Clinical dextran purified by fractional ultrafiltration coupled with water washing

Dextran with extremely narrow molecular weight distribution (MWD) is demanded for clinical use. To elucidate the effect of fractional ultrafiltration on the purification of clinical dextran, a range of ultrafiltration membranes with 100, 30, 5 and 1 kDa molecular weight cut-off (MWCO) were applied. High Performance Gel Permeation Chromatography (HPGPC) analysis indicated that the MWD of ultrafiltrated dextran fractions are wide, with polydispersity index (D) between 2.2 and 5.4, suggesting that the MWD of dextran is hard to be strictly controlled by fractional ultrafitration. However, when coupled with water washing during ultrafiltration process, the homogeneity of dextran was greatly improved. An ultrafiltration fraction of 5-30 kDa (Mw ≈ 35 kDa) with narrow MWD (D = 1.2) was obtained after 5 times of water washing. The results show that fractional ultrafiltration coupled with water washing can be used as a simple and effective method to improve the quality of clinical dextran.     

Temperature dependence of binding of hyaluronate oligomer to bovine nasal proteoglycan

An ultrafiltration technique was used to study the temperature coefficient of the association constant K for 1:1 binding of proteoglycan to a hyaluronate oligosaccharide fraction containing an average of about 16 monosaccharide units. The proteoglycan was concentrated during the filtration experiment in order to provide minimal disturbance of the equilibrium in the retained solution. Analytical results calculated from assay of ³H-labeled hyaluronate in the filtrate fractions were extrapolated back to initial equilibrium cell conditions. At 10 °C values of K obtained in this way from ultrafiltration agreed within experimental error with those from equilibrium dialysis. Apparent K values obtained with both techniques tended to decrease somewhat with increasing proteoglycan concentration, due probably in part to excluded volume effects. Values of K obtained by ultrafiltration over the temperature range 5 to 40 °C were used to estimate the enthalpy of binding ΔH° as ?17.5 (±1.5) kcal mol^?1 and the entropy of binding ΔS° as ?50 (±5) cal K^?1 mol^?1 (based on a 1 μm standard state). The dilute solution value of K at 37 °C is sufficiently large to suggest that most of the proteoglycan monomers having a binding site are complexed under tissue conditions.     

Expression of recombinant HAO3 from an Iranian isolate of Hyalomma anatolicum anatolicum in Pichia pastoris and evaluation of its antigenicity

Hyalomma anatolicum anatolicum tick is considered as one of the main problem of ruminants’ productivity in endemic countries such as parts of Africa, the Middle East and India. The disease is economically important and hence, its control and eradication is a priority. This problem reinforces the need for alternative approach like vaccine to control tick infestations instead of continuous application of acaricide which led to the natural selection of the acaricide-resistant ticks. Therefore, the present study provided evidence for the construction of transformant containing the chromosomally integrated multi-copy expression cassettes of HAO3, its successful and efficient expression in Pichia pastoris yeast and purification of the secreted protein by ultrafiltration (UF) system in a high level yield and purity.The result of antigenicity assay for the rHAO3 protein pointed well toward its capability for the elicitation of antibody response in immunized rabbits. Interestingly, the results indicated that the expressed HAO3 protein reacted well with mid gut antigen (MGAg) and rBm86 (Gavac) antisera in ELISA and western blot assays making it evident that the epitopes present in expressed protein are well recognized by the antibodies against MGAg and rBm86 proteins. Moreover, the presence of cross-reactive epitopes between rHAO3 protein with its native antigen from mid gut cells was also determined.     

Effect of salts and Triton X-100 on ultrafiltration purification and properties of extracellular glucose oxidase from <Emphasis Type="Italic">Penicillium adametzii</Emphasis> LF F-2044.1

We compared the effectiveness of glucose oxidase isolation from the culture fluid of Penicillium adametzii LF F-2044.1 in the presence of ammonium sulfate, ammonium chloride, and Triton X-100. Ammonium chloride inhibited glucose oxidase in the culture fluid. This compound increased K _M (by 1.2–1.3 times), but decreased V _max for D-glucose oxidation (by 1.7–1.8 times). Ammonium sulfate had little effect on kinetic parameters. Combined treatment with salts and Triton X-100 was followed by a significant increase in the effectiveness of ultrafiltration purification of the culture fluid. The samples of glucose oxidase were electrophoretically characterized. The dependence of kinetic parameters on glucose oxidase concentration during oxidation of D-glucose was evaluated. The catalytic constant and k _cat/K _M ratio for glucose oxidase samples from the culture fluid isolated in the presence of additives significantly surpassed those for enzyme samples, which were obtained by ultrafiltration of the culture fluid with no additives and chromatography on aluminum oxide. The activity of glucose oxidase isolated from the culture fluid in the presence of ammonium chloride was lower compared to that of the enzyme obtained in the presence of ammonium sulfate. This agent is preferable for ultrafiltration of the culture fluid.     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

Vaccination against the nematode Trichostrongylus colubriformis. II. Attempts to protect guinea-pigs with worm acetylcholinesterase.

Soluble material extracted from T. colubriformis fourth stage larvae was fractionated by membrane ultrafiltration, gel filtration and ion-exchange chromatography. The acetylcholinesterase (AChE) peak obtained by gel filtration protected guinea-pigs against infection but it was contaminated by worm allergen. However, there was no relationship between the AChE content of fractions obtained by membrane ultrafiltration and ion-exchange chromatography and their ability to stimulate protective immunity. Purified T. colubriformis AChE, prepared by ion-exchange chromatography and free from demonstrable allergen did not stimulate protective immunity whereas another fraction, containing less than one-thousandth the amount of AChE, was effective in doing so.     

Ultrafiltration recovery of entomotoxicity from supernatant of Bacillus thuringiensis fermented wastewater and wastewater sludge

The study investigates the recovery of active components (crystal proteins, spores and other factors of virulence) of Bacillus thuringiensis (Bt) based biopesticides from centrifuged supernatant, by ultrafiltration. The centrifuged fermented broths comprised, starch industry wastewater, non-hydrolyzed and hydrolyzed wastewater sludge and semi-synthetic soya medium (as control). The ultrafiltration membrane of 5 kDa gave highest recovery of the active components and increased the entomotoxicity in the retentates by 7.9%, 10.5%, 9.0%, 5.7%, for semi-synthetic soya medium, starch industry wastewater, non-hydrolyzed and hydrolyzed wastewater sludge, respectively. However, the retention of suspended solids on the membrane (measured via mass balance) varied with the type of fermented broths and was very high for hydrolyzed sludge (soya-15%; starch industry wastewater-12%; non-hydrolyzed sludge-7% and hydrolyzed sludge-68%). This reflected the deposit on the membrane. In the given context, scale-up of the ultrafiltration process will give better efficacy for non-hydrolyzed sludge and starch industry wastewater in comparison to soya and hydrolyzed sludge medium.     

Giardia Cysts and Cryptosporidium Oocysts in Membrane-Filtered Municipal Wastewater Used for Irrigation

A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.     

Development of a Rapid Method for Simultaneous Recovery of Diverse Microbes in Drinking Water by Ultrafiltration with Sodium Polyphosphate and Surfactants

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.5-μm microspheres, Salmonella enterica serovar Typhimurium, Bacillus globigii endospores, and echovirus 1. Ten-liter tap water samples were concentrated to ~250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2, C. parvum, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.     

The isolation and partial characterization of a protective antigen from developing larvae of Ascaris suum.

An antigen (ACF antigen) obtained by in vitro cultivation of third-stage larvae of Ascaris suum to the fourth stage in defined medium induced significant protection in guinea pigs. The antigen was further characterized by ultrafiltration and gel filtration and was shown to be a single component by gel precipition and immunoelectrophoresis. It induced active cutaneous anaphylaxis in sensitized animals. The ACF antigen is estimated to contain about 79% protein and 22% carbohydrate and to have a molecular weight of approx 67,000.     

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS

Background : Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR).Methods: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies.Results: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC₅₀ ≤ 6.07 μM.Conclusion: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.     

Study of the antioxidant capacity of Miscanthus sinensis lignins

The present work studied the antioxidant capacity of the lignin obtained from Miscanthus sinensis. As the lignin structure is very complex, extraction and separation processes highly influence obtained lignin structure and its properties, including the antioxidant activity. Lignin fractions were obtained from black liquor resulting from different fractionating and autohydrolysis processes of M. sinensis. Different lignin fractions with specific molecular weight were separated by ultrafiltration using different cut-off ceramic membranes (10 and 15 kDa). The physico-chemical properties, phenolic groups content and the antioxidant capacity of each lignin sample were studied. It was found that antiradical activity of the analyzed lignin samples was closely related with the used fractionating process (soda, organosolv and autohydrolysis treatments). The ultrafiltration process allows obtaining lignins with different antioxidant capacity, improving this property as the used cut-off was greater.     

Mass separation and in vitro immunological activity of membrane-fractionated polysaccharides from fruiting body and mycelium of Agaricus subrufescens

Membrane technology has been applied to separate polysaccharides from Agaricus subrufescens (ASPs). The membrane-retained fractions and unfractionated preparations have been tested for in vitro immunological activity. Both the microfiltration (MF) and ultrafiltration (UF1) membranes were able to separate high-molecular weight polysaccharides from fruiting body (ASP-FB) and submerge-fermented mycelium (ASP-SmF) extracts. All fractions showed immunostimulatory effects on RAW 264.7 macrophages, measured by TNF-??, iNOs gene expression, and NO production. In contrast, antibody and proliferation levels in B lymphoblastoid SKW 6.4 cells were significantly increased after treatment with ASP-FB, but did not with ASP-SmF preparations. The ASPs- and LPS-induced stimulation could be differentiated by the finding that polymyxin B, a specific inhibitor of LPS, did not significantly affect the immunoactivating response and proliferation activity of ASPs on macrophages and B cells, respectively. Furthermore, the ASP-FB treatment was unable to induce IL-6 production by B cells unlike LPS activation, sustaining distinct signaling pathways for ASP-FB and LPS. The overall results provided additional information about the action of ASPs on the immune system and support the membrane method to separate and concentrate high-molecular weight ASPs for immunopharmacological and biotechnological applications.     

Properties of extracellular polymer having an effect on expression of activated sludge

Extracellular polymer in activated sludge was found to affect expression of these sludge by raising the value of the average specific resistance (α_av) during filtration and lowering the value of the modified consolidation coefficient (C_e) during consolidation. Further, the addition of extracellular polymer to the sludges caused higher values of α_av and lower values of C_e. The logarithmic plots of α_av and C_e against the quantity of extracellular polymer was linear. When these two values (α_av and C_e) of each activated sludge were plotted against the quantity of extracellular polymer extracted from each sludge, they were correlated by a single straight line with two exceptions. These were the return sludge of a textile factory and the anaerobically digested sludge of a sewage treatment plant. These exceptions indicate that the values of α_av of these sludges are higher than those of other sludges containing the same amount of extracellular polymer and that of C_e are lower than those of other sludges. It was confirmed that the extracellular polymer of these two sludges contains a lot of polysaccharides of the molecular weight 600–100,000 (with a standard of polyethylene glycol) by gel chromatography. The effects of the flocculant on dewatering were investigated. The effects of the cationic polymer flocculant addition to the extracellular polymer were analyzed using an ultrafiltration cell (molecular weight cut-off 10,000). The ultrafiltration rate of extracellular polymer was much improved by adding a cationic polymer flocculant. The removal of extracellular polymer by adding cationic polymer flocculant was ascertained by measuring the molecular weight distribution of the polysaccharides. On the other hand, no flocculations of extracellular polymer and sludge by adding anionic and nonionic polymer flocculant could be observed.     

Isolation and identification of RANKL-induced osteoclast differentiation inhibitor from Pleurotus citrinopileatus

The purpose of this study was to investigate the effects of water extracts from edible mushroom, Pleurotus citrinopileatus fruiting body on RANKL-induced osteoclast differentiation. The osteoclast differentiation inhibitor from water extracts of P. citrinopileatus was purified by ultrafiltration, size exclusion column chromatography, ethanol precipitation, and treatment with various enzymes. The water extracts from P. citrinopileatus showed significant osteoclast differentiation inhibitory activity, and its 50 kDa above fraction from ultrafiltration showed the highest osteoclast differentiation inhibitory activity. Fractions 2 and 3 from size exclusion column chromatography clearly inhibited RANKL-induced osteoclast differentiation. When these fractions were treated by ethanol precipitation, the precipitates showed high osteoclast differentiation inhibitory activity. Finally, we obtained the precipitates as the purified osteoclast differentiation inhibitor, and it was identified as β-glucan. However, further studies are required to be used as candidate of anti-osteoporotic agent.     

Rat brain hexokinase: glucose and glucose-6-phosphate binding sites and C-terminal amino acid of the purified enzyme

The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined K_m for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed.The binding of glucose-6-P (but not glucose) is prevented in the presence of P_i. This is in accord with a previously suggested model in which binding of P_i is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P.Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.     

Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein,rVP2H,in insect larvae

This study describes an alternative approach to produce rVP2H protein using insect larvae of the cabbage looper Trichoplusia ni as hosts for the expression of the protein. The chimeric rVP2H protein, having an extra six histidine residues at the C-terminus of the VP2, a structural protein of infectious bursal disease virus (IBDV), is a vaccine candidate for the prevention of infectious bursal disease. The chimeric rVP2H protein was expressed in insect larvae in form of virus-like particles, in which they maintain their native immunogenic properties. The expression level of rVP2H protein in T. ni larvae was estimated to be approximately 0.4 mg/g of larvae or 0.2 mg/larvae. The rVP2H particles have a uniform morphology of dodecahedral structure with a size of 23 nm in diameter, and the particles could be affinity-purified in one step with immobilized metal-ion affinity chromatography (IMAC) from the larvae homogenate. The recovery of rVP2H protein was approximately 55% following IMAC and the protein was obtained with a purity of around 90%. An additional purification step of ammonium sulphate precipitation was added to speed up the process of microfiltration and ultrafiltration of the homogenate prior to IMAC. This step enhanced the final purity of rVP2H protein to 99%, demonstrating that the purification protocol developed herein was a powerful strategy for obtaining highly pure rVP2H protein from insect larvae. The immunogenicity and protective properties of the larvae-derived rVP2H protein were evaluated using a chicken protection assay. When larvae-derived rVP2H protein was intramuscularly injected into specific-pathogen-free chickens (20 μg/bird), high titres of virus-neutralizing antibodies were induced and the chickens were protected from the infection of a very virulent strain of IBDV isolated locally.     

Alteration of Gene Expression Profile in Kidney of Spontaneously Hypertensive Rats Treated with Protein Hydrolysate of Blue Mussel (Mytilus edulis) by DNA Microarray Analysis

Marine organisms are rich sources of bioactive components, which are often reported to have antihypertensive effects. However, the underlying mechanisms have yet to be fully identified. The aim of this study was to investigate the antihypertensive effect of enzymatic hydrolysis of blue mussel protein (HBMP) in rats. Peptides with in vitro ACE inhibitory activity were purified from HBMP by ultrafiltration, gel filtration chromatography and reversed-phase high performance liquid chromatography. And the amino acid sequences of isolated peptides were estimated to be Val-Trp, Leu-Gly-Trp, and Met-Val-Trp-Thr. To study its in vivo action, spontaneously hypertensive rats (SHRs) were orally administration with high- or low-dose of HBMP for 28 days. Major components of the renin-angiotensin (RAS) system in serum of SHRs from different groups were analyzed, and gene expression profiling were performed in the kidney of SHRs, using the Whole Rat Genome Oligonucleotide Microarray. Results indicated although genes involved in RAS system were not significantly altered, those related to blood coagulation system, cytokine and growth factor, and fatty acids metabolism were remarkablely changed. Several genes which were seldom reported to be implicated in pathogenesis of hypertension also showed significant expression alterations after oral administration of HBMP. These data provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of HBMP, and will contribute towards increased value-added utilization of blue mussel protein.     

The effects of cell recycling on the production of 1,3-propanediol by Klebsiella pneumoniae

The effects of both biomass age and cell recycling on the 1,3-propanediol (1,3-PDO) production by Klebsiella pneumoniae were investigated in a membrane-supported bioreactor using hollow-fiber ultrafiltration membrane module in two separate experiments. It was determined that older cells have a negative effect on 1,3-PDO production. The concentrations of by-products, such as acetic acid and ethanol, increased in cultures with older cells, whereas the concentrations of succinic acid, lactic acid and 2,3-butanediol decreased. The effect of cell recycling was comparatively studied at a cell recycling ratio of 100 %. The results showed that cell recycling had also negative effects on 1,3-PDO fermentation. It was hypothesized that both cell recycling and biomass age caused metabolic shifts to undesired by-products which then inhibited the 1,3-PDO production. On the other hand, the use of hollow-fiber ultrafiltration membrane module was found to be very effective in terms of removal of cells from the fermentation broth.     

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.