Molecular Sieve Chromatography Research Articles

Hemagglutinin from the Root Tuber of Dioscorea preussii Pax.

In this study, Dioscorea preussii root tuber hemagglutinin was purified and its physicochemical properties were determined. The antioxidative and anti-hemolytic activities of the hemagglutinin were also investigated. The hemagglutinating assay was used to detect the presence of lectin in the phosphate-buffered saline extract of the D. preussii root tuber. The lectin was purified using ammonium sulfate fractionation and molecular sieve chromatography. The optimum pH and temperature were determined. In addition, antioxidant activity was assessed using 2,2 diphenylpicrylhydrazyl (DPPH) radical scavenging, metal chelating, ferric reducing antioxidant power (FRAP), and lipid peroxidation inhibition assays. Red blood cells subjected to oxidative damage caused by H2O2 were employed to evaluate their antihemolytic ability. Starch inhibited hemagglutinin activity. Dioscorea preussii hemagglutinin (DPH) maintained full hemagglutinating activity from 30 °C to 60 °C and pH 5-13. Ethylene diamine tetraacetic acid did not affect the hemagglutinating activity of hemagglutinin. All denaturing agents (Guanidine-HCl, urea, and β-mercaptoethanol) reduced the hemagglutinating activity of the hemagglutinin to different degrees. The hemagglutinin scavenged the DPPH radical and chelated iron metal with half maximum inhibitory concentration (IC50) of 0.727 ± 0.035 mg/mL and 0.583 ± 0.078 mg/mL, respectively, while the FRAP assay showed that it contained 76 mg of ascorbic acid equivalent per gram of the purified hemagglutinin. In the absence of hemolytic agents and at lower concentration tested, hemagglutinin showed positive membrane integrity protection. This study provides information on the antioxidant properties of D. preussii root tuber hemagglutinin as well as its cell membrane protective ability. The lectin is a starch-binding, which makes it a novel lectin.

Novel antioxidant peptides in fermented pork sausage: Purification, characterization, and cytoprotective functions on Caco-2 cells.

In this study, crude peptides from fermented sausages inoculated with Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 were initially separated by ultrafiltration and molecular-sieve chromatography. The obtained fractions with high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and ferric-reducing antioxidant power values (MWCO-1 and fraction A) were used in Caco-2 cells to evaluate their cytoprotective effect on oxidative damage triggered by H2O2. MWCO-1 and A showed slight cytotoxicity. Increased glutathione peroxidase, catalase, and superoxide dismutase activities and decreased malondialdehyde content were observed in the peptide-treated groups. Fraction A was further purified using reversed high-performance liquid chromatography. Eighty potential antioxidant peptides were identified by liquid chromatography with tandem mass spectrometry, and fourteen antioxidant peptides were synthesized. SDEEVEH and FAGDDAPR showed strong DPPH radical scavenging activity, whereas ALELDSNLYR and QEYDESGPSIVHR presented strong ABTS+· scavenging activity. These peptides might have great potential for food and pharmacological applications.

Purification and characterization of acidophilic xylanase from Bacillus sp. with potential application as fruit juice clarifier

Two bacterial isolates from the mangrove’s soil were isolated and screened for xylanase production. Screened isolates designated as AS1 and AS2 were identified by sequencing and found to be Bacillus altitudinis and Bacillus licheniformis. The highest xylanase activity recorded from Bacillus altitudinis is 82.664 units per ml and from Bacillus licheniformis is 153.59 unitsper ml at pH 4.0. Purification of the crude enzyme was done by molecular sieve chromatography and molecular weight was estimated by the SDS PAGE method. The molecular weight of xylanase was noted as 43 and 40 kDa respectively. Temperature 40°C and pH 4.0 was found to be optimum for purified xylanase enzyme. Purified xylanase was active in broad pH and temperature range. Purified xylanase activity was repressed upon incubation with FeSO4 solution. Application of purified xylanase was done in clarifying different fruit juices.The results of fruit juice clarification indicate the escalation in the sugar concentration and transparency in the juices. Percent transmittance of fruit juices showed 25.11%, 75.82%, 52.40%, 40.09%, and 30.21% for apple, orange, mousambi, pineapple, and kiwi respectively, with Bacillus altitudinis xylanase. while 44.31%, 80.12%, 72.11%, 33.00%, and 21.42 for apple, orange, mousambi, pineapple, and kiwi respectively, with Bacillus licheniformis Xylanase. The application studies show the potential of purified xylanase as a clarifier in food Industries. Keywords: Acidophiles; Xylanase; Fruit juice clarification; Profiling; Bacillus sp

A secretory form of Parkin-independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo.

We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP‐4T1 syngeneic murine cancer model to study EVs in‐transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV‐associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission‐induced mitophagy preferentially populated the CD81+/PD‐L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin‐independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC‐1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti‐CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.

Development and biological activity of long-acting recombinant human interferon-\u03b12b

BackgroundThe type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research.ResultsTwo novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b.ConclusionsCHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.

A brief review on: Separation techniques chromatography

Chromatography is an important biophysical process, allowing the components of a mixture to be isolated, classified and extracted for qualitative and quantitative analysis. Proteins can be purified based on features such as size and shape, total charge, surface hydrophobic groups and stationary phaset binding ability. Four separation strategies based on molecular characteristics and form of interaction Four separation techniques based on molecular characteristics and form of interaction use ion exchange mechanisms, surface adsorption, partition, and exclusion of the size. Column chromatography is one of the most common methods of protein purification Chromatography is based on the theory of separation of fluid stationary phase (stable phase) when traveling with the support of a mobile phase. The variables that are involved in this separation process include adsorption (liquid-solid), partition (liquid-solid), and affinity or variations between their molecular weights. Due of these variations, certain components of the mixture stay in the stationary phase longer, and travel gradually in the chromatography system, while others move rapidly through the mobile phase, leaving the system faster. Based on this method, three components form the basis of the chromatography technique. Stationary phase: is a phase consisting of a phase or a surface adsorbed Mobile phase: is a phase consisting of a or a part. Separate molecules which form the interaction between stationary phase, mobile phase and substances contained in the mixture are the basic component that is successful in separating molecules from the partition-based chromatography methods are very effective in separating small molecules and distinguishing them as amino acids, carbohydrates and fatty acids. In the separation of macromolecules as nucleic acids and proteins, however, affinity chromatography’s (i.e. ion-exchange chromatography) are more efficient. Paper chromatography is used in protein separation and protein synthesis studies; gas-liquid chromatography is used to isolate alcohol, ethanol, lipid, and amino groups, and to analyze enzymatic interactions, while molecular-sieve chromatography is used in particular to evaluate molecular protein weights. Agarose gel chromatography is used to purify RNA, DNA particles, and viruses. The chromatography stationary phase is a solid phase or liquid phase that is sprayed on a solid phase surface. A gaseous or liquid phase is a mobile phase which flows through the stationary phase. If the mobile phase is liquid, it is called liquid chromatography, and if it is gas, it is called gas chromatography. of gases and mixtures of volatile liquids, and solid material, gas chromatography is used. Liquid chromatography is used in particular for thermal unstable and non-volatile materials. The purpose of applying chromatography, which is used as a quantitative analysis tool apart from its separation, is to record a sufficient separation within an acceptable time interval. Various chromatography methods have been developed to that end. Some of these have chromatography on columns, thin layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, liquid high pressure chromatography, and affinity chromatography.

Peptide profile and free radical scavenging activity of the low molecular weight peptide fraction from whole body extracts of <i>Tympanotonus fuscatus</i> var radula (Linnaeus) and <i>Pachymelania aurita</i> (Muller)

Low molecular weight peptides are among the most active bioactive components that have been isolated from marine molluscs. Currently, peptides isolated from molluscs as well as their synthetic structural analogues are in clinical trials as anticancer compounds and have been approved for use in pain management. These Bioactive peptides have also been demonstrated to exhibit antioxidant activities. This study aimed at isolating and profiling the low molecular weight peptide fractions from extracts of two common marine molluscs; P. aurita and T. fuscatus obtained from the Niger-Delta region of Nigeria. This study also investigated the free radical scavenging activity of the isolated fraction. Fourier Transform Infrared Spectroscopy analysis was carried out to determine the chemical entities and common functional groups found in the extracts. The Low molecular weight peptides were isolated using Molecular sieve chromatography, while the peptide profiles were established using SDS-PAGE and Thin Layer Chromatography (TLC). The free radical scavenging potential of the peptides was also assessed qualitatively using TLC, with DPPH as stain. The results show the presence of several low molecular weight peptides in the extracts of both P. aurita and T. fuscatus. Yellow spot on a purple background, indicating antioxidant activity was observed in the TLC plates sprayed before the chromatographic run. This activity was lost on further separation, indicating a synergistic effect. Further studies to effectively separate these fractions into individual peptides, investigate the synergistic effect observed as well as further explorations of possible bioactivity of these peptides is required.© 2019 International Formulae Group. All rights reserved.Keywords: Tympanotonus fuscatus var radula, Pachymelania aurita, peptides, FT-IR, free- radical scavenging

A novel antimicrobial protein of the endophytic Bacillus amyloliquefaciens and its control effect against Fusarium chlamydosporum

Fusarium chlamydosporum is the causal agent of stem rot in Jacaranda acutifolia. Bacillus amyloliquefaciens is an endophytic bacterium that shows promising biological control activity against F. chlamydosporum. In this study, B. amyloliquefaciens was cultured in a fermentation broth, and the main antimicrobial substance in the fermentation broth was isolated using the ammonium sulfate precipitation method and purified by DEAE sepharose fast flow weak anion exchange chromatography and sephadex G-50 molecular sieve chromatography. Polyacrylamide gel electrophoresis revealed a single antimicrobial protein with a molecular weight of approximately 29.0 kDa. The antimicrobial protein is a strong antagonist against F. chlamydosporum, leading to mycelial deformity, unclear mycelial septa, and isolated and granulated intracellular protoplasm. The N-terminal eight-amino acid sequence, NH2-Gly-Arg-Pro-Leu-Pro-Leu-Ala-Ala, was determined using the automatic Edman degradation method. BLAST analysis indicated that the amino acid residue sequence at positions 154–161 shows 100% homology to the hypothetical protein of Chromobacterium sp. LK1. However, no known homologous antimicrobial protein was identified, indicating that we have isolated a novel antimicrobial protein.

New approaches to old problems: Removal of phospholipase A2 results in highly active microsomal membranes from the honey bee, Apis mellifera

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.

Isolation and purification of a novel antimicrobial peptide from Porphyra yezoensis.

We aimed to isolate antimicrobial peptides from Porphyra yezoensis. Enzymatic hydrolysate of P. yezoensis was purified by ultrafiltration, molecular sieve chromatography, and ion exchange chromatography sequentially. A novel peptide with strong antimicrobial activity against Staphylococcus aureus was isolated and the amino acid sequence was identified to be Thr-Pro-Asp-Ser-Glu-Ala-Leu (TPDSEAL). Physical and chemical properties and antimicrobial activity of the peptide were determined. The antimicrobial mechanism was studied. The antimicrobial activity of TPDSEAL kept stable under acidic or basic conditions, high temperature, and ultraviolet radiation. The antimicrobial mechanism of antimicrobial peptides may damage the cell wall and membrane, and enhance the permeability of cells, which leads to the outflow of intracellular substances and death of bacteria. This study provides novel insight into the preparation of marine-derived antimicrobial peptides. PRACTICAL APPLICATIONS: Antimicrobial peptides, which act as defensive weapons against microbes, have been broadly used as food additives in food industry. Due to the limited amount of natural antimicrobial peptides in organisms and the high cost of chemical synthesis, producing novel natural antimicrobial peptides with bioengineering methods has become an urgent task. In the present study, we prepared a novel antimicrobial peptide from pepsin-digested hydrolysate of Porphyra yezoensis using ultrafiltration, molecular sieve chromatography, ion exchange chromatography, and mass spectrometry analysis. A novel peptide with strong antimicrobial activity against Staphylococcus aureus was isolated and the amino acid sequence was identified to be Thr-Pro-Asp-Ser-Glu-Ala-Leu (TPDSEAL). The identified peptide exhibits great stability under acidic or basic conditions, high temperature, and ultraviolet radiation. Mechanism revealed that TPDSEAL treatment may damage the cell wall and membrane, enhance the permeability of cells, and lead to the death of bacteria. Our study provides the novel insight into the preparation of marine-derived antimicrobial peptides.

The formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding

BackgroundAlthough the wound response of plants has been extensively studied, little is known of the rapid occlusion of wounded cell itself. The laticifer in rubber tree is a specific type of tissue for natural rubber biosynthesis and storage. In natural rubber production, tapping is used to harvest the latex which flows out from the severed laticifer in the bark. Therefore, study of the rapid wound-occlusion of severed laticifer cells is important for understanding the rubber tree being protected from the continuously mechanical wounding.ResultsUsing cytological and biochemical techniques, we revealed a biochemical mechanism for the rapid occlusion of severed laticifer cells. A protein-network appeared rapidly after tapping and accumulated gradually along with the latex loss at the severed site of laticifer cells. Triple immunofluorescence histochemical localization showed that the primary components of the protein-network were chitinase, β-1,3-glucanase and hevein together with pro-hevein (ProH) and its carboxyl-terminal part. Molecular sieve chromatography showed that the physical interactions among these proteins occurred under the condition of neutral pH. The interaction of β-1,3-glucanase respectively with hevein, chitinase and ProH was testified by surface plasmon resonance (SPR). The interaction between actin and β-1,3-glucanase out of the protein inclusions of lutoids was revealed by pull-down. This interaction was pharmacologically verified by cytochalasin B–caused significant prolongation of the duration of latex flow in the field.ConclusionsThe formation of protein-network by interactions of the proteins with anti-pathogen activity released from lutoids and accumulation of protein-network by binding to the cytoskeleton are crucial for the rapid occlusion of laticifer cells in rubber tree. The protein-network at the wounded site of laticifer cells provides not only a physical barrier but also a biochemical barrier to protect the wounded laticifer cells from pathogen invasion.

Partial purification and characterisation of cytosolic Fructose-1, 6-bisphosphatase from Drymaria cordata

Drymaria cordata is an important ethnomedicinal plant from which many important secondary metabolites have been reported. Partial purification of the enzyme, fructose 1,6-bisphosphatase was carried out following the methods of homogenization, streptomycin sulphate precipitation, ammonium sulphate cut and molecular sieve chromatography through Bio-Gel A-0.5m column. Biochemical characterization experiments were performed by standard methods with the enzyme preparation as purified from the column. Cytosolic fructose 1,6-bisphosphatase from the leaves of Drymaria cordata was purified to about 27-fold with 77% of recovery over homogenate fraction. The enzyme was highly specific to D-fructose-1,6-bisphosphate. With increase of protein concentration upto 300µg and incubation time upto120 minutes, the enzyme activity increased linearly. The metal ions Mg2+ or Mn2+ strongly stimulated the enzyme activity on the other hand Li+, Hg2+ and Zn2+ were potent inhibitors. The D. cordata enzyme showed temperature maxima at 40˚C while the optimum pH was at 8.0. The Km value of the enzyme for its substrate, Fructose 1,6-bisphosphate was 1.11µM proving its strong affinity.

A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells.

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

A new strategy for the passive skin delivery of nanoparticulate, high molecular weight hyaluronic acid prepared by a polyion complex method

Restoring hyaluronic acid (HA) content is important for maintaining the function of photo-aged skin. This study aimed to evaluate the passive delivery into skin of HA nanoparticles formed by the polyion complex method. Nanoparticles were prepared by mixing and stirring anionic HA with a cationic polymer, protamine, at the charge ratio 55:45. The permeation of fluorescently-labelled HA nanoparticles (HANP) or free HA through hairless mouse skin was characterized in vitro. HANP or free HA was applied to ultraviolet (UV)-irradiated mice in vivo, and their transepidermal water loss (TEWL) was measured after 4 days. HA that had been delivered into skin was separated and characterized by molecular sieve chromatography. HANP were able to deliver HA into the dermis both in vitro and in vivo, whereas free HA penetrated no further than the stratum corneum. Following HANP application, HA within the skin was present in the form of free HA rather than nanoparticles. When applied in vivo, HANP significantly reduced the TEWL caused by UV irradiation. Thus, although free HA does not penetrate into the skin by passive diffusion, HA can be effectively delivered by nanoparticles. HA is then released from the nanoparticles and can contribute to barrier recovery following UV irradiation.

Immunological features and efficacy of the recombinant subunit vaccine LTB-EMY162 against Echinococcus multilocularis metacestode.

Alveolar echinococcosis is a zoonotic disease caused by the infection of the larval stage Echinococcus multilocularis with worldwide distribution especially in the northwest China. It is important to develop a well-tolerated immunoprophylaxis against E. multilocularis for alveolar echinococcosis control. In this study, a prokaryotic expression system for recombinant immunogen LTB-EMY162 was established, and the immunological features, sensitized lymphocyte, IL-4/IFN-γ secreted, prophylactic effect, and therapeutic effect were also evaluated. Arctic Express (DE3) system, Ni2+-charged and molecular sieve chromatography were used to obtain a high-purity 29kDa protein. The ELISA and lymphocyte proliferation assay showed that LTB-EMY162 induced high-titer specific IgG against EMY162 and E. multilocularis protoscoleces protein in BALB/c mice and promoted sensitized T lymphocyte cell proliferation, and LTB-EMY162 stimulated Th cell to secrete IL-4 and IFN-γ and induced a Th1/Th2 mixed type immunological response. We also found that LTB-EMY162 significantly inhibited the cysts formation by challenging with 1000 E. multilocularis protoscoleces. The growth of protoscoleces and cysts were also significantly decreased by treating with LTB-EMY162 in 1000 protoscoleces intraperitoneal injection therapeutic mice model. In conclusion, we have constructed a subunit vaccine LTB-EMY162 which has prevention and therapeutic effect against E. multilocularis infection.

Immunodominant antigens that induce Th1 and Th17 responses protect mice against Helicobacter pylori infection.

Helicobacter pylori has infected more than half of the world's population, causing gastritis, gastric ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The oral recombinant Helicobacter pylori vaccine currently used has made great progress in addressing this problem, however, its efficacy and longevity still need to be improved. Th1 and Th17 cells play essential roles in local protection against Helicobacter pylori in the stomach mucosa. Additionally, protective immunodominant antigens are the preferred for a vaccine. In this work, Helicobacter pylori whole cell lysate was separated into 30 groups based on molecular weight by molecular sieve chromatography. The group best promoting CD4 T cells proliferation was selected and evaluated by immunization. The detail proteins were then analyzed by LC-MS/MS and expressed in Escherichia coli. Eleven proteins were selected and the dominant ones were demonstrated. As a result, three protective immunodominant antigens, inosine 5'-monophosphate dehydrogenase, type II citrate synthase, and urease subunit beta, were selected from Helicobacter pylori whole cell. Two of them (inosine 5'-monophosphate dehydrogenase and type II citrate synthase) were newly identified, and one (urease subunit beta) was confirmed as previously reported. The mixture of the three antigens showed satisfactory protective efficiency, with significant lower H. pylori colonization level (P < 0.001) and stronger Th1 (P < 0.001) and Th17 (P < 0.001) responses than PBS control group. Thus, inosine 5'-monophosphate dehydrogenase, type II citrate synthase, and urease subunit beta are three protective antigens inducing dominant Th1 and Th17 responses to defend against Helicobacter pylori infection.

Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson's Disease.

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.

IFN-\u03bb1 in CHO cells: its expression and biological activity

BackgroundMany studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.ResultsA full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 106 IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.ConclusionThe Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

Purification of a Fe-SOD excreted by Leishmania braziliensis for specific antibodies detection in Mexican human sera: Cutting-edge the knowledge

Clinical diagnosis of leishmaniasis is highly complex, presenting a wide range of clinical manifestations, sometimes non-specific, and thus the epidemiological study and diagnostic need specific molecular markers for each Leishmania species. Leishmania spp. posses different Fe-SOD isoforms, one of which is excreted into the external milieu and, presenting immunogenic characteristics, is a very reliable molecular marker. Superoxide dismutases (SODs) are antioxidant metal-enzymes responsible for the dismutation of superoxide ion into hydrogen peroxide and molecular oxygen, and it is considered an important virulence factor. In this manuscript we have purified the iron(Fe)-SOD excreted by Leishmania braziliensis using ion-exchange and molecular-sieve chromatography and we have studied it as an antigen in serodiagnostic analyses in ELISA and Western blot techniques, testing 213 human sera from Mexico. Indeed, L. braziliensis Fe-SODe has been purified 123.26 times with a specific activity of about 893.66U/mg of protein. Applying the purified enzymes in serological tests we found 17.84% sera positive. We have demonstrated that the purified enzyme is more sensitive than the non-purified ones and we also demonstrated, for the first time, the presence of antibodies against L. braziliensis, not the main species in the country, in human population from Hidalgo and Nuevo Leon States.

PBT1, a novel antimicrobial protein from the biocontrol agent Bacillus subtilis XF-1 against Plasmodiophora brassicae

Plasmodiophora brassicae is a Plasmodiophorid and obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. It causes clubroot disease of cruciferous crops by producing resting spores that can survive in the soil for more than ten years. Although up to date there is no effective chemical control but now a strain of the bacterium Bacillus subtilis XF-1, isolated from China, has been found to inhibit P. brassicae and the mycelial growth of other plant-pathogenic fungi, such as Magnaporthe oryzae, Curvularia lunata,Fusarium oxysporum and so on. The antimicrobial activity of this biocontrol bacterium lies in its water-soluble protein (PBT1) which was isolated and purified by means of molecular sieve and affinity chromatography. The single band of the purified protein has a molecular mass of 25 kDa, estimated by a sodium dodecyl sulfate- polyacrylamide gel electrophoresis and it was heat resistant. The result of the sequencing proved PBT1 to be a new protein. Recombinant plasmid pQE81L-PBT1 was constructed using the coding gene of Pbt1 and plasmid PQE81-L, and expressed in Escherichia coli BL21. The fusion protein of BL21 exhibited significant detrimental effects on P. brassicae spores. In addition, pQE81L-PBT1 was successfully inserted into Pseudomonas fluorescens strain 2P24 which developed antagonistic effect to the clubroot pathogen that was not suppressed by 2P24 alone. The results showed that PBT1 protein might play a key role in the biocontrol of clubroot disease of crucifers.