Pellet Fraction Research Articles

Structural features and anticancer activity in vitro of fucoidan derivatives from brown alga Saccharina cichorioides

A fucoidan ScF from brown alga Saccharina cichorioides was extracted, purified and partially depolymerized by autohydrolysis at 37°C for 24, 48 and 72h. Supernatant (SN) and pellet (PL) fractions were obtained by ethanol precipitation of each sample. Unlike spectral data of ScF, NMR of PL derivatives clearly suggested the structure: 1,3-linked α-l-Fucp-4-OSO3− repeating unit. Molecular weights (MWs) of PL fractions were 30, 26 and 18kDa for 24, 48 and 72h of autohydrolyis, respectively. MALDI-TOFMS, size-exclusion HPLC and carbohydrate polyacrylamide-gel electrophoresis (C-PAGE) indicated a similarity of SN mixtures. They consisted mainly of a polysaccharide part (MW 6kDa, C-PAGE data) with a structure similar to PL components (NMR data) and monosaccharides α-l-Fucp-4-OSO3−, α-l-Fucp-2,4-di-OSO3−. PL fractions exhibited almost identical antiproliferative activity in vitro as native fucoidan, while an SN sample for 72h of autohydrolysis was slightly more active against colony formation of colorectal carcinoma cells HT-29.

Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions

Background: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes.Objective: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs).Methods: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light scattering, quantitative polymerase chain reaction, and density measurements.Results: A large quantity of dairy milk Bos taurus microRNA-223 (bta-miR-223) and bta-miR-125b (∼109–1010 copies/300 mL milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important decrease in microRNA amounts. A large quantity of these 2 microRNAs (∼108–109 copies/300 mL milk) was detected in the upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 × g pellet fraction that was positive for the exosomal markers tumor susceptibility gene-101 (TSG101), apoptosis-linked gene 2–interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of ALIX−, HSP70−/low, and TSG101−/low EVs larger than exosomes and 2–6 times more enriched in bta-miR-223 and bta-miR-125b (P < 0.05).Conclusions: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist digestion and are associated mostly with ALIX−, HSP70−/low, and TSG101−/low EVs. Our results support the existence of interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the sole carriers of milk-derived microRNAs.

Aggregation of soy protein-isoflavone complexes and gel formation induced by glucono-δ-lactone in soymilk.

This study investigated the glucono-δ-lactone (GDL)-induced aggregation of isoflavones and soy proteins in soymilk. High-performance liquid chromatography (HPLC) analysis indicated that isoflavones mixed with β-conglycinin (7S) and glycinin (11S) proteins formed 7S-isoflavone and 11S-isoflavone complexes in soymilk supernatant fraction (SSF). Most of the soy protein-isoflavone complexes then precipitated into the soymilk pellet fraction (SPF) following the addition of 4 mM GDL, whereupon the pH value of the soymilk dropped from 6.6 to 5.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC analysis suggest that the addition of 4 mM GDL induced the aggregation of most 7S (α’, α and β subunits), 11S acidic and 11S basic proteins as well as isoflavones, including most aglycones, including daidzein, glycitein, genistein and a portion of glucosides, including daidzin, glycitin, genistin, malonyldaidzin and malonylgenistin. These results provide an important reference pertaining to the effects of GDL on the aggregation of soy protein-isoflavone complexes and could benefit future research regarding the production of tofu from soymilk.

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Coupling of gel-based 2-DE and 1-DE shotgun proteomics approaches to dig deep into the leaf senescence proteome of Glycine max

Leaf senescence is the last stage of leaf development that re-mobilizes nutrients from the source to sink. Here, we have utilized the soybean as a model system to unravel senescence-associated proteins (SAPs). A comparative proteomics approach was used at two contrasting stages of leaf development, namely mature (R3) and senescent (R7). Selection criteria for these two stages were the contrasting differences in their biochemical parameters – chlorophyll, carotenoids and malondialdehyde contents. Proteome analysis involved subjecting the total leaf proteins to 15% poly-ethylene glycol (PEG) pre-fractional method to enrich the low-abundance proteins (LAPs) and their analyses by gel-based 2-DE and 1-DE shotgun proteomics approaches. 2-DE profiling of PEG-supernatant and -pellet fractions detected 153 differential spots between R3 and R7 stages, of which 102 proteins were identified. In parallel, 1-DE shotgun proteomics approach identified 598 and 534 proteins in supernatant and pellet fractions of R3 and R7 stages, respectively. MapMan and Gene Ontology analyses showed increased abundance and/or specific accumulation of proteins related to jasmonic acid biosynthesis and defense, while proteins associated with photosynthesis and ROS-detoxification were decreased during leaf senescence. These findings and the generated datasets further our understanding on leaf senescence at protein level, providing a resource for the scientific community. Biological significanceLeaf senescence is a major biological event in the life cycle of plants that leads to the recycling of nutrients. However, the molecular mechanisms underlying leaf senescence still remain poorly understood. Here, we used a combination of gel-based 2-DE and 1-DE shotgun proteomics approaches to dig deeper into the leaf senescence proteome using soybean leaf as a model experimental material. For the identification of low-abundance proteins, polyethylene glycol (PEG) fractionation was employed and both PEG-supernatant and -pellet fractions were utilized for 2-DE and shotgun proteomic analysis. A total of 1234 (102 from 2-DE and 1132 from 1-DE shotgun proteome analysis) proteins were identified which were functionally annotated using GO and MapMan bioinformatics tools. Our results also emphasize the role of jasmonic acid in soybean leaf senescence.

Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.

Comparison of initial stream urine samples and cervical samples for detection of human papillomavirus.

Uterine cervical cancer is a treatable and preventable cancer. Medical efforts to reduce rates of cervical cancer focus on the promotion of human papillomavirus (HPV) vaccination and the promotion of routine cervical cancer screening done by cervical cytology and cervical HPV testing. Urine-based HPV testing would be simple and noninvasive approach to screen for cervical cancer. Two biospecimens (clinician-taken sample from cervix and initial stream urine sample) were provided from a total of 240 healthy women attending for cancer screening provided for HPV testing. We have assessed the HPV detection rates among cervical samples and pellet fraction of urine samples using HPV test (Anyplex™ II HPV28 Detection kit, Seegene, Korea). Among 240 samples screened, HPV prevalence was 42.9% in pellet fractions of urine samples. The agreement between the two kinds of samples was 98.4%, k=0.792. Discordant results were observed in 27 cases; 5 were positive only by urine samples and 22 were positive only by smear samples. Sensitivity and specificity for all HPV DNA in pellet fractions of urine using cervical samples as reference was 68.4% and 99.9%. Comparing methodologies of collection of samples for HPV detection, they showed the higher agreements for almost genotypes between cervical samples and pellet fractions of urine samples. These results suggest that urine could be a good noninvasive tool to monitor HPV infection in women. Additional research in a larger and general screening population would be needed.

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC-eseE Operon.

Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ΔeseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.

The common G60G variant (c.180C > T) of CTRC is associated with increased risk of chronic pancreatitis in children

s / Pancreatology 16 (2016) S1eS130 S6 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Norway Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital of Pittsburgh of UPMC, Pit, United States Introduction: We have previously identified disease-associated mutations in the last exon of the CEL gene, localized in a variable number of tandem repeats (VNTR) domain. Two single base deletions (DEL1 and DEL4) lead to frameshifts, predicting a new C-terminus of the CEL protein. Patients with these mutations suffer from exocrine pancreatic dysfunction and diabetes. Aims: To understand the role of the C-terminal domain of CEL in human pancreatic disease by investigating expression, secretion and intracellular localization of different protein-variants in cellular models. Materials & methods: cDNAs of CEL-wild-type, CEL-DEL1, CEL-DEL4 and CEL-TRUNC (lacking the VNTR) were cloned into pcDNA3.1 vectors, with or without epitope tags (V5/His), and transfected into HEK293 cells. The expressed CEL-proteins were studied by Western-blotting, immunostaining and confocal imaging. Results: Forty-eight hours post-transfection all CEL-variants were detectable in the lysate, pellet and medium fractions of HEK293 cells. The amount of CEL protein in each fraction varied between variants and also depended upon the presence of epitope tags. Comparing with V5/Histagged protein variants, we observed a higher amount of untagged proteins in the pellet and less in the medium. Significantly more of the disease-causing CEL-variants were detected in the pellet as compared with the wild-type protein. When co-expressing FLAGand V5/His-tagged CELvariants, we observed that CEL-WT could be detected in the pellet fraction together with CEL-DEL1. Conclusion: The cellular fate of CEL protein variants is highly influenced by the C-terminus of the protein. Epitope tags may cause increased solubility of disease-causing CEL variants, leading to underestimation of their propensity to aggregate in cellular model systems. Abstract ID: 1346, Oral-20.ID: 1346, Oral-20. The common G60G variant (c.180C > T) of CTRC is associated with increased risk of chronic pancreatitis in children Agnieszka Magdalena Rygiel , Alicja Monika Grabarczyk , Grzegorz Oracz , Aleksandra Kujko , Katarzyna Wertheim-Tysarowska , Elwira Kolodziejczyk , Dorota Koziel , Artur Kowalik , Stanislaw Gluszek , Jerzy Bal 1 Department of Medical Genetics, Institute of Mother and Child Warsaw, Poland Department of Gastroenterology, Hepatology and Feeding Disorders, The Children's Memorial Health Institute, Warsaw, Poland Department of Surgery and Surgical Nursing, Jan Kochanowski University of Kielce, Poland Department of Molecular Diagnostics, Oncology Center of the Holy Cross, Kielce, Poland Introduction: Loss-of-function variants in chymotripsin C (CTRC) increase the risk for chronic pancreatitis (CP). The most commonly found pathogenicmutations of CTRC include K247_R254del, and R254W but their frequencies are low. Studies with adults indicated the association of G60G (c.180C>T) common variant of CTRC with CP but the available data are limited and obscure. Aims: To evaluate the risk of CP associated with CTRC mutations and G60G variant in case (CP children)-control study. Patients & methods: We sequenced exones 2-7 of the CTRC in 136 children including idiopathic CP (ICP, n1⁄461) and CP with various etiological factors (n1⁄475).Children (median age at onset 8 years) with CP verified by imagingmethods were included. Control group (n1⁄4401, median age 45) of healthy volunteers was screened by HRM-PCR and detected mutations were confirmed by direct sequencing. Results: In CP, we identified R254W(4,5%) and K247_R254del (4,5%) mutations, and G60G variant (c.180 CT or TT, 49%). Compared with controls, CTRC mutations are associated with 7 times increased risk of CP (9% vs.1.2%, OR1⁄47.95% CL:2.5-19.7, P1⁄48,6*10-6). The c.180 CT and TT genotype is associated with 1,8 (OR1⁄4 1.8; 95% CL:1.3-3.2) and 15 (OR1⁄414.7;95% CL:5.1-42.3) times increased risk of CP with frequencies of 35% vs.18% (P1⁄43,5*10-4) and 15% vs.0.9% (P1⁄41,5*10-9), respectively. This association was also significant when ICP and control group were compared (c.180 CT: OR1⁄41,9; 95% CL (1.28-2.8), P1⁄40,0039 and c.180 TT: OR1⁄414.8;95% CL: 4.6-46.5, P1⁄43,2*10-6). Conclusion: We showed, for the first time that G60G variant of CTRC, especially c.180 TT genotype is strongly associated with idiopathic CP in children.

Highly potent intracellular membrane-associated Aβ seeds.

An early event in Alzheimer’s disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ.

Bio-refining of perennial ryegrass (Lolium perenne): evaluation of aqueous extracts for plant defence elicitor activity using French bean cell suspension cultures

There is growing interest in the bio-refining of foliage grasses to yield a range of industrial raw products. The aim of this research was to evaluate if aqueous extracts from grasses have the potential to act as crop bio-stimulants by assessing their defence elicitor activity. Field-grown foliage (500 g fresh weight) from the Lolium perenne varieties Tyrella, Dromara, Abermagic, and Spelga were homogenised in water (2 l) and the run-off liquid filtered through Whatman No. 1 filter paper before freezing. Thawed filtrate was centrifuged at 21,000 rpm for 15 min, to give pellet and supernatant fractions, which were assayed separately for defence elicitor activity by adding them to cell suspension cultures of Phaseolus vulgaris in the early exponential phase of growth (7–10 days into a 14 days culture passage). Elicitor activity was evaluated 24 h after treatment by assessing the extent of necrosis (browning) in the P. vulgaris cells. Supernatant and pellet fractions of all four L. perenne varieties were found to have potent cell defence elicitor activity, although there were relatively small but significant differences between the varieties. In a second series of extractions, homogenisation of tissues in water was compared with screw-pressed juice using the varieties, Malone, Seagoe, and Copeland. Varietal differences in elicitor activity of these extracts were removed if they were first equalised for protein content prior to eliciting the bean cells, although the screw-pressed juice was marginally more active as an elicitor than the homogenised extract. Autoclaving the extracts in attempts to solubilise/release additional elicitor compounds neither increased nor decreased the elicitor activities of the supernatant or pellet fractions. Elicitor activity of the extract was partially reduced by treating with a cation exchange resin, almost entirely removed by adsorption onto activated carbon and completely abolished by ashing at 650 °C. The L. perenne defence elicitor(s) is therefore organic rather than inorganic, heat-stable, cationic at neutral pH, and readily soluble in water at room temperature.

Dairy milk microRNAs resist in vitro digestion in a gastro‐intestinal tract model

MicroRNAs are small, gene regulatory non‐coding RNA species that are present in large amounts in milk where they seem to be protected against degradative conditions, presumably because of their association with exosomes. We monitored the relative stability of commercial dairy milk microRNAs, under the gastrointestinal (GI) tract conditions that prevails during digestion, and examined their association with extracellular vesicles (EVs). To do so, we used a computer‐controlled TNO in vitro GI model (TIM‐1) and analyzed, by quantitative PCR (qPCR), the level of two milk microRNAs within all GI tract compartments at different time points. MicroRNA‐containing EVs within digested and non‐digested samples were studied by immunoblotting, dynamic light scattering, qPCR and density measurements. We observed that an important proportion of dairy milk bta‐miR‐223 and bta‐miR‐125b withstand digestion under simulated GI tract conditions, with the stomach causing the greater loss in microRNAs. A significant amount of these two microRNAs (~108–109 copies/300 ml milk) was detected in the upper small intestine compartments, which supports their bioavailability. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 g pellet fraction positive for the exosomal marker Tumor susceptibility gene‐101 (TSG‐101) and containing bta‐miR‐223 and bta‐miR‐125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of TSG‐101 negative EVs that are larger than exosomes and more enriched in bta‐miR‐223 and bta‐miR‐125b. Our findings indicate that commercial dairy milk contains numerous microRNAs that can resist digestion and are associated mostly with non‐exosomal EVs. Our results strengthen the idea that milk microRNAs are bioactive compounds in dairy milk, support the existence of interspecies transfer of microRNAs based on milk consumption, and challenge our current view of exosomes as the sole carriers of milk‐derived microRNAs.Support or Funding InformationThis work was supported by Grants No. 319618 and 327522 from the Canadian Institutes of Health Research (CIHR), through the Institute of Genetics (to P.P.).

&lt;em&gt;In Vitro&lt;/em&gt; Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

16K Fractionation of 3T3-L1 Adipocytes to Produce a Crude GLUT4-Containing Vesicle Fraction.

The insulin-sensitive pool of glucose transporter isoform 4 (GLUT4) can be isolated from total cell membranes using the 16K fractionation protocol, described here. This method produces a light membrane-containing supernatant that includes the insulin-sensitive pool of GLUT4 in GLUT4 storage vesicles. The 16K pellet fraction contains the heavy membranes (including the plasma membrane, mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum). The distribution of proteins between the two fractions is determined via immunoblotting. By subjecting insulin-stimulated versus unstimulated cells to this protocol, the mobilization of proteins out of the insulin-sensitive GLUT4 pool can be assessed.

Group B streptococcus alters properties of vaginal epithelial cells in pregnant women

Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage.

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.

During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.

Selective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8

ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.

Protein identification and in vitro digestion of fractions from Tenebrio molitor

The nutritional value of insect protein is evaluated not only in amino acid composition, but also in protein digestibility. The general amino acid composition of Tenebrio molitor has been reported before, but limited knowledge is available on its digestibility. The objective of this study was to investigate in vitro protein digestibility of whole T. molitor larvae, a water-soluble fraction (supernatant) and water-insoluble fractions (pellet and residue), and to identify which proteins were present in the fractions studied. The digestibility of the supernatant fraction (~80 %) was much higher than that of pellet (~50 %) and residue (~24 %) after in vitro gastroduodenal digestion as was determined using the o-phthaldialdehyde (OPA) method. More proteins were digested after pepsin/pancreatin digestion than after only pepsin digestion. The most abundant proteins in the supernatant were hemolymph protein (~12 kDa), alpha-amylase (~50 kDa, a putative allergen), and muscle proteins (e.g. actin 30–50 kDa) in the pellet fraction as determined from LC–MS/MS and SDS-PAGE. In conclusion, the proteins in the soluble fraction that contained hemolymph proteins were more easily digestible than the insoluble, muscle protein-containing fractions.

Coacervation of β-conglycinin, glycinin and isoflavones induced by propylene glycol alginate in heated soymilk

This study investigated the propylene glycol alginate (PGA)-induced coacervation of β-conglycinin (7S), glycinin (11S) and isoflavones in heated soymilk. The addition of 0.9% PGA caused 7S, 11S, daidzein and genistein to coacervate following a 1h incubation period. SDS–PAGE showed that the protein bands corresponding to the 7S α′, 7S α, 7S β, 11S A3, and 11S acidic subunits and the 11S basic proteins in the soymilk supernatant fraction (SSF) decreased to 37.7±12.7%, 24.7±3.9%, 4.9±1.8%, 8.5±2.7%, 18.1±1.8% and 6.0±1.6%, respectively. In addition, isoflavones including daidzein and genistein were also coacervated from the SSF into the soymilk pellet fraction (SPF) following incubation with 0.9% PGA for 1h. The amounts of daidzein and genistein in the SSF decreased to 8.6±1.6% and 2.0±1.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound to the 7S and 11S proteins. These results suggested that daidzein and genistein were co-precipitated with the 7S and 11S proteins into the SPF by 0.9% PGA. Our results demonstrated that PGA is a potent coagulant for the coacervation of 7S, 11S, daidzein and genistein.

REFOLDING PROTEIN, NIES39_A07830 FROM Arthrospira platensis AND ITS L-ASPARAGINASE ACTIVITY

Ponte Academic JournalDec 2016, Volume 72, Issue 12 REFOLDING PROTEIN, NIES39_A07830 FROM Arthrospira platensis AND ITS L-ASPARAGINASE ACTIVITYAuthor(s): Asep A. Prihanto ,Happy Nursyam, Center for Coastal and marine Studies, Institute of Community Service, Brawijaya UniversityJ. Ponte - Dec 2016 - Volume 72 - Issue 12 doi: 10.21506/j.ponte.2016.12.14 Abstract:L-Asparaginase (E.C. 3.5.1.1) is an amidohydrolase enzyme which catalyze L-asparagine to L-aspartate and generate ammonia. This enzyme has an important application on medicine and food processing aid. In this research, L-asparaginase from food grade microorganism, Arthrospira platensis was cloned and expressed in Escherichia coli system. The expression of cloned gene resulted in the inclusion body form. It was found that deduced protein was located and accumulated in pellet fraction, inclusion bodies. Hence, to achieve active form of the enzyme, it need to be correctly folded with steps as follow solubilization and refolding. Here, we report the recovering activity of the enzyme throughout our refolding process. Several denaturant agents were used to completely denature a protein from inclusion body. Urea, triton-X 100, and sarkosyl are able to denature the protein. The refolding process was conducted by dilution and dialysis methods. After experienced refolding process, L-asparaginase activity was detected. The highest activity was achieved from dialysis methods (64 U). We, therefore, reported the activity in the refolded enzyme even though the process needs to be optimized. Download full text:Check if you have access through your login credentials or your institution Username Password