used to filter the YE solutions decreased. The source of YE had a much greater effect than UF on the growth of LAB. This was also the case for the YEF contents in total and alpha-amino nitrogen. Growth curves of the LAB showed that maximum growth rate (mumax) data were on average 30% higher with bakers' YE than with brewers' YE, while maximum optical density (ODmax) values were on average 16% higher with bakers' YE. This could be related to the higher nitrogen content of the bakers' YE used in this study. Modification by UF of the YE had no significant influence on the growth of 4 of the 9 LAB strains. The three strains of Lactobacillus casei were negatively influenced by UF, as they did not grow as well in the media containing the YEF obtained after filtering with 1 and 3 kDa membranes. On a total solids basis, the 2.5 x retentates from the 10 kDa membrane gave, on average, 4% lower mumax and 5% lower ODmax values as compared to cultures where the corresponding YEF was used as medium supplement. This could also be partially related to the different nitrogen contents of the filtrates and retentates.

A study of a polysulfone membrane for use in an in-situ tunable microdialysis probe during monitoring of starch enzymatic hydrolysates

We report a study made on a 5 kDa molecular weight cut-off (MWCO) polysulfone membrane (SPS 4005) for use in microdialysis sampling of starch enzymatic hydrolysates on-line coupled to column liquid chromatography with integrated pulsed electrochemical detection. Membrane characteristics were evaluated by examining both the membrane and membrane support layer using a scanning electron microscope (SEM). This study was made so as to elucidate the mechanism or mode by which a membrane exposed to different bioprocess conditions is either fouled or destroyed. The correlation of extraction fraction data with SEM was a confirmatory test for the observed change in the membrane characteristics. Examinations were made after keeping the membrane in pure water for 5, 10 and 30 min at room temperature or after perfusing the membrane with pure water continuously at room temperature, 60 and 90°C for 24 h. Extraction fractions were evaluated at these temperatures to see applicability of the SPS 4005 membrane to high temperature bioprocesses. Scanning electron microscopy studies were also made on membranes used for sampling and sample clean-up during on-line monitoring of the hydrolysis of soluble starch (according to Zulkowsky) for 32 h, and the hydrolysis of wheat starch at room temperature, 60 and 90°C for 6 h. Non-specific/directed protein-membrane interactions were evaluated by sampling maltoheptaose with a microdialysis probe fitted with a 10 mm SPS 4005 membrane, before and after treatment with an enzyme solution of Termamyl (endo-1,4-α- d-glucan glucanohydrolase EC 3.2.1.78).

Modulation of Ia+ Langerhans cell numbers in vivo by cultured epidermis derived supernatants and by GM‐CSF

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.