Polysaccharides from Hericium erinaceus Fruiting Bodies: Structural Characterization, Immunomodulatory Activity and Mechanism.

Five fractions from crude Hericium erinaceus polysaccharides (HEPs), including HEP-1, HEP-2, HEP-3, HEP-4 and HEP-5, were obtained through column chromatography with a DEAE Cellulose-52 column and Sephadex G-100 column. The contents of total carbohydrates and uronic acid in HEPs were 53.36% and 32.56%, respectively. HEPs were mainly composed of Fuc, Gal and Glu in a molar ratio of 7.9:68.4:23.7. Its chemical structure was characterized by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. HEP-1 contains the backbone composed of (1→6)-linked-galactose with branches attached to O-2 of some glucose. The immunological activity assay indicated that HEP-1 significantly promoted the production of nitric oxide, interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α and the phosphorylation of signaling molecules. Collectively, these results suggested that HEP-1 could improve immunity via NF-κB, MAPK and PI3K/Akt pathways. Hericium erinaceus polysaccharides might be explored as an immunomodulatory agent for use in dietary supplements.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns).

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations. HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity. Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal. These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Structure elucidation and bioactivities of a new polysaccharide from Xiaojin Boletus speciosus Frost.

A new water-soluble polysaccharide named BSF-X was extracted and purified from the fruiting bodies of Boletus speciosus Frost which had a molecular weight of 141,309 Da. The structure identification results showed that BSF-X was mainly composed of ß-d-glucose and α-D-galactose. BSF-X had a backbone of 1, 4-linked ß-d-glucose of which branches were mainly composed of two 1, 6-linked α-D-galactose residue and a 4-linked ß-d-glucose at the end of the branches. Antitumor activities results showed that BSF-X could inhibit the proliferation of L929 cells in vitro and S180 tumor cells in vivo. Immunoregulatory activities results showed that BSF-X could promote the proliferation of T cells, B cells and macrophages by promoting the cells into S phase from G0/G1 phase. Polysaccharide BSF-X can also enhance the phagocytes is and cytokine secretion of macrophages. This study introduced the new polysaccharide BSF-X as a valuable source which exhibit unique antitumor and immunoregulatory properties.

Physicochemical properties and cell-based bioactivity of Pu'erh tea polysaccharide conjugates.

Polysaccharide conjugates were prepared from Pu'erh tea and fractionated by DEAE-cellulose DE-52 column chromatography to yield one unexplored polysaccharide-conjugate fraction termed TPC-P with a molecular weight of 251,200Da. DVS (dynamic vapour sorption) result discovered that the humidity condition of long-term preservation for TPC-P is below 70% RH. Although it contained proteins, TPC-P could not bind to the Coomassie Brilliant Blue dyes G250 and R250. The "shoulder-shaped" ultroviolet absorption peak in TPC-P UV-vis scanning spectum ascribe theabrownins that inevitably adsorbed the polysaccharide conjugate. Zeta potential results demonstrated TPC-P aqueous solution merely presented the negative charge properties of polysaccharides instead of acid-base property of its protein section, and had more stability in greater than pH 5.5. No precipitation or haze occurred in the three TPC-P/EGCG aqueous mixtures during their being stored for 12h. The phase separation was observed in aqueous mixtures of TPC-P and type B gelatin. TPC-P possessed the fine stability as a function of temperature heating and cooling between 0 and 55°C. It is proposed that some properties of the covalent binding protein of TPC-P were "shielded" by its polysaccharide chains.

Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 µM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 µM and 2.5 µM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 µM.

Fermentation of rice bran hydrolysate to ethanol using Zymomonas mobilis biofilm immobilization on DEAE-cellulose

Background The major challenges associated with the fermentation of lignocellulosic hydrolysates are the reduction in the operating cost and minimizing the complexity of the process. Zymomonas mobilis biofilm has been emerged to resolve these complexities. Biofilm has been reported to tolerate to the toxic inhibitors and easily manipulated toward the cell recycle through the cell immobilization. Results Z. mobilis ZM4 and TISTR 551 were able to develop biofilms on DEAE cellulose under the differences in the morphologies. Z. mobilis ZM4 developed homogeneous biofilm that brought DEAE fiber to be crosslinking, while Z. mobilis TISTR 551 developed heterogeneous biofilm in which crosslinking was not observed. Ethanol production under batch and repeated batch fermentation of rice bran hydrolysate containing toxic inhibitors were compared between these two biofilms. TISTR 551 biofilm produced the maximum yield (Y P/S) of 0.43 ± 0.09 g ethanol/g glucose (83.89% theoretical yield). However the repeated batch could not be proceeded due to the bacterial detachment. Z. mobilis ZM4 biofilm produced the maximum yield (Y P/S) of 0.177 ± 0.05 g ethanol/g glucose (34.74% theoretical yield) in the batch culture and the biofilm remained intact to proceed along the repeated batch. The highest ethanol yield (Y P/S) in the repeated batch of Z. mobilis ZM4 was 0.354 ± 0.07 g ethanol/g glucose (69.51% theoretical yield). Conclusions Homogeneous biofilm structure of Z. mobilis provided more recycle beneficial over the heterogeneous biofilm structure for the ethanol production from lignocellulosic hydrolysate.

3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].

BACKGROUND: Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS: hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. RESULTS: hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. CONCLUSION: This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions.

Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein-metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel 'tentacle' carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0-8.0 and temperature optimum at 25°C. Immobilized PPO shows almost 100% of activity at 0°C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8h duration.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

A peptide with potent antifungal and antiproliferative activities from Nepalese large red beans.

An antifungal defensin-like peptide with a molecular mass of 7.1kDa was isolated from dried Nepalese large red beans (Phaseolus angularis). The purification protocol employed included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antifungal peptide was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and SP-Sepharose. The antifungal peptide inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 1.4 and 1.8 microM, respectively. It did not inhibit HIV-1 reverse transcriptase when tested up to 200 microM. It exerted an antiproliferative action on L1210 leukemia cells and MBL2 lymphoma cells with an IC(50) of 15 and 60 microM, respectively.

High-resolution mapping of points of site-specific replication stalling.

Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.

Decontamination of polymerase chain reaction reagents using DEAE-cellulose.

Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity. We also show the suitability of the proposed approach for decontamination of the Taq polymerase, monoclonal antibodies to Taq polymerase, and Moloney murine leukemia virus (M-MLV) reverse transcriptase.

Purification of matrix metalloproteinases by column chromatography.

Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin-Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes approximately 7-10 d.

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata).

The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.

Reversed-phase LC/MS method for polyphosphoinositide analyses: changes in molecular species levels during epidermal growth factor activation in A431 cells.

In studies on lipid metabolomics, liquid chromatography/ mass spectrometry (LC/MS) is a robust and popular technique. Although effective reversed-phase (RP) LC/ MS methods enabling the separation of phospholipid molecular species have been developed, RPLC methods to analyze phosphatidylinositol phosphates (PIPs) have not been reported. In this study, we developed conditions suitable for PIP analysis. Coupled with (diethylamino)ethyl (DEAE)-cellulose pretreatment, at least 1 pmol each of phosphatidylinositol monophosphates (PIP1), bisphosphates (PIP2), and triphosphates standards per approximately 6 x 10(6) cultured cells could be measured. Using these methods, we detected elevated concentrations of more than 12 PIP1 species in epidermal growth factor (EGF)-stimulated A431 cells, a human epidermoid carcinoma cell line. The PIP2 species detected were not elevated after stimulation. We also detected EGF-induced increases in the levels of several phosphatidic acid species using another set of methods. Our method sensitively determined PIPs within a biological sample and is thus suitable for analysis of phoisphoiniositide metabolism.

A hemagglutinin with mitogenic activity from dark red kidney beans.

A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. 'Dark Red Kidney Bean'. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 degrees C and 70 degrees C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 degrees C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells.

Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.

Extracorporeal plasma treatment for the removal of endotoxin in patients with sepsis: clinical results of a pilot study.

Despite the advances in therapeutic approaches in the management of inflammatory conditions, the incidence of sepsis is on increase in the intensive care units (ICU). In a pilot study, we investigated whether the use of an apheresis system based on DEAE-cellulose is capable of reducing the plasma concentration of endotoxin in patients with severe sepsis. We enrolled 15 intensive care patients with severe sepsis and plasma endotoxin concentrations >0.3 EU/mL. In addition to standard ICU therapy, a total of 83 apheresis treatments were performed. About 1.7 volumes of plasma (6000 mL) were treated at each apheresis session. A significant reduction in plasma endotoxin levels from a median of 0.61 to 0.39 EU/mL (-35%) could be achieved (P < 0.001). Long-term comparison of the initial and post-treatment levels after a series of five to six individual apheresis treatments also showed a statistically significant decline in circulating endotoxin, interleukin (IL)-6, C-reactive protein (CRP), fibrinogen, and an increase in cholesterol levels. Except for a transient and reversible increase of prothrombin time, no adverse events were observed in patients undergoing this new adsorption apheresis treatment. Our data show that reduction of endotoxin by extracorporeal DEAE-cellulose-based plasma treatment may prove a promising therapeutic tool for patients suffering from bacterial sepsis and proven endotoxemia.

Isolation of cicadin, a novel and potent antifungal peptide from dried juvenile cicadas.

A single-chained antifungal protein with a molecular weight of 6.5 kDa and displaying a novel N-terminal sequence was isolated from dried juvenile cicadas which are used in traditional Chinese medicine, by using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and then gel filtration on a Superdex peptide column. The peptide, designated cicadin, exerted potent antifungal activity with IC(50) values at nonomolar concentrations against a variety of fungi including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus. Cicadin suppressed the activity of HIV-1 reverse transcriptase and stimulated the proliferation of murine splenocytes.

Anti-platelet fraction from Galega officinalis L. inhibits platelet aggregation.

A fraction from crude extract of Galega officinalis L. was purified by gel filtration on Sephadex G-25, Sepharose 4B, and ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The fraction with molecular weight 100-140 kDa appears to have a polysaccharide nature, including protein. The fraction inhibits platelet aggregation initiated by 25 microM adenosine 5'-diphosphate (ADP), 100 microg/ml collagen, and 0.8 U/ml thrombin with the 50% inhibiting concentration (IC(50)) being 11.2 microg/ml for ADP, and the IC(100) being 15.1 microg/ml for collagen and IC(100) 19.6 microg/ml for thrombin.