Gel High-performance Liquid Chromatography Research Articles

In vitro study on antioxidant and lipid-lowering activities of tobacco polysaccharides

Tobacco polysaccharides were extracted by hot water extraction, and purified and separated using DEAE-52 cellulose chromatography columns, and three purified polysaccharide fractions, YCT-1, YCT-2, and YCT-3, were finally obtained. The physicochemical properties of the three fractions were analyzed by ultraviolet spectroscopy, high-performance liquid chromatography and high-performance gel chromatography. The in vitro antioxidant activity of tobacco polysaccharides was compared among different fractions by using DPPH radical, hydroxyl radical scavenging assay and potassium ferricyanide method. The in vitro hypoglycemic activity was compared using α-amylase and α-glucosidase activity inhibition assay. And the in vitro hypolipidemic activity were investigated by using pancreatic lipase activity inhibition assay and HepG-2 intracellular lipid accumulation assay. All the results showed that the constituent monosaccharides of the three tobacco polysaccharide fractions were similar, but the molar percentages of each monosaccharide were different. The average molecular weights of the three components were 27,727 Da, 27,587 Da, and 66,517 Da, respectively, and the scavenging activities on DPPH radicals and hydroxyl radicals were at a high level with good quantitative-effect relationships. The reducing power were much lower than that of the positive control VC, and the three polysaccharide fractions had a weak inhibitory ability on α-amylase activity, but showed excellent inhibitory ability on α-glucosidase and pancreatic lipase activity. In addition, the results of cellular experiments showed that all three fractions were able to inhibit lipid over-accumulation in HepG-2 cells by increasing the mRNA expression levels of PPAR-α, CPT-1A, and CYP7A1 genes, and the tobacco polysaccharide YCT-3 showed the best effect. The mechanism by which YCT-3 ameliorated the over-accumulation of intracellular lipids in HepG-2 cells was found to be related to its influence on the expression of miR-155-3p and miR-17-3p in the exosomes of HepG-2 cells.Graphical

Chemical Constituents from the Roots of Jasminum sambac (L.) Ait. and their Cytotoxicity to the Cancer Cell Lines.

The roots of J. sambac is the Traditional Chinese Medicine (TCM) with analgesic and anesthetic effects. However, relatively fewer studies on the chemical compositions and the biological activities of the roots of J. sambac have been carried out till now. We studied the chemical compositions of the roots of J. sambac planted in Fujian Province to discover new compounds from this TCM to develop new drugs or drug candidates. This work aims to find the new compounds from the roots of Jasminum sambac (L.) Ait. (J. sambac) for the development of new drugs or drug candidates. The dichloromethane (DCM) extract was selected to isolate over silica gel column chromatography to obtain different polar fractions. Several similar fractions were combined according to Thin Layer Chemotherapy (TLC) or High-Performance Liquid Chromatography (HPLC) analysis. The combined fractions were reisolated by silica gel column chromatography, preparative TLC or HPLC to obtain nine pure compounds (1-9). The purity of the isolated compounds was detected by HPLC, and their structures were determined by 1D, 2D NMR, and HRESIMS analysis. The in vitro anticancer activity was evaluated using Cell Counting Kit-8 (CCK8) method. Nine compounds were isolated in this work. Compounds (1-3) are new compounds, while compounds (4-9) were isolated for the first time from the roots of J. sambac. Their structures were elucidated by 1D, 2D NMR, and HRESIMS analysis. The biological evaluation showed that compound 7 exhibited potent cytotoxic efficacy against MCF-7 cell lines with IC50 values of 148.3 μM for 24 hs and 35.94 μM for 48 hs, respectively; compound 1 displayed significant cytotoxic potential against MCF-7 cell lines with IC50 value of 38.5 μM for 24 hs; while compound 3 and 4 displayed potent cytotoxic effects against MCF-7 cell lines with IC50 values of 161.1 μM and 243.7 μM for 48 hs, respectively. We discovered new compounds from the roots of J. sambac. and several compounds exhibited potent cytotoxity to MCF-7 cell lines. This work encourages us to further study the chemical constituents and their biological activities from the roots of J. sambac.

Characterization of structure and antioxidant activity of exopolysaccharides from endophytic Lysinibacillus sphaericus Ya6 under acid-base stress

In this study, the structure and antioxidant activity of exopolysaccharides (EPS) produced by Lysinibacillus sphaericus Ya6 under different acid-base stresses were investigated. The structure of EPS was analyzed by UV-visible and mid-IR, and the monosaccharide composition and the molecular weight were determined by High-performance liquid chromatography (HPLC) and high-performance gel permeation chromatography respectively. The findings indicated that EPS produced by Lysinibacillus sphaericus Ya6 was a homogenous polysaccharide with a molecular weight of 79.25 kDa that was composed of mannose and glucose. The molecular weight and monosaccharide composition of polysaccharides changed under acid-base stress conditions. With the addition of the acid-base stress level, the logarithmic growth period of Lysinibacillus sphaericus Ya6 was prolonged for a short while, but later growth rates and EPS production under stress conditions were all higher than under the control. In vitro determinations found that acid-base stress boosted the antioxidant activity of EPS. Particularly under conditions of alkaline stress, DPPH radical scavenging capacity and superoxide anion scavenging of EPS were higher than those of the control, and their capacities to scavenge DPPH was increased from 39.30% (the control) to 95.65% at pH 10.0, which was close to ascorbic acid's (95.77%) as a positive control.

Polypeptide from Moschus Suppresses Lipopolysaccharide-Induced Inflammation by Inhibiting NF-κ B-ROS/NLRP3 Pathway.

To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus (PPM) in lipopolysaccharide (LPS)-induced THP-1 macrophages and BALB/c mice. The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice. In LPS-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays; the proinflammatory cytokines and reactive oxygen species (ROS) were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively; and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR), respectively. In LPS-induced BALB/c mice, the proinflammatory cytokines were measured, and lung histology and cytokines were observed by hematoxylin and eosin (HE) and immunohistochemical (IHC) staining, respectively. The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10-26 kD. In vitro, PPM reduced the production of interleukin 1β (IL-1β), IL-18, tumor necrosis factor α (TNF-α), IL-6 and ROS in LPS-induced THP-1 macrophages (P<0.01). Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factor κB (NF-κB) pathway and thioredoxin interacting protein (TXNIP)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome pathway by reducing protein expression of phospho-NF-κB p65, phospho-inhibitors of NF-κB (Iκ Bs) kinase α/β (IKKα/β), TXNIP, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1 (P<0.05 or P<0.01). In addition, qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP, NLRP3, ASC, and caspase-1 (P<0.05 or P<0.01). Furthermore, in LPS-induced BALB/c mice, PPM reduced TNF-α and IL-6 levels in serum (P<0.05 or P<0.01), decreased IL-1β and IL-18 levels in the lungs (P<0.01) and alleviated pathological injury to the lungs. PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway, and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.

A New Galactoglucomannan from the Mycelium of the Medicinal Parasitic Fungus Cordyceps cicadae and Its Immunomodulatory Activity In Vitro and In Vivo.

A new galactoglucomannan (C-0-1) was purified from the medicinal parasitic fungus of Cordyceps cicadae using an anion-exchange column and gel permeation column. The results of high-performance liquid chromatography and high-performance gel permeation chromatography indicated that C-0-1 consists of galactose, glucose, and mannose in a ratio of 5:1:4 and has a molecular weight of 23.3 kDa. The combined structural elucidation analysis methods including partial acid hydrolysis, methylation analysis, and NMR experiments revealed that C-0-1 was a comb-like polysaccharide with a core structure including (1→2)-α-D-Manp residues in the backbone and branches at O-6 of the main chain. (1→4)-α-D-Glcp, (1→2)-β-D-Galf, (1→2,6)-β-D-Galf, and terminal β-Galf were located at the side chains. An in vitro experiment using RAW 264.7 cells indicated that C-0-1 exhibits good immunomodulatory activity by enhancing inducible nitric oxide synthase secretion and the production of some major inflammatory cytokines. On inhibiting the cytokine production using anti-pattern recognition receptors antibodies, it was revealed that the activation of macrophages is mainly carried out by C-0-1 through the mannose receptor. Toll-like receptor 4 and Toll-like receptor 2 were also involved in this identification process. An in vivo experiment on immunosuppressive mice treated with cyclophosphamide indicated that C-0-1 improves the secretion of serum-related cytokines (IFN-γ, TNF-α, IL-2, IL-4, and IL-10) and affects the balance of T helper cells Th1/Th2. Given the structural and bioactivity similarity between Cordyceps cicadae and Cordyceps sinensis, we can conclude that Cordyceps cicadae could be used as an important medicinal fungus like Cordyceps sinensis.

Codelivery of TGF-β1 and anti-miR-141 by PLGA microspheres inhibits progression of intervertebral disc degeneration

BackgroundCervical and lumbar pain is usually caused by degeneration of the nucleus pulposus (NP). As a powerful therapeutic strategy, tissue engineering can effectively restore the normal biological properties of the spinal unit. Previous studies suggested that poly(lactic-co-glycolic acid) (PLGA) microspheres are effective carriers of cells and biomolecules in NP tissue engineering. This study aims to explore the therapeutic effect of PLGA microspheres coloaded with transforming growth factor-β1 (TGF-β1) and anti-miR-141 on intervertebral disc degeneration (IDD).MethodsPLGA microspheres were characterized by scanning electron microscopy, a laser particle size analyzer, and laser confocal microscopy. The in vitro release rate of biomolecules from the microspheres was analyzed by reversed-phase high-performance liquid chromatography and agarose gel electrophoresis. The rat NP cells (NPCs) treated with the solutions released from microspheres for different lengths of time were assigned to a control group (Ctrl), an empty PLGA microsphere group (Mock microsphere, MS), a TGF-β1-loaded PLGA microsphere group (TMS), an anti-miR-141-loaded PLGA microsphere group (AMS), and an anti-miR-141 + TGF-β1-loaded PLGA microsphere group (ATMS). The proliferation and apoptosis of NPCs were observed by alamar blue and flow cytometry. The gene and protein expression of cartilage markers COL2A1 and ACAN were observed by RT-qPCR and Western blot. The rat model of IDD was established by tail puncture. Rats were divided into a control group (Ctrl), a mock operation group (Mock), a TGF-β1 microsphere group (TMS), an anti-miR-141 microsphere group (AMS), and an anti-miR-141 + TGF-β1 microsphere group (ATMS). The degree of rat tail IDD was assessed in each group through magnetic resonance imaging (MRI), safranin O-fast green staining, immunohistochemistry, and Western blotting.ResultsPLGA microspheres were stably coloaded and could sustainably release TGF-β1 and anti-miR-141. The results of in vitro cell experiments showed that the release solution of PLGA microspheres significantly enhanced the proliferation of NPCs without inducing their apoptosis and significantly upregulated cartilage markers in NPCs. The effect of microspheres was greater in the ATMS group than that in the TMS group and AMS group. In vivo experiments showed that IDD could be effectively inhibited and reversed by adding microspheres coloaded with TGF-β1 and/or anti-miR-141, and the effect was greatest in the ATMS group.ConclusionPLGA microspheres coloaded with TGF-β1 and anti-miR-141 can reverse IDD by inhibiting the degeneration of NPCs.

Peruranolides A-D, four new withanolides with potential antibacterial and cytotoxic activity from Physalis peruviana L.

Many drugs for anti-tumour have been developed, nevertheless, seeking new anticancer drug is the focus of ongoing investigation. Withanolides have been reported to possess potent antiproliferative activity. Literature findings revealed that a diversity of withanolides were obtained from Physalis peruviana, however, the antitumor activity of these bioactive compounds is still unclear. The EtOAc fraction of P. peruviana were decolorized on Middle Chromatogram Isolated (MCI) Gel column, repeatedly subjected to column chromatography (CC) over sephadex LH-20, preparative High Performance Liquid Chromatography (HPLC) and silica gel to afford compounds. Their chemical structures of the new isolates were elucidated through analyzing spectroscopic and HRESIMS data. All these obtained metabolites were appraised for their potential antiproliferative activity against the human breast cancer cell line MCF-7 by MTT assay, and in vitro antibacterial activity of the isolated compounds (1-7) were evaluated against E. coli, B. cereus and S. aureus. Results: Four new withanolides, including one withaphysalin-type withanolide (peruranolide A, 1), two 13,14-seco-withaphysalins (peruranolides B-C, 2-3), as well as one normal withanolide (peruranolide D, 4), were purified and separated from P. peruviana L.. Compound 5 was discovered to exhibit potent cytotoxic effect with an IC50 value of 3.51 μM. In vitro antibacterial activities, compounds 1-7 had no obvious inhibitory activity against E. coli, but had moderate inhibitory activities against B. cereus and S. aureus. Our findings might offer valuable clues for the utilization of withanolides as lead compounds for antineoplastic or antibacterial drug development.

Application of Polysaccharide-Based Chiral High-Performance Liquid Chromatography Columns for the Separation of Regio-, E/Z-, and Enantio-Isomeric Mixtures of Allylic Compounds.

Daicel Chiralpak IA, IB, and IC, which are the polysaccharide-based chiral stationary phase (CSP) columns for high-performance liquid chromatography (HPLC), were applied in the separation of the non-enantiomeric isomeric mixtures obtained by the various allylation reactions and were highly effective in separating the regio- and (E)/(Z)-isomers in the allylation products. Due to the close structural similarity of the isomeric allylic compounds in the reaction mixtures, separations of the isomers are laborious and could not be accomplished by the conventional methods such as silica gel column chromatography, silica gel HPLC, preparative GPC, distillation, and so forth. This study has shown potential advantages of using the polysaccharide-based CSP columns in the separation of not only enantiomeric but also non-enantiomeric isomeric mixtures.

Enzyme-Assisted Method for Phycobiliproteins Extraction from Porphyra and Evaluation of Their Bioactivity

Due to the poor protein availability of algae in their unprocessed form, development of extraction methods for phycobiliproteins is of great significance. This study aimed to extract phycoerythrin (PE) and phycocyanin (PC) from Porphyra via bacterial enzymatic hydrolysis and to evaluate their bioactivity. To induce enzyme production, Porphyra powder was added into the culture medium of two marine bacterial strains. The pH and enzyme activity of the cultured supernatant, namely crude enzyme solution, were significantly raised. For PE and PC extraction, Porphyra were incubated within crude enzyme solution with homogenization and ultrasonication followed by ultrafiltration process. After distinguishing by fast performance liquid chromatography (FPLC), three major fractions were observed and identified as R-PE, R-PC and small molecular PE by high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) analysis. With respect to bioactivity, these three fractions exhibited free radical scavenging and antioxidant activities in a various degree. In addition, the angiotensin-converting-enzyme (ACE) inhibitory activity of both R-PE and R-PC fractions was observed in a concentration-dependent manner. Taken together, the employed process of bacterial enzymatic hydrolysis is suggested to be a feasible method to obtain PE and PC from Porphyra without limiting their bioactivity.

ISOLATION OF POTENTIAL ANTIMICROBIAL METABOLITE FROM ENDOPHYTIC BACILLUS AMYLOLIQUEFACIENS DL06 OF CARNIVOROUS PLANT DROSERA BURMANNII VAHL.

Objectives: Exploitation of bacterial endophytes for production of antimicrobial substances has led to the discovery of novel natural metabolites of diverse chemical nature. The present study focuses attention toward optimization of cultural conditions for production of antimicrobial compound(s) by an endophytic bacterium DL06 followed by its extraction and partial purification.&#x0D; Methods: The leaf endophytic bacterium Bacillus amyloliquefaciens DL06 (GenBank Accession no. MK696415, Microbial Culture Collection Accession no. 4186) isolated from carnivorous plant Drosera burmannii has been identified as a potent producer of antimicrobial metabolite following agar cup assay against several test bacterial and fungal strains. Cultural conditions for production of antimicrobials were optimized by “one variable at a time” method. The active fraction was isolated and purified partially using solvent extraction, thin-layer chromatography, and high performance liquid chromatography (HPLC) analysis.&#x0D; Results: B. amyloliquefaciens DL06 produced maximum antimicrobial compound in tryptic soy broth and Davis–Mingioli’s medium when grown under shake culture. Production of the antimicrobial metabolite has been optimized for the inoculum density, aeration, temperature, pH as well as carbon, and nitrogen sources. The antimicrobial metabolite was extracted from the cell-free culture filtrate in butanol and partially purified by silica gel column chromatography and HPLC.&#x0D; Conclusions: The antimicrobial metabolite, tentatively identified as quercetin showed broad spectrum bioactivity affecting several fungi and a number of Gram-positive and Gram-negative bacteria.

Proteomics, toxicity and antivenom neutralization of Sri Lankan and Indian Russell's viper (Daboia russelii) venoms.

Background: The western Russell’s viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell’s viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity.Methods: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol.Results: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately.Conclusion: Considering that a large amount of venom can be injected by Russell’s viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.

Operational Laboratory Methods for GDGTs Groups Separation

Glycerol dialkyl glycerol tetraethers (GDGTs), specific membrane lipids synthesized mainly by bacteria and archaea, can be divided into isoprenoids and methyl branched alkyl GDGTs (iGDGTs and brGDGTs, respectively). Three GDGTs groups (iGDGTs, brGDGTs, and other membrane lipids) in a peat sample were separated and collected in this study using semi-preparative high-performance liquid chromatography (HPLC) and silica gel column chromatography. The obtained recoveries for the whole analytical procedure were 85%−55% using semi-preparative HPLC and 70%−20% using gel column chromatography. In addition, in each method, the recoveries of brGDGTs and iGDGTs were similar, regardless of the huge difference in their contents. High purity was found in the fractionated groups, determined based on ether cleavage and reduction. Moreover, the semi-preparative HPLC method could realize a better separation efficiency than the silica gel method, but it was time-consuming and required expensive equipment, while the silica gel chromatography method featured merits of time saving and convenient operation at the cost of a slight reduction in separation efficiency. The advantages of the silica gel method make it an operational laboratory method for batch experiments and isotopic studies.

Detection of Haemoglobin S using Multiplex Ligation-Dependent Probe Amplification and Flow-through Hybridization Techniques: Experience in a Tertiary Hospital

Haemoglobin S (HbS, α2β26GluVal) is a variant haemoglobin resulted from GAGGTG mutation on codon 6 of HBB gene. HbS haemoglobinopathy is uncommon in Malaysia and mainly seen in immigrants. However, Malaysian Indians and Malays are rarely affected. This study reviewed the laboratory findings of patients with HbS and the utilization of multiplex ligation-dependent probe amplification (MLPA) and flow-through hybridization (FTH) in the detection of HbS mutation. HbS was identified and quantified by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and cellulose acetate gel electrophoresis. Molecular analysis was performed using MLPA, FTH and Sanger Sequencing. Two Africans, three Malays and two Indian individuals aged between 2-31 years were identified from our laboratory. Five patients were homozygous HbS, one was compound heterozygous HbS/β-thalassemia and one was a carrier of HbS. The patients with homozygous HbS had their haemoglobin (Hb) ranging from 7.4-10.2 g/dL with HbS and HbF levels of 58.3-94.7% and 1.5-35.5%, respectively. The Hb of compound heterozygous HbS/β-thalassaemia patients was 5.8 g/dL and was normal in heterozygous HbS. HbS, HbF and HbA2 levels for the HbS/β-thalassaemia and the carrier were 67%, 27.2% and 4.2%, and 38.6%, 0.1% and 2.8%, respectively. Both MLPA and FTH successfully detected HbS mutation in all cases but only FTH could identify the zygosity of the HbS mutation together with underlying concomitant β-thalassaemia in a single test.

Proteomics and antivenom immunoprofiling of Russell's viper (Daboia siamensis) venoms from Thailand and Indonesia.

BackgroundThe Eastern Russell’s viper, Daboia siamensis, is a WHO Category 1 medically important venomous snake. It has a wide but disjunct distribution in Southeast Asia. The specific antivenom, D. siamensis Monovalent Antivenom (DsMAV-Thailand) is produced in Thailand but not available in Indonesia, where a heterologous trivalent antivenom, Serum Anti Bisa Ular (SABU), is used instead. This study aimed to investigate the geographical venom variation of D. siamensis from Thailand (Ds-Thailand) and Indonesia (Ds-Indonesia), and the immunorecognition of the venom proteins by antivenoms.Methods: The venom proteins were decomplexed with reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by in-solution tryptic digestion, nano-liquid chromatography-tandem mass spectrometry and protein identification. The efficacies of DsMAV-Thailand and SABU in binding the various venom fractions were assessed using an enzyme-linked immunosorbent assay optimized for immunorecognition profiling. Results: The two most abundant protein families in Ds-Thailand venom are phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitor (KSPI). Those abundant in Ds-Indonesia venom are PLA2 and serine protease. KSPI and vascular endothelial growth factor were detected in Ds-Thailand venom, whereas L-amino acid oxidase and disintegrin were present in Ds-Indonesia venom. Common proteins shared between the two included snaclecs, serine proteases, metalloproteinases, phosphodiesterases, 5’nucleotidases and nerve growth factors at varying abundances. DsMAV-Thailand exhibited strong immunorecognition of the major protein fractions in both venoms, but low immunoreactivity toward the low molecular weight proteins e.g. KSPI and disintegrins. On the other hand, SABU was virtually ineffective in binding all fractionated venom proteins. Conclusion: D. siamensis venoms from Thailand and Indonesia varied geographically in the protein subtypes and abundances. The venoms, nevertheless, shared conserved antigenicity that allowed effective immunorecognition by DsMAV-Thailand but not by SABU, consistent with the neutralization efficacy of the antivenoms. A specific, appropriate antivenom is needed in Indonesia to treat Russell’s viper envenomation.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells.

Background: Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. Methods: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 μg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate).Results: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. Conclusion: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.

Solubility variation of wheat dough proteins: A practical way to track protein behaviors in dough processing

To understand wheat dough protein behavior under dual mixing and thermal treatment, solubility of Mixolab-dough proteins were investigated using nine extraction buffers of different dissociation capacities. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) demonstrated that overall changes of protein fractions and dynamic responses of specific proteins during dough processing were well reflected by their solubility variations. After starch pasting, the abundance of 0.5 M NaCl extractable proteins were decreased except for six protein groups including α-amylase inhibitors and superoxide dismutase (SOD). The solubility loss of glutenin proteins at C3 (32 min; 80 ℃) was mainly ascribed to the un-extractable HMW-GSs, LMW-GSs, globulin and triticin, while the extract yield of α-, β-, γ-gliadins and avenin-like proteins (ALPs) increased after starch pasting. Differential responses of dough proteins to extraction systems provides the basis for further exploring wheat protein dynamics in processing.

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

Improvement in emulsifying properties of whey protein–Rhamnolipid conjugates through short-time heat treatment

Whey protein–rhamnolipid (WP–rhamnolipid) conjugates were prepared via Maillard reaction by heating in liquid systems. Protein-saccharide conjugates generally exhibit better technical functional properties. To optimize the reaction conditions, mixtures of whey protein and rhamnolipid at varying weight ratios, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8, were heated at 80 ℃ for 10, 20, 40 and 60 min. Emulsifying properties, zeta potential and interfacial activity of the conjugates were evaluated, and results showed that conjugates prepared in some conditions showed significantly improved emulsifying activity, the 1:1 conjugates heated for 10 min had the highest emulsifying activity; the zeta potential decreased significantly in the early stage of heat treatment and then stabilized, indicating that the colloidal stability and electrostatic repulsion were enhanced; an increase in the rhamnolipid component could increase the interfacial activity of conjugates. The formation of conjugates and changes in reaction process were studied by browning value, free amino analysis, fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). WP–rhamnolipid conjugates formed by short-time heat treatment have great potential as highly efficient bioemulsifiers.

Wheat storage proteins: changes on the glutenins after wheat infection with different isolates of Fusarium graminearum.

Wheat gluten proteins are decisive for the industrial properties of flour, so alterations resulting from grain infection with Fusarium graminearum produce changes in the glutenin content that affect the baking properties. This work analyzes the high-molecular-weight glutenin changes from wheat flour with different degrees of F. graminearum infection at field, since these proteins are determinant for the quality properties of flour. Wheat cultivars-on field trials-infected with F. graminearum isolates of diverse aggressiveness showed severity values between 9.1 and 42.58% and thousand kernel weight values between 28.12 and 32.33g. Negative correlations between severity and protein content and positive correlations between yield and protein content were observed, employing reversed-phase high-performance liquid chromatography and polyacrylamide gel electrophoresis. Furthermore, the protein signal changes were in agreement for both methodological approaches. Also, the degree of disease observed and the protein changes on infected wheat cultivars varied in relation with the aggressiveness of the isolate responsible for the infection. The principal component analysis showed a close arrangement among protein values obtained by HPLC. For each cultivar, two principal components were obtained, which explained 80.85%, 88.48%, and 93.33% of the total variance (cultivars Sy200, AGP Fast, and Klein Tigre respectively). To our knowledge, the approaches employed for the analysis of protein changes according to the degree of disease, as well as the thorough statistical analysis, are novel for the study of Fusarium Head Blight.