To see the other types of publications on this topic, follow the link: Selective isolation and concentration of proteins.
Journal articles on the topic 'Selective isolation and concentration of proteins'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Selective isolation and concentration of proteins.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Stratford, C. A., and S. S. Brown. "Isolation of an actin-binding protein from membranes of Dictyostelium discoideum." Journal of Cell Biology 100, no. 3 (March 1, 1985): 727–35. http://dx.doi.org/10.1083/jcb.100.3.727.
Full textAbstract:
We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.
2
Bouvier, M., S. D. Miszczycha, X. Guillarme, C. Tadla, L. Collinet, and D. Thevenot Sergentet. "Comparative Evaluation of a Novel Phage Protein Ligand Assay and Immunomagnetic Separation Method To Isolate the Seven Top Serogroups of Escherichia coli (O157, O26, O103, O145, O111, O45, and O121) in Foods at Risk." Journal of Food Protection 80, no. 12 (November 13, 2017): 1973–79. http://dx.doi.org/10.4315/0362-028x.jfp-16-479.
Full textAbstract:
ABSTRACT The presence of Shiga toxin–producing Escherichia coli (STEC) in food is a major concern for food safety authorities and industries. Methods for detecting these pathogenic bacteria are crucial. Enrichment of foods for STEC identification has been optimized, but selective concentration of bacteria before isolation still needs to be improved. In the present study, we tested the performance of the VIDAS ESPT detection method against that of the immunomagnetic separation (IMS) method. A preenrichment inoculation was performed to provide a realistic scenario of the contamination that occurs in foods, and the methods were then compared. Results obtained were then confirmed in naturally contaminated foods. Preenrichment inoculation assays revealed that the novel concentration method using phage recombinant proteins or the selective capture of the target top seven STEC serogroups is as specific and sensitive as IMS. Subsequent evaluation of naturally contaminated samples confirmed that the novel concentration method and IMS are equivalent in performance under the conditions tested.
3
Athauda, Senarath B. P., Katsuji Yoshioka, Tadayoshi Shiba, and Kenji Takahashi. "Isolation and characterization of recombinant Drosophila Copia aspartic proteinase." Biochemical Journal 399, no. 3 (October 13, 2006): 535–42. http://dx.doi.org/10.1042/bj20060800.
Full textAbstract:
The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15−–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.
4
Haddad, F., R. R. Roy, H. Zhong, V. R. Edgerton, and K. M. Baldwin. "Atrophy responses to muscle inactivity. I. Cellular markers of protein deficits." Journal of Applied Physiology 95, no. 2 (August 2003): 781–90. http://dx.doi.org/10.1152/japplphysiol.00317.2003.
Full textAbstract:
The goal of this study was to use the model of spinal cord isolation (SI), which blocks nearly all neuromuscular activity while leaving the motoneuron muscle-fiber connections intact, to characterize the cellular processes linked to marked muscle atrophy. Rats randomly assigned to normal control and SI groups were studied at 0, 2, 4, 8, and 15 days after SI surgery. The slow soleus muscle atrophied by ∼50%, with the greatest degree of loss occurring during the first 8 days. Throughout the SI duration, muscle protein concentration was maintained at the control level, whereas myofibrillar protein concentration steadily decreased between 4 and 15 days of SI, and this was associated with a 50% decrease in myosin heavy chain (MHC) normalized to total protein. Actin relative to the total protein was maintained at the control level. Marked reductions occurred in total RNA and DNA content and in total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These findings suggest that two key factors contributing to the muscle atrophy in the SI model are 1) a reduction in ribosomal RNA that is consistent with a reduction in protein translational capacity, and 2) insufficient mRNA substrate for translating key sarcomeric proteins comprising the myofibril fraction, such as MHC and actin. In addition, the marked selective depletion of MHC protein in the muscles of SI rats suggests that this protein is more vulnerable to inactivity than actin protein. This selective MHC loss could be a major contributor for the previously reported loss in the functional integrity of SI muscles. Collectively, these data are consistent with the involvement of pretranslational and translational processes in muscle atrophy due to SI.
5
Farhana, Mashuri Nurul, Huey Ling Tan, Ying Pei Lim, and Siti Noor Suzila Maqsood-Ul-Haque. "Isolation of Antimicrobial Peptide from Food Protein Hydrolysates: An Overview." Key Engineering Materials 797 (March 2019): 168–76. http://dx.doi.org/10.4028/www.scientific.net/kem.797.168.
Full textAbstract:
Antimicrobial resistance (AMR) is one of the most serious public health threats that results mostly from the selective pressure exerted by antibiotic use and abuse. AMR has become a major problem with global human deaths due to antibiotic resistant infections predicted to reach 10 million by 2050. Antimicrobial peptides (AMPs) has been discovered to have capabilities to kill microorganisms and can take other roles as an alternative to antibiotics which is favorable to the need of minimizing the usage of antibiotics as they lead to the increasing of AMR. AMP can be found naturally in almost all domains of life as part of the innate immune system to combat virus, bacteria, fungi and even cancer cells. It can also be extracted from food proteins using enzymatic hydrolysis. High antimicrobial properties/activities are depend on the degree of hydrolysis (DH) of peptides, peptide source/origin, and type of enzyme used for the hydrolysis process. There are several other variables that can be manipulated to optimum condition to obtain high DH. Variables such as temperature, pH, enzyme concentration and hydrolysis time have proven to bring impact to the DH of peptides. Other bioactive peptides that have been discovered during the process have great potential to bring benefit in medicinal and nutraceutical areas.
6
Sjolin, C., and C. Dahlgren. "Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI." Blood 87, no. 11 (June 1, 1996): 4817–23. http://dx.doi.org/10.1182/blood.v87.11.4817.bloodjournal87114817.
Full textAbstract:
Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti- cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.
7
Platt, R. M., G. G. Geesey, J. D. Davis, and D. C. White. "Isolation and partial chemical analysis of firmly bound exopolysaccharide from adherent cells of a freshwater sediment bacterium." Canadian Journal of Microbiology 31, no. 8 (August 1, 1985): 675–80. http://dx.doi.org/10.1139/m85-128.
Full textAbstract:
Cells of a freshwater sediment bacterium produced firmly bound extracellular polymers in laboratory cultures which, at the ultrastructural level, resembled those produced by natural sediment bacterial populations. Production of the exopolymers during subculture was maintained by using as a source of inoculum the population of cells which adhered to each other and to the wall of the glass culture vessel. The exopolymers were selectively released from the cells by blending and centrifugation in the presence of EDTA. Evaluation of glucose-6-phosphate dehydrogenase activity and 2-keto-3-deoxyoctonate concentration indicated that only small amounts of intracellular and cell wall components were released from the cells during exopolymer removal. Chemical analysis of the isolated crude exopolymer material indicated that it contained protein, polysaccharide, and DNA. The treatment promoted the selective isolation of firmly bound polymers from the surface of adherent cells.
8
Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918.
Full textAbstract:
The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
9
Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918-1924.1985.
Full textAbstract:
The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
10
Balmayor, Elizabeth R., Iva Pashkuleva, Ana M. Frias, Helena S. Azevedo, and Rui L. Reis. "Synthesis and functionalization of superparamagnetic poly- ɛ -caprolactone microparticles for the selective isolation of subpopulations of human adipose-derived stem cells." Journal of The Royal Society Interface 8, no. 59 (January 5, 2011): 896–908. http://dx.doi.org/10.1098/rsif.2010.0531.
Full textAbstract:
There has been a growing interest in using biofunctionalized magnetic particles for cell isolation. This paper describes the synthesis and characterization of magnetite-polymer (Fe 3 O 4 -poly- ɛ -caprolactone, magnetite-PCL) microparticles surface functionalized with amino and epoxy groups allowing easy covalent attachment of specific antibodies and subsequent ability to bind target cells. Particles with different sizes (4–135 µm), spherical shape and superparamagnetic behaviour (magnetite content of about 13 wt%) were obtained. The functionalized microparticles presented high protein-binding capacity (coupling efficiency of 47% for epoxy- and 71% for amino-functionalized particles) with a low level of non-specific binding. We have further investigated the influence of initial protein concentration, pH, ionic strength, temperature and incubation time on the capacity of amino-functionalized particles to bind protein molecules. The results showed that maximum protein coupling is rapidly achieved (≤5 h) at pH 5.5 and low ionic strength (0.05 M NaCl). Furthermore, when cultured in direct contact with osteoblast-like cells (Saos-2) or human-derived adipose stem cells (ASCs), the amino-functionalized particles did not affect the proliferation and morphology of the cells. As a proof of principle for the application of magnetic microparticles for cell isolation, CD105 (endoglin) antibody was coupled to the magnetic particle surface to bind subpopulations of human ASCs expressing the CD105 antigen. The isolation of CD105+ ASCs from a heterogeneous cell population was confirmed by flow cytometry analysis. Given the demonstrated potential of functionalized magnetite-PCL microparticles for selective cell isolation, we expect that these particles may be further applied in immuno-magnetic cell separation owing to their versatility and ease of surface modification.
11
Gillig, Kent J. "Gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry: bovine insulin and ubiquitin." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 374, no. 2079 (October 28, 2016): 20150368. http://dx.doi.org/10.1098/rsta.2015.0368.
Full textAbstract:
Ion mobility-mass spectrometry (IMMS) is a very attractive method for studies in structural biology because of the ability of rapid isolation by nearly simultaneous m/z characterization and size separation, leading to an emergence of IMMS as a complimentary biochemical tool. Earlier, we developed a method based on varying the protein concentration in solution prior to electrospray ionization (ESI) with subsequent m/z selection and dissociation of protein multimers by IMMS of cytochrome c. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z for well-studied proteins bovine ubiquitin and insulin. These proteins were chosen due to their large difference in solution phase structures: insulin tightly bound by disulfide linkages, and ubiquitin—a protein that may adopt a range of states from compact to extended. Our preliminary results, as with cytochrome c reveal false negatives for protein oligomer formation and false positives for protein conformational states. In addition, these results will be couched in terms of the need for quantification of IMMS analysis of proteins given the total area under IMMS peaks can also distinguish conformation versus aggregation as higher order oligomers have more mass per ion. This article is part of the themed issue ‘Quantitative mass spectrometry’.
12
Shtam, T. A., R. A. Samsonov, A. V. Volnitskiy, R. A. Kamyshinsky, N. A. Verlov, M. S. Kniazeva, E. A. Korobkina, et al. "Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods." Biomeditsinskaya Khimiya 64, no. 1 (January 2018): 23–30. http://dx.doi.org/10.18097/pbmc20186401023.
Full textAbstract:
Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O “cushion”, precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.
13
Thiemann, Jutta, and Wolfgang Barz. "Photoautotrophic Chenopodium rubrum Cell Suspension Cultures Resistant against Photosynthesis-Inhibiting Herbicides I. Selection and Characterization." Zeitschrift für Naturforschung C 49, no. 3-4 (April 1, 1994): 186–94. http://dx.doi.org/10.1515/znc-1994-3-405.
Full textAbstract:
Abstract For establishing metribuzin-resistant, photoautotrophic Chenopodium rubrum cell cultures plated cells, callus cultures or suspension cultures were subjected to selection proce dures. The most effective procedure was the stepwise increase in the concentration of the herbicide from 0.01 μᴍ to 10 μᴍ in suspension cultures, which resulted in the isolation of eight different metribuzin-resistant photoautotrophic cell lines. Conjugation metabolism or a decrease in the uptake and translocation of the selective agent were not responsible for resist ance, which was stable in the absence of the inhibitor over numerous growth cycles. Meas urements of the photosynthetic electron transport, analyses of fluorescence induction kinetics and determination of the binding properties of 14C-labelled metribuzin to isolated thylakoids indicated that resistance of the cell lines is based on an alteration in the photosystem II her bicide-binding protein (D 1 protein). RFLP analysis of the psbA gene of the eight resistant cell lines demonstrated that none of them possess an amino acid exchange in position 264 of the D1 protein leading to altered herbicide-binding properties.
14
Barber, D. A., S. R. Michener, S. C. Ziesmer, and V. M. Miller. "Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 1 (January 1, 1996): H65—H71. http://dx.doi.org/10.1152/ajpheart.1996.270.1.h65.
Full textAbstract:
Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.
15
Daniels, M. P. "Localization of actin, beta-spectrin, 43 × 10(3) Mr and 58 × 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes." Journal of Cell Science 97, no. 4 (December 1, 1990): 615–26. http://dx.doi.org/10.1242/jcs.97.4.615.
Full textAbstract:
I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4–6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 × 10(3) Mr and 58 × 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.
16
Hoang, Phuong Ha, Thi Ngoc Mai Cung, Thi Minh Nguyen, Thi Lien Do, Lan Phuong Do, and Thi Nhi Cong Le. "Isolation and selection of probiotic bacteria capable of forming biofilm for fermenting soybean meal." Journal of Vietnamese Environment 9, no. 2 (July 16, 2018): 99–105. http://dx.doi.org/10.13141/jve.vol9.no2.pp99-105.
Full textAbstract:
Soybean meal (SBM) is residua product after oil extraction, the SBM with 48% protein is used for poultry, cattle. The SBM contains significant amount of anti-nutritional factors. Degradation of most antigenic proteins and protease inhibitors in SBM fermented by fungal, yeast and bacterial strains. Soybean fermented products are used as feed for livestock or aquaculture. Recently, biofilm forming microorganisms were broadly applied for fermentation process using substrates such as rice bran, corn, soybean meal ... to produce probiotics. In this study, we isolated and selected beneficial microbial strains that are capable of well biofilm forming, produce digestive enzymes and resist pathogenic microorganisms to ferment of soybean meal. The result showed that, four microorganism strains including NA5.3; TB2.1; TB4.3 TB4.4 had ability of forming higher biofilm, producing digestive enzymes such as amylase, protease and cellulose. Among them, NA5.3 and TB 4.4 strains had anti-pathogenic bacteria capacity such as Vibrio parahaemolyticus; Enterococcus faecalis; Bacillus cereus and Escherichia coli. Four selected strains were checked effection of pH, temperature, NaCl and bile salt concentration to their biofilm formation. The result indicated suitable conditions for forming biofilm at pH 6-8 range; temperature range 30-37°C; NaCl concentration of 0-3%, bile salt concentrtion of 0.5-2%. The selected strains grew well during solid fermentation process, achieved 1011 CFU/gram.
Khô đậu nành là sản phẩm còn lại từ quá trình ép dầu chứa tới 48% protein thô và thường được sử dụng làm thức ăn cho gia cầm, gia súc. Nhưng trong khô đậu nành còn chứa một lượng đáng kể một số chất ức chế dinh dưỡng, các chất ức chế này lại được phân hủy bởi quá trình lên men nhờ một số loài vi khuẩn, nấm mốc hay nấm men. Sản phẩm lên men khô đậu tương được sử dụng làm thức ăn cho gia cầm, gia súc hay nuôi trồng thủy sản. Trong những năm gần đây, các vi sinh vật tạo màng sinh học đã được ứng dụng để lên men các cơ chất như cám gạo, ngô, khô đậu nành… tạo sản phẩm probiotics. Trong nghiên cứu này, chúng tôi đã phân lập và tuyển chọn một số vi sinh vật có lợi tạo màng sinh học cao, sinh các enzyme tiêu hóa và kháng lại một số vi khuẩn gây bệnh cho mục đích lên men khô đậu nành. Kết quả đã lựa chọn được 4 chủng vi khuẩn NA5.3; TB2.1; TB4.3 TB4.4 có khả năng tạo màng sinh học cao, sinh các enzyme như amylase, protease và cellulose. Trong đó,hai chủng NA5.3 và TB4.4 có khả năng kháng lại một số vi khuẩn gây bệnh như Vibrio parahaemolyticus; Enterococcus faecalis; Bacillus cereus và Escherichia coli. Bốn chủng vi khuẩn lựa chọn được nghiên cứu ảnh hưởng của các điều kiện lên khả năng tạo màng sinh học của chúng, chúng thích hợp ở pH 6-8; nhiệt độ 30-37°C; NaCl 0-3%, muối mật 0,5-2%. Sử dụng các chủng vi khuẩn này cho quá trình lên men rắn khô đậu tương, mật độ vi khuẩn sau khi lên men đạt 1011 CFU/gram.
17
Mozumder, N. H. M. R., M. Akhtaruzzaman, M. A. Bakr, and F. Tuj Zohra. "Study on Isolation and Partial Purification of Lactase (β-galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt." Journal of Scientific Research 4, no. 1 (December 26, 2011): 239. http://dx.doi.org/10.3329/jsr.v4i1.8478.
Full textAbstract:
Lactase has many applications in dairy industry including for the treatment of lactose intolerance. The present study was conducted to identify the activity of lactase enzyme produced by Lactobacillus bacteria isolated from yogurts available in Dhaka city. The strains were identified to be gram positive, catalase negative, fermentative and lactase producer when cultured on selective MRS agar media by using standard bacteriological procedures and techniques. The study revealed that enzymes produced by lactobacilli were capable to produce glucose from substrate lactose in lactose modified media using lactase assay Kit Glu IB and their highest protein concentration (17.25 mg/ml) was observed in the supernatant of culture media isolated from L. lactis. Highest total activity (850.69 U/l) and specific activity (50.04 U/mg) of lactase enzyme was observed in the strain of L. bulgaricus. The crude extract which showed highest activity was further purified by ammonium sulphate precipitation followed by anion exchange column chromatography (DEAE cellulose). Final specific activity and fold purification of lactase enzyme reached to 62.80 U/mg and 1.47 respectively. The highest physic-chemical properties (Effect of pH and temperature) of lactase enzyme were observed at PH 6.0 which was 43.98 U/mg of protein and at 70°c temperature which was 111.11 U/mg of protein.Keywords: β-Galactosidase; Specific activity; DEAE cellulose; Fold purification; Yogurt.© 2012 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.doi: http://dx.doi.org/10.3329/jsr.v4i1.8478J. Sci. Res. 4 (1), 239-249 (2012)
18
Bayraktar, V. N. "COMPARATIVE ASSESSMENT OF THE LABORATORY SELECTED AND ACTIVE DRIED SACCHAROMYCES CEREVISIAE YEAST CULTURE IN BIOTECHNOLOGY OF THE BRANDY PRODUCTION." Biological Bulletin of Bogdan Chmelnitskiy Melitopol State Pedagogical University 5, no. 01 (January 30, 2015): 48. http://dx.doi.org/10.15421/2015003.
Full textAbstract:
<p>Samples from different industrial grape cultivars were collected during the vintage season from the vineyard of the winery (the «Shabo» winery Company, located in the Odesa region, Ukraine). The following industrial cultivars of grapes were selected for the research: Chardonnay, Cabernet Sauvignon, Merlot, Sauvignon, Riesling Rhenish, Aligote, Rkatsiteli, Bastardo, Traminer, Telti Kuruk, Grinosh.</p> <p>The grape cultivars were cultivated on the sandy soils in the district located between the Black Sea and the Dnestrovsky estuary. Grape must derived from different grape cultivars was placed into sterile glass flasks to half of the 450ml flask volume. Each flask was carefully closed with a rubber stopper with an injection needle in it. During the fermentation process, it was necessary to remove carbon dioxide, which was present as a result of active anaerobic fermentation processes in the grape must. At the end of grape must fermentation, pure yeast cultures were isolated using traditional microbiological methods by consistent inoculation of a sample into a Petri dish with a few modifications of nutrient selective agar for yeast isolation and cultivation. Primary yeast isolation was carried out using Inhibitory Mold Agar medium (Becton Dickinson Company, USA).</p> <p>The yeast culture morphological properties were analyzed after the primary yeast culture isolation. Yeasts were identified by polymerase chain reaction (PCR) using universal yeast primers. After yeast culture identification, the next step in yeast cultivation was carried out on Wort Agar medium (Becton Dickinson Company, USA). Each isolated, and identified yeast culture was deposited in the Genebank of Japan, MAFF culture Collection, Tsukuba, Ibaraki, Japan and (NCYC) - Yeast Culture Collection (National Collection of Yeast Cultures, Institute of Food Research, Norwich, United Kingdom). Each yeast culture was tested for technological characteristics such as growth resistance to high temperature (+42°C) and low temperature (+6°C), growth at low pH 2.6–3.0 (acid resistance), growth in the presence of 5, 10, and 15% ethanol (ethanol resistance), and growth in the presence of high concentration potassium bisulfite (bisulfite resistance). Hydrosulfide synthesis (H<sub>2</sub>S gassing production) was studied in addition.</p> <p>Parameters of cellular metabolism in yeast suspension, such as concentration of nitrogen, protein, triglicerides, enzymatic activity and total sugar (which include glucose, fructose, and galactose) were determined. Macro- and micro-element concentrations in fermented grape must, which contained pure yeast culture was determined and included: potassium, sodium, calcium, phosphorus, magnesium, iron, chlorides. In addition to identifying parameters of macro- and micro- element concentration in grape must during and following fermentation based on a principle of photometric analysis, carried out using a biochemical analyser Respons-920 (DiaSys Diagnostic Systems GmbH, Germany).</p> <p>Laboratory selected <em>Saccharomyces cerevisiae </em>wine yeast showed high enzymatic activity with short lag phase. Since of fermentation started on third day concentration of Triglicerides, Protein (total), Potassium and Sodium increased and then level of Protein (total) on the 5th day of fermentation twice decreased. Trigliceride concentration on the 5th day of fermentation continued to increase. Concentration of Iron on the 5th day of fermentation increase in geometrical progression, concentration increase in 4-5 times. Contrary Chloride concentration on the 5th day of fermentation decreased in 3-4 times. Enzymatic activity on 3rd day of fermentation maximal for Lactate Dehydrogenase, Alanine aminotransferase, Aspartate aminotransferase, Phosphatase. Since of 5th day of fermentation Enzymatic activity for Lactate Dehydrogenase, Alanine aminotransferase, Aspartate aminotransferase 3-4 times. Especially level of Phosphatase activity very decreased in 6-7 times. Comparative assessment between our Laboratory selected <em>Saccharomyces cerevisiae</em> yeast culture and Dry active commercial <em>Saccharomyces cerevisiae</em> yeast culture did not showed any difference in enzymatic activity. Both groups showed high enzymatic activity on the third day from the start of fermentation and decreasing on the fifth day since of fermentation started.</p> <p><em> Key words: wine yeast, enzymatic activity, cellular metabolism</em><em>, </em><em>Saccharomyces cerevisiae</em><em>.</em><em></em></p>
19
Stelzer, W., and J. Jacob. "A Study of Campylobacter in Sewage, Sewage Sludge and in River Water." Water Science and Technology 24, no. 2 (July 1, 1991): 117–20. http://dx.doi.org/10.2166/wst.1991.0040.
Full textAbstract:
This study is aimed at determining the occurrence of campylobacters in waste water, sewage sludge and in a river system. The selective medium for the isolation of campylobacter consists of blood agar supplemented with the antibiotics vancomycin (10 µg/ml), cephalexin (15 µg/ml), trimethoprim (5 µg/ml), polymycin B (2.5 µg/ml) and rifampicin (5 µg/ml). Raw sewage samples contained about 103 Campylobacter/100 ml while the effluent showed an average concentration of 1.3 × 102/100 ml. Raw sewage from an oxidation pond treatment plant contained an average of 51 Campylobacter/100 ml while none were found in the effluent. No Campylobacter could be found in digested, conditioned sludge. The organism could be detected in 82.1% of river waters examined with the majority showing <10/100 ml. The presence of waterfowl and the faecal contamination from a poultry farm resulted in higher Campylobacter levels. About 50 % of the isolates typed as C. coli were really confirmed as C. jejuni by electrophoretic pattern (whole cell protein profiles).
20
Ananthanarayanan, M., J. C. Bucuvalas, B. L. Shneider, C. J. Sippel, and F. J. Suchy. "An ontogenically regulated 48-kDa protein is a component of the Na(+)-bile acid cotransporter of rat liver." American Journal of Physiology-Gastrointestinal and Liver Physiology 261, no. 5 (November 1, 1991): G810—G817. http://dx.doi.org/10.1152/ajpgi.1991.261.5.g810.
Full textAbstract:
Recent evidence suggests that the Na(+)-coupled carrier mechanism for bile acids on the hepatocyte basolateral plasma membrane is a polypeptide in the molecular weight range of 48,000-50,000. In this study we used a strategy for the identification and isolation of this transport protein based on the observation that Na(+)-dependent transport activity is abruptly expressed in fetal rat liver just before birth [Suchy et al. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G665-G673, 1986]. Analysis of basolateral plasma membranes by SDS-PAGE revealed that a protein of apparent molecular weight 48,000 was absent from fetal rat liver on day 19 of gestation, barely detectable on day 20, and thereafter increased progressively with postnatal development. Monospecific, polyclonal antibodies raised against the 48-kDa protein but not preimmune antibodies significantly inhibited the initial rate of Na(+)-dependent taurocholate uptake by isolated rat hepatocytes. In contrast, Na(+)-independent taurocholate transport and uptake of another anion, 35SO4(2-), were not affected by antibody treatment. When an extract containing the total complement of basolateral proteins was incorporated into asolectin liposomes, Na+ gradient-dependent uptake of taurocholate was observed, including a 2- to 2.5-fold accumulation of substrate above its equilibrium concentration (overshoot). However, if the membrane extract was first selectively depleted of the 48-kDa protein by immunoprecipitation with the anti-48-kDa antibody before reconstitution, Na(+)-dependent stimulation of taurocholate transport was completely abolished. These studies indicate that an ontogenically regulated 48-kDa protein is a component of the basolateral Na(+)-dependent transport system for bile acids.
21
Boorse, Graham C., Erica J. Crespi, Frank M. Dautzenberg, and Robert J. Denver. "Urocortins of the South African Clawed Frog, Xenopus laevis: Conservation of Structure and Function in Tetrapod Evolution." Endocrinology 146, no. 11 (November 1, 2005): 4851–60. http://dx.doi.org/10.1210/en.2005-0497.
Full textAbstract:
Several corticotropin-releasing factor (CRF) family genes have been identified in vertebrates. Mammals have four paralogous genes that encode CRF or the urocortins 1, 2, and 3. In teleost fishes, a CRF, urotensin I (a fish ortholog of mammalian urocortin 1) and urocortin 3 have been identified, suggesting that at least three of the four mammalian lineages arose in a common ancestor of modern bony fishes and tetrapods. Here we report the isolation of genes orthologous to mammalian urocortin 1 and urocortin 3 from the South African clawed frog, Xenopus laevis. We characterize the pharmacology of the frog peptides and show that X. laevis urocortin 1 binds to and activates the frog CRF1 and CRF2 receptors at picomolar concentrations. Similar to mammals, frog urocortin 3 is selective for the CRF2 receptor. Only frog urocortin 1 binds to the CRF-binding protein, although with significantly lower affinity than frog CRF. Both urocortin genes are expressed in brain, pituitary, heart, and kidney of juvenile frogs; urocortin 1 is also expressed in skin. We also identified novel urocortin sequences in the genomes of pufferfish, zebrafish, chicken, and dog. Phylogenetic analysis supports the view that four paralogous lineages of CRF-like peptides arose before the divergence of the actinopterygian and sarcopterygian fishes. Our findings show that the functional relationships among CRF ligands and binding proteins, and their anorexigenic actions mediated by the CRF2 receptor, arose early in vertebrate evolution.
22
Duthie, Garry G. "Determination of activity of antioxidants in human subjects." Proceedings of the Nutrition Society 58, no. 4 (November 1999): 1015–24. http://dx.doi.org/10.1017/s0029665199001330.
Full textAbstract:
Evidence from biochemical and animal models suggests that nutritional antioxidants should inhibit the development of diseases such as CHD and certain cancers. This evidence is not clearly corroborated by intervention studies in human subjects, due, in part, to inadequacies in current analytical methodologies. Althoughin vitroassays can give useful information on the attributes required by a compound to act as an antioxidant, results may have little nutritional relevance due to limited bioavailability. The determination of antioxidants in blood is often used as a measure of antioxidant statusin vivo, but may not necessarily reflect concentrations in target tissues where oxidative stress is greatest. In addition, the accumulation of antioxidants in selective tissues may not be apparent from plasma measurements. Participation in quality-control schemes for antioxidant determination by HPLC allows inter-laboratory comparison of results. Moderation of indices of oxidative damage to lipids, proteins and DNA can provide information on the effectiveness of compounds as nutritional antioxidants. However, most current methods of assessing oxidative stress are subject to confounding factors of non-oxidative origin. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. They give different results, should never be used in isolation, and results should be interpreted with caution. Until more is known about the activity and metabolic fate of antioxidants, caution should be exercised in the consumption of large amounts of commercially-available antioxidant preparations.
23
Lee, Hyunwoo, Nora Vázquez-Laslop, Katya A. Klyachko, and Alex A. Neyfakh. "Isolation of Antibiotic Hypersusceptibility Mutants of Acinetobacter spp. by Selection for DNA Release." Antimicrobial Agents and Chemotherapy 47, no. 4 (April 2003): 1267–74. http://dx.doi.org/10.1128/aac.47.4.1267-1274.2003.
Full textAbstract:
ABSTRACT Isolation of bacterial mutants hypersusceptible to antibiotics can reveal novel targets for antibiotic potentiators. However, identification of such mutants is a difficult task which normally requires laborious replica plating of thousands of colonies. The technique proposed here allows for the positive selection of genetic knockout mutants leading to hypersusceptibility. This technique, designated SDR (selection for DNA release), involves introduction of random insertions of a marker gene into the chromosome of a highly transformable bacterial species, followed by treatment of the obtained library with an antibiotic at subinhibitory concentrations. DNA released by lysing bacteria is collected and used to transform fresh bacteria, selecting for insertion of the marker gene. These selection cycles are repeated until variants with a hypersusceptibility phenotype caused by insertion of the marker begin to dominate in the library. This approach allowed for isolation of a number of mutants of the gram-negative opportunistic pathogen Acinetobacter sp. susceptible to 4- to 16-times-lower concentrations of ampicillin than wild-type bacteria. The mutations affected proteins involved in peptidoglycan turnover and, surprisingly, proteins involved in exopolysaccharide production. A further modification of the SDR technique is described which allows for selecting mutants hypersensitive to agents that affect bacterial physiology but do not cause cell lysis, e.g., inhibitors of translation. This application of SDR is illustrated here by identification of several mutants of Acinetobacter sp. with increased susceptibility (two- to fivefold decrease in the MIC) to erythromycin. The same technique can be used to identify prospective targets for potentiators of many other antibacterial agents.
24
Wang, Fanqiang, Shelby Kashket, and Eva R. Kashket. "Maintenance of ΔpH by a butanol-tolerant mutant of Clostridium beijerinckii." Microbiology 151, no. 2 (February 1, 2005): 607–13. http://dx.doi.org/10.1099/mic.0.27587-0.
Full textAbstract:
The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane ΔpH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The ΔpH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the ΔpH component of the protonmotive force) and supplemented with glucose and Mg2+. The ΔpH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a ΔpH of 1·4 when butanol was added to a concentration of 1·5 % (w/v), while the wild-type ΔpH was reduced to 0·1. The ΔpH of both strains was completely dissipated with 1·75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the ΔpH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0·8 %) prevented this ΔpH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in ΔpH occurred more slowly than in the mutant, and sparing of the ΔpH by 0·8 % butanol was less pronounced. The authors interpret these data to mean that the ΔpH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.
25
Aguilera, C., D. Veraguas, C. Henriquez, A. Velasquez, F. O. Castro, and L. Rodriguez-Alvarez. "80 Evaluation of extracellular vesicles from culture medium of human embryos as a possible method of pre-implantation genetic diagnosis." Reproduction, Fertility and Development 32, no. 2 (2020): 166. http://dx.doi.org/10.1071/rdv32n2ab80.
Full textAbstract:
Noninvasive methods are the clue to increase the efficiency of invitro-derived embryo selection without decreasing their competence. Embryos selection based on their morphology is the most used method but only 40% of selected embryos are able to implant and develop correctly. In humans, pre-implantation genetic diagnosis increases the efficiency of selection by excluding embryos with chromosomal abnormalities. However, pre-implantation genetic diagnosis needs embryonic cells, which might compromise embryo viability. On the other hand, embryos release extracellular vesicles (EVs: microvesicles and exosomes) to the culture medium that contain biological cargo-like proteins and mRNA lipids, and might contain genomic DNA (gDNA). For this study we evaluated the culture medium from embryos generated by intracytoplasmic sperm injection in a certified fertility clinic. Embryos were cultured in Global Total serum-free medium. The embryos were assessed at Day 3 of development and classified in three categories: top, fair, and poor quality. Corresponding medium was collected for isolation of EVs. The nature of EVs was confirmed by their size and concentration using nanoparticle tracking analysis (NTA), presence of surface markers (CD9, CD63, CD81, and CD40L), and morphology using transmission electron microscopy. A correlation analysis between NTA results (EV size and concentration) and embryo quality was performed. To evaluate chromosomal abnormalities of gDNA present in isolated EVs from embryo culture medium, microarray-based comparative genomic hybridization (aCGH) assay was performed. In a second experiment, aCGH analysis was performed and compared between arrested embryos and EVs isolated from corresponding culture medium. Isolated nanoparticles from embryo culture medium were positive to all markers CD9 (30.9%), CD63 (27.2%), CD81 (31.7%), CD40L (8.7%) and had a morphology accordingly to exosomes. The analysis of NTA data indicated that top-quality embryos had EVs with higher diameter (mean: 112.17nm, mode: 91.74nm) than embryos classified as fair (mean: 108.02nm, mode: 89.67nm) and poor quality (mean: 102.78nm, mode: 88.17 nm; P<0.05). The aCGH analysis showed the representation of the 23 pairs of chromosomes in EVs from culture medium and the chromosomal abnormalities were detected in chromosome 4 (C4: 6/15 (40%)) and chromosome 13 (C13: 6/15 (40%)). In the second experiment, the aCGH assay also showed abnormalities in different chromosomes from samples of EVs from culture medium (24.9%) and were more frequent than those observed in the arrested embryos (8.7%; P=0.03). However, the rate of similitude in chromosomal abnormalities between EVs and their respective embryo was 70-80%. In conclusion, the size and gDNA of EVs from culture medium might be an alternative to evaluate the competence of human embryos. This research was supported by FONDECYT-1170310 and Corfo 17Cote-72437, Chile.
26
Atasheva, Svetlana, Rodion Gorchakov, Robert English, Ilya Frolov, and Elena Frolova. "Development of Sindbis Viruses Encoding nsP2/GFP Chimeric Proteins and Their Application for Studying nsP2 Functioning." Journal of Virology 81, no. 10 (February 28, 2007): 5046–57. http://dx.doi.org/10.1128/jvi.02746-06.
Full textAbstract:
ABSTRACT Sindbis virus (SINV) is one of almost 30 currently known alphaviruses. In infected cells, it produces only a few proteins that function in virus replication and interfere with the development of the antiviral response. One of the viral nonstructural proteins, nsP2, not only exhibits protease and RNA helicase activities that are directly involved in viral RNA replication but also plays critical roles in the development of transcriptional and translational shutoffs in the SINV-infected cells. These multiple activities of nsP2 complicate investigations of this protein's functions and further understanding of its structure. Using a transposon-based approach, we generated a cDNA library of SINV genomes with a green fluorescent protein (GFP) gene randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. Recombinant SIN viruses encoding nsP2/GFP chimeric protein were capable of growth in tissue culture and interfering with cellular functions. SINV, expressing GFP in the nsP2, was used to isolate nsP2-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsPs, all of the cellular proteins that we previously coisolated with SINV nsP3, and some additional protein factors that were not found before in detectable concentrations. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous proteins, can be applied for mapping the domain structure of the viral nonstructural and structural proteins, cloning of peptide tags for isolation of the protein-specific complexes, and studying their formation by using live-cell imaging. This approach may also be applicable to presentation of additional antigens and retargeting of viruses to new receptors.
27
Onida, Francesco, Cinzia Scavullo, Federica Servida, Giorgia Saporiti, Pierfausto Seneci, Gianluigi Reda, Wilma Barcellini, Agostino Cortelezzi, Domenico Delia, and Giorgio Lambertenghi Deliliers. "New SMAC-Mimetic Compounds Strongly Induce In Vitro Apoptosis of Human Lymphocytes From Patients with Chronic Lymphocytic Leukemia." Blood 116, no. 21 (November 19, 2010): 1835. http://dx.doi.org/10.1182/blood.v116.21.1835.1835.
Full textAbstract:
Abstract Abstract 1835 Defective apoptosis has a central role both in development and progression of chronic lymphocytic leukemia (CLL). Indeed in this hematological malignancy the expression of inhibitor of apoptosis proteins (IAPs) has been shown to be significantly up-regulated. The most potent among IAPs is probably the X-linked IAP (XIAP), which is characterized by three highly conserved tandem BIR domains (BIR1-3) able to block various members of the family of cysteine proteases (caspases) known as the main effectors of the apoptotic process. BIR3 domain selectively targets caspase 9, an initiator caspase, whereas the linker region between BIR1 and BIR2 binds with executioner caspases 3 and 7, thus inhibiting the activity of both initiator and effector of apoptosis. Smac-DIABLO (Second Mitochondria-derived Activator of Caspases - Direct IAp Binding protein with LOw pI) is a protein released from the mitochondria antagonizing the activity of XIAP. Therefore, small molecules mimicking Smac (Smac-mimetics) can induce apoptosis in tumor cells by displacing the interaction between XIAP and caspases. At the University of Milan, Center for biomolecular Interdisciplinary Studies and Industrial applications (CISI), new cell permeable Smac-mimetic compounds (monomers and dimers) binding to XIAP with high affinity were recently synthetized. Here we report the activity of a selection of these new agents in human lymphocytes from 32 CLL patients and from 6 healthy controls. The patient population included only subjects with active disease (11 females and 21 males; median age 72 years; Rai-Binet stage 0-A to II-B), who, at the time of samples collection, were either untreated or out of therapy since several months. Patients were characterized for the mutational IGVH status, cytogenetics (by FISH analysis), and the expression of both CD38 and ZAP 70. Following isolation from peripheral blood samples by density gradient centrifugation, mononuclear cells were treated for 16 hours at 37°C and 5% CO2 with low micromolar concentrations (1-10 microM) of a selection of our new Smac mimetic compounds, including 4 monomers (34, 55, 66 and 73), and 2 dimers (83, 85) which were have been previously shown to be active in leukemic cell lines overexpressing XIAP. Combination treatments with the proteasome inhibitor bortezomib (10nM) and with Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) (50 ng/ml) were also tested. Apoptosis rate was evaluated by flow cytometric analysis following annexin V and propidium iodide staining. Overall, the monomers were more cytotoxic than the dimeric compounds on leukemic cells. The most active agent was the monomer Smac66, which, after 16 hours of treatment at a 10 microM concentration promoted apoptosis in up to 72,5% (median value 40% ranging from 11% to 72,5%) of CLL lymphocytes. In comparison to normal lymphocytes from healthy controls, where the median rate of apoptosis was 10.1% (range1.2% to 18%), the cytotoxic effect of the Smac66 compound was significantly higher (p=0.0004) in CLL cells. No synergistic effect was observed in combined treatment with bortezomib nor with TRAIL. Rate of apoptosis did not correlate with clinical parameters, cytogenetics, mutational IGVH status, and expression of CD38 and ZAP70 was observed, but the limited size of our patient population should be taken into account. In conclusion, among our Smac mimetics selected molecules seem be very active in CLL lymphocytes, suggesting a promising therapeutic potential in this malignancy. Further molecular investigations as well as in vivo studies in animal models are currently ongoing respectively to better elucidate the mechanism of action of these compounds and to promote their preclinical development. Disclosures: No relevant conflicts of interest to declare.
28
Toschi, P., D. Iuso, D. A. Anzalone, M. Czernik, G. Ptak, and P. Loi. "343 DIRECT EXPRESSION OF PLURIPOTENCY MARKERS IN CULTURED SOMATIC CELLS BY SMALL REPROGRAMMING MOLECULES." Reproduction, Fertility and Development 27, no. 1 (2015): 260. http://dx.doi.org/10.1071/rdv27n1ab343.
Full textAbstract:
The differentiate state of the cell may be reversed by a process called reprogramming. To date, a totipotent status is conferred to a somatic cell by nuclear transfer (SCNT) and a condition of pluripotency is conferred by induced expression of defined factors (iPSC). While the restoration of full totipotency by SCNT is rarely achieved, pluripotency by Yamanaka's factors (Oct4, c-myc, Sox-2, Klf4) is inducible, although with low efficiency, in a large set of cell in different animal models. However, the isolation of iPSC requires complex technical skills and time-consuming protocols. In our laboratory we have observed that the simple expansion of fibroblasts in culture switches on pluripotency markers such as Oct4 and Nanog (Anzalone et al. 2015 Reprod. Fertil. Dev. IETS Abstract 344). CHIR99021 is a small molecule, targeting the Wnt/β-catenin signalling pathway, which is used for stem cell culture (Li et al. 2009). CHIR99021 acts as selective inhibitor of both isoform of GSK3 α/β regulating cellular proliferation and differentiation. In this work we tested the hypothesis that the exposure to a small reprogramming molecule (CHIR99021) induces pluripotency marker expression in primary cultures of somatic cells. Sheep and mouse primary fibroblasts cultured in low oxygen and induced to enter GO (low serum, 0.5% FBS for 5 days, <3% cell proliferation in our conditions) were treated with different CHIR concentrations (from 2.5 to 5 µM) for different time periods (from 1 to 5 days) in order to test the proper concentration and to exclude any cytotoxic effects. Nuclear reprogramming was assessed in treated and control cells by analysing β-catenin and oct4, nanog, sox2, klf4, and c-myc expression by immuno-detection and PCR. We found that CHIR interferes with β-catenin pathway in both sheep and mouse fibroblast in a time- and dose-dependent manner; the best results were obtained using 3 µM of CHIR for 3 days. Western blot analysis confirmed that CHIR treatment leads to an increased cellular level of β-catenin; furthermore, pluripotency marker expression (protein and mRNA) was increased (P = 0.023 nonparametric Mann-Whitney test) in CHIR-treated cells compared to controls. These observations, confirmed in both the experimental models, indicate that treatment with a small molecule inhibitor interfering with glucose metabolism induces the expression of pluripotency marker in somatic cells.
29
Ota, Kazuhisa, Keiji Kito, Shun-ichiro Iemura, Tohru Natsume, and Takashi Ito. "A parallel affinity purification method for selective isolation of polyubiquitinated proteins." PROTEOMICS 8, no. 15 (August 2008): 3004–7. http://dx.doi.org/10.1002/pmic.200800271.
Full text
30
Boyiadzis, Michael, Chang Sook Hong, and Theresa L. Whiteside. "Natural Killer Cell Derived Exosomes As a Novel Therapeutic for Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 5226. http://dx.doi.org/10.1182/blood-2018-99-115385.
Full textAbstract:
Abstract Introduction: Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. NK cells have been evaluated for safety and efficacy in acute myeloid leukemia (AML), both in the transplant and non-transplant settings. Exosomes, small, 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells, have recently emerged as a universal intercellular communication system. Exosomes are released by virtually all cells and carry proteins, lipids, and nucleic acids from the parent to recipient cells at short and long distances. The exosome molecular cargo reflects the content of the parent cell and is delivered to recipient cells in a membrane-protected vesicle. We hypothesize that exosomes produced by human activated NK cells carry the machinery necessary for the killing of leukemic cells. Methods: Venous blood (20-50 mL) was obtained from healthy donors (n=10). NK cells were isolated using Ab-based immunomagnetic selection from the recovered peripheral blood mononuclear cells. NK cells were cultured in the presence of interleukin-2 and interleukin-15, and NK-cell supernatants were used for exosome isolation by size exclusion chromatography. Protein levels, numbers and size (qNano), and exosome morphology (transmission electron microscopy) were determined. Exosome cargos were studied by Western blots and/or flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for K562 targets, AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays. Results: Activated human NK cells produced large quantities of exosomes. PKH-26-labeled NK cell-derived exosomes were avidly taken up by leukemic blasts. NK cell derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors and PD-1. NK cell derived exosomes mediated anti-leukemia activity against K562 targets, AML cell lines and primary leukemia blasts. Lysis of leukemic blasts by NK cell-derived exosomes was exosome concentration dependent. Conclusion: We report that NK cell derived exosomes have anti leukemia activity in vitro. These data provide a foundation for the future development of new therapeutic strategies using NK cell-derived exosomes for the elimination of leukemia. Disclosures No relevant conflicts of interest to declare.
31
Badger, Murray R., G. Dean Price, and Jian Wei Yu. "Selection and analysis of mutants of the CO2-concentrating mechanism in cyanobacteria." Canadian Journal of Botany 69, no. 5 (May 1, 1991): 974–83. http://dx.doi.org/10.1139/b91-125.
Full textAbstract:
The selection and analysis of mutants of the CO2-concentrating mechanism in cyanobacteria have greatly advanced our understanding of the physiological and genetic components of this mechanism. This paper reviews the processes for the creation of mutants and the properties of mutants that have been produced so far. In addition to this, consideration is given to developing new mutant selection protocols, based on our current knowledge of the operation of the CO2-concentrating mechanism. As a result, new screens are suggested for the isolation of transport mutants that are defective in either HCO3− or CO2 transport activity, and putative carboxysomal mutants that have altered carbonic anhydrase activities. The procedures for physiological analysis of mutants are also reviewed, with a conclusion that there is a great need for the further development of in vitro assays, particularly for the inorganic carbon transport process and carboxysome function. Particular consideration is given to the in vitro carboxysome assay, and it is concluded that many of the properties of a carboxysomal Rubisco may be difficult to resolve owing to increases in carboxysomal leakiness during isolation and the higher ratio of [CO2] to [HCO3−] experienced during assays compared with the in vitro situation. Finally, consideration is given to the genetic analysis of cyanobacterial mutants and the progress that will need to be made in the future, discovering what are the functions of the proteins coded for by the genes that are isolated. Key words: cyanobacteria, mutants, photosynthesis, carboxysome, CO2-concentrating mechanism.
32
Rafiee, Mahmoud-Reza, Charles Girardot, Gianluca Sigismondo, and Jeroen Krijgsveld. "Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins." Molecular Cell 64, no. 3 (November 2016): 624–35. http://dx.doi.org/10.1016/j.molcel.2016.09.019.
Full text
33
Shu, Yang, Xu-Wei Chen, and Jian-Hua Wang. "Ionic liquid–polyvinyl chloride ionomer for highly selective isolation of basic proteins." Talanta 81, no. 1-2 (April 15, 2010): 637–42. http://dx.doi.org/10.1016/j.talanta.2009.12.059.
Full text
34
Liu, Jia-Wei, Ting Yang, Lin-Yu Ma, Xu-Wei Chen, and Jian-Hua Wang. "Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins." Nanotechnology 24, no. 50 (November 22, 2013): 505704. http://dx.doi.org/10.1088/0957-4484/24/50/505704.
Full text
35
Goyal, Arun, Jens Thielmann, and N. E. Tolbert. "Isolation of chloroplast envelopes from Dunaliella tertiolecta." Canadian Journal of Botany 76, no. 6 (June 1, 1998): 1146–52. http://dx.doi.org/10.1139/b98-064.
Full textAbstract:
Techniques have been developed to obtain Dunaliella chloroplasts and their outer and inner envelopes in high yield. With purified envelopes, we have identified two integral membrane proteins that are specifically induced by low CO2. These two proteins are located in the inner envelope, which is the proposed site for the active dissolved inorganic carbon transporter in unicellular green algae.Key words: algae, carbon concentration mechanism, chloroplast envelopes, dissolved inorganic carbon, Dunaliella, low CO2 induced proteins.
36
Heitner, Tara, Noboru Satozawa, Kirk Mclean, David Vogel, Ronald R. Cobb, Bing Liu, Mithra Mahmoudi, et al. "Obligate Multivalent Recognition of Cell Surface Tomoregulin following Selection from a Multivalent Phage Antibody Library." Journal of Biomolecular Screening 11, no. 8 (December 2006): 985–95. http://dx.doi.org/10.1177/1087057106293841.
Full textAbstract:
A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.
37
Kuwahara, Daichi, Takahiro Hasumi, Hajime Kaneko, Madoka Unno, Daisuke Takahashi, and Kazunobu Toshima. "A solid-phase affinity labeling method for target-selective isolation and modification of proteins." Chem. Commun. 50, no. 98 (2014): 15601–4. http://dx.doi.org/10.1039/c4cc06783e.
Full textAbstract:
Solid-phase affinity labeling of target proteins with the specifically designed chemical tools selectively and effectively furnished the labeled target proteins without the need for tedious manipulations.
38
Rudoman, H., V. Balatskyi, and V. Nor. "ANALYSIS OF POLYMORPHISM IN MUCIN 4 GENE ASSOCIATED WITH ANIMAL RESISTANCE TO COLIBACTERIOSIS IN PIGS OF DOMESTIC AND FOREIGN SELECTIONS." Animal Breeding and Genetics 51 (March 28, 2018): 200–205. http://dx.doi.org/10.31073/abg.51.27.
Full textAbstract:
Nowadays one of the most common and critical problem in pig breeding is colibacteriosis. This infectious disease has acute course and it is caused by enteropathogenic strains of bacteria Escherichia coli. One of the recent and effective approaches to prevent colibacteriosis is using markers of selection; it involves pig genotyping by genome loci. The chosen loci are associated with animal sensitivity to the disease and selection of animals with increased resistance by the results of genotyping. According to several researches, one of such loci is Mucin 4 (MUC4) gene. MUC4 gene is located in the13-th chromosome (SSC13q41). Mucins (MUC) are macromolecular glycoproteins synthesized by goblet enterocytes and play main role in protecting the intestinal epithelium from pathogens, including adhesive strains of Escherichia coli. As a result of spot replacement g.1849 G>С in intron 7, structure of mucin protein encoded by gene is changing which leads to changes in the sensitivity of the intestinal mucosa to pathogenic Escherichia coli. G allele and respectively GG genotype determine the resistance of animals to colibacteriosis, while СС і GС genotypes are susceptible to this disease. In Ukraine, the studies of polymorphism MUC4 g.1849 G> C were held fragmentally and only at certain populations of Ukrainian Meat and Large White. The aim of the work was to determine the genetic structure of pig breeds of domestic and foreign selection for MUC4 gene and to establish the possibility of organizing marker associated selection for genetic improvement of resistance to colibacteriosis. For research the samples of blood and hair were used from the animals of Large White of English selection, Large White of Ukrainian selection Type 1 and Type 3, Red White-Belt, Mirgorod, Poltava Meat and Landrace breeds. DNA isolation from the samples of biomaterial were carried out using ion exchange resin Chelex-100. Genotyping was performed by PCR-RFLP by Jorgensen methods (2006) with own modifications, concerning the selection of primer annealing temperature and optimum concentration of the gel to separate the restriction fragments. Genetic structure was determined using DNA analysis of MUC4 locus at seven breeds and intrabreed types. Predominance of potentially beneficial G allele frequency over undesirable C allele was established in all the populations of the studied pigs. The highest frequencies of G allele were characterized for Mirgorod (0.795), Poltava Meat (0.740) and Red White-Belt (0.820) breeds. Analysis of the distribution of genotypes showed domination of genotypes GG and GC in all analysed populations. Positive Wright fixation index and predominance of expected heterozygosity (0.484) over actual one (0.460) for Large White of Ukrainian selection Type 3 indicate the existence of moderate inbreeding and selective pressure in this herd. Other populations were characterized by a negative value of fixation index, which is indicative of an excess of heterozygotes, these breeds are in outbreeding depression. Statically significant deviation of actual frequencies of genotypes from expected ones was identified using Hardy-Weinberg criterion for pig population of Large White of English selection and Large White of Ukrainian selection Type 1. This may indicate that these populations aren’t in condition of equilibrium and about inclusion of the chosen gene to the selection process. The valuation was performed by calculating the PIC (polymorphic information content) of the marker. Based on the calculated PIC index MUC4 locus could be perspective to be used in marker associated selection with improving genetic resistance to colibacteriosis. The data of Mucin 4 gene polymorphism at Ukrainian pig populations of different origins and productive direction show the possibility of marker selection to improve the genetic resistance of animals to colibacteriosis regardless of their belonging to breed. This creates prerequisites for the establishment and implementation of the early molecular diagnosis of carriers of harmful C allele in MUC4 gene in pig breeding.
39
Trinh, Thuong Minh, Mai Thi Xuan Huynh, and Hieu Van Tran. "Generation and selective isolation of IgG antibodies against Jurkat T-cell membrane proteins." Science and Technology Development Journal 19, no. 1 (March 31, 2016): 26–35. http://dx.doi.org/10.32508/stdj.v19i1.541.
Full textAbstract:
Currently, Graft-versus-Host Disease (GvHD) has been the major barrier to the application of allogeneic bone marrow transplantation for hematopoietic disorders treatment, especially leukemia. GvHD is caused by donor mature T cells’ attack on recipient’s tissues and organs. Recently, T cell depletion using immunomagnetic nanoparticles is one of the most effective methods to prevent GvHD. In this present study, polyclonal antibodies against Jurkat T cell membrane were generated as a component of immunomagnetic particle. Firstly, Jurkat T cell membrane was fractionated by cloud point extraction using Triton X-114. Subsequently, the fractionated membrane was used to subcutaneously immunize rabbits for polyclonal antibodies production with a dose of 50 μg for priming and 25 μg for the next four boostings. Rabbit serum was collected and partially precipitated in 50 percent of saturated ammonium sulfate solution. Next, precipitated polyclonal antibodies were purified by using protein-A affinity chromatography column and the purity was determimed by SDS-PAGE. Afterwards, the purified antibodies were used in immune-fluorescent stainings and the recognition to Jurkat T cells was evaluated via fluorescent microscopic imaging. Finally, the purified antibodies were negatively selected by incubating with TF-1 cell, a hematopoietic stem cell, to eliminate cross-reacting antibodies. The immunocytofluorescent staining results showed that the purified and selected polyclonal antibodies weakly cross-reacted with TF-1 cells and strongly bound to Jurkat T cell
40
Fukushi, K., S. Kato, T. Antsuki, and T. Omura. "Isolation of copper-binding proteins from activated sludge culture." Water Science and Technology 44, no. 2-3 (July 1, 2001): 453–59. http://dx.doi.org/10.2166/wst.2001.0801.
Full textAbstract:
Six copper-binding microbial proteins were isolated from activated sludge cultures grown on media containing copper at various concentrations. Molecular weights among isolated proteins were ranged from 1.3k to 174k dalton. Isolated proteins were compared for their copper binding capabilities. Proteins isolated from cultures grown in the presence of copper in the growth media exhibited higher copper binding capabilities than those isolated from the culture grown in the absence of copper. The highest metal uptake of 61.23 (mol copper/mol protein) was observed by a protein isolated from a culture grown with copper at a concentration of 0.25 mM. This isolated protein (CBP2) had a molecular weight of 24k dalton. Other protein exhibited copper binding capability of 4.8-32.5 (mol copper/mol protein).
41
Lin, Shu-Ling, Yuanyuan Li, H. Dennis Tolley, Paul H. Humble, and Milton L. Lee. "Tandem electric field gradient focusing system for isolation and concentration of target proteins." Journal of Chromatography A 1125, no. 2 (September 2006): 254–62. http://dx.doi.org/10.1016/j.chroma.2006.05.041.
Full text
42
J. LANE, NANCY, and STEPHEN M. DILWORTH. "Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties." Journal of Cell Science 93, no. 1 (May 1, 1989): 123–31. http://dx.doi.org/10.1242/jcs.93.1.123.
Full textAbstract:
Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.
43
Whedon, Samuel D., Marissa K. Parker, Elizabeth L. Tyson, Tobias Ritterhoff, Patrick M. M. Shelton, and Champak Chatterjee. "A clickable glutamine (CliQ) derivative for the traceless reversible modification of peptides and proteins." Chemical Communications 55, no. 14 (2019): 2043–45. http://dx.doi.org/10.1039/c8cc09404g.
Full text
44
Hanušová, J., M. Mihulová, L. Diblíková, and L. Čurda. "Influence of salts on selective coagulation of whey proteins and their application in the isolation of β-lactoglobulin." Czech Journal of Food Sciences 32, No. 1 (February 18, 2014): 77–81. http://dx.doi.org/10.17221/524/2012-cjfs.
Full textAbstract:
Whey proteins are an important constituent of milk, especially whey from cheese manufacture and have many valuable functional properties such as foaming and emulsifying ability or gel formation. Some whey proteins are sensitive to salt content in a solution. High or low salt content may lead to selective coagulation of these proteins. A part of whey proteins was precipitated by addition of 7% (wt) NaCl and β-lactoglobulin and caseinomacropeptide remained in the supernatant. It was necessary to demineralise the supernatant by electrodialysis for the selective coagulation of caseinomacropeptide from this material. Subsequently, ethanol was added and pH was adjusted. This reduction of the ionic strength and the addition of ethanol induced the selective precipitation of caseinomacropeptide (91.4% from the original amount of CMP). β-lactoglobulin of 91% purity remained in the solution.
45
Chaga, Grigoriy S., Bo Ersson, and Jerker O. Porath. "Isolation of calcium-binding proteins on selective adsorbents application to purification of bovine calmodulin." Journal of Chromatography A 732, no. 2 (May 1996): 261–69. http://dx.doi.org/10.1016/0021-9673(95)01277-x.
Full text
46
Kenry, Kenry, Kian Ping Loh, and Chwee Teck Lim. "Selective concentration-dependent manipulation of intrinsic fluorescence of plasma proteins by graphene oxide nanosheets." RSC Advances 6, no. 52 (2016): 46558–66. http://dx.doi.org/10.1039/c6ra04978h.
Full text
47
D'Amico, L., N. J. Ajami, J. A. Adachi, P. R. C. Gascoyne, and J. F. Petrosino. "Isolation and concentration of bacteria from blood using microfluidic membraneless dialysis and dielectrophoresis." Lab on a Chip 17, no. 7 (2017): 1340–48. http://dx.doi.org/10.1039/c6lc01277a.
Full text
48
Forstner, Gordon G., Shirly M. Vesely, and Peter R. Durie. "Selective Precipitation of 14 kDa Stone/Thread Proteins by Concentration of Pancreaticobiliary Secretions." Journal of Pediatric Gastroenterology and Nutrition 8, no. 3 (April 1989): 313–20. http://dx.doi.org/10.1097/00005176-198904000-00009.
Full text
49
Hiwatari, Mitsuteru, Jingqiu Dai, Wei Liu, Yu-Dong Zhou, Dale G. Nagle, and Stephan W. Morris. "Cytotoxicity Assessment of Quassinoids Neosergeolide and Isobrucein B toward Hematopoietic and Solid Malignancies Identifies STAT3 Activation To Be Predictive of Antitumor Response." Blood 110, no. 11 (November 16, 2007): 1394. http://dx.doi.org/10.1182/blood.v110.11.1394.1394.
Full textAbstract:
Abstract Quassinoids are natural product compounds known to possess tumor cytotoxicity and antimalarial activity. Neosergiolide and isobrucein B are two quassinoids previously isolated from roots and stems of Picrolemma sprucei. In screening studies to identify inhibitors that target STAT3, we discovered neosergeolide and isobrucein B as active compounds. Approximately 5000 plant-derived extracts were screened using a cell line that stably expresses a STAT3-dependent luciferase reporter and NPM-ALK, which constitutively induces STAT3 transcriptional activity. Of 25 total hits, a P. sprucei extract was potent and selective for STAT3 inhibition, and bioassay-guided isolation identified neosergeolide and isobrucein B as the inhibitory compounds. Western blot analysis confirmed that neosergeolide and isobrucein B not only inhibit the tyrosine phosphorylation and activation of STAT3 but also decrease total STAT3 protein levels via a mechanism due in part to enhanced proteasome-mediated degradation. Small-molecule proteasome inhibitors such as MG132 and ALLN reversed the ability of the two quassinoids to decrease STAT3 protein levels; furthermore, simultaneous incubation of various hematopoietic malignancy cell lines with either neosergeolide or isobrucein B and MG132 or ALLN antagonized the cytotoxic activity of the quassinoids. Assessment of neosergiolide and isobrucein B antitumor effects using an XTT assay revealed both compounds to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-leukemias/lymphomas being especially responsive. For example, mycosis fungoides (MF)- and Sezary syndrome (SS)-derived cell lines, as well as non-MF/SS cutaneous T-cell lymphoma (CTCL) lines, were potently inhibited by both quassinoids (neosergiolide IC50 values: MAC-1, 11.6 nM; MAC-2A, 6.9 nM; Hut-78, 6.6 nM; HH, 4.3 nM; MJ, 7.0 nM; isobrucein B IC50 values: MAC-1, 31.9 nM; MAC-2A, 72.3 nM; Hut-78, 23.5 nM; HH; 20.3 nM; MJ, 13.5 nM). Non-hematopoietic cell lines representing various solid tumors also exhibited potent cytotoxic responses to the quassinoids (e.g., gastric carcinoma line AGS [neosergiolide IC50: 16.9 nM; isobrucein B IC50: 114.9 nM]). With rare exceptions, the cytotoxicity of the quassinoids against a specific tumor cell line correlated with STAT3 activation status; for example, breast cancer line MCF7 with inactive STAT3 was resistant to both quassinoids even at the maximum concentration tested (6.25 μM), whereas breast cancer lines MDA-MB-468 and MDA-MB-435s with activated STAT3 were inhibited by both compounds at low concentrations (neosergiolide IC50: MDA-MB-435s, 31.3 nM; MDA-MB-468, 29.9 nM; isobrucein B IC50: MDA-MB-435s, 209.3 nM; MDA-MB-468, 356.8 nM). The in vitro antitumor activity of the two quassinoids could also be demonstrated in vivo. For example, isobrucein B (1.0 mg/kg IP once q 3d x 5 doses) could be safely administered and potently inhibited the growth in SCID mice of the CD30+ primary CTCL MAC-1 cell line; mice at treatment day 16 showed average subcutaneous tumor volumes of 3839 ± 863 (s.e.) mm3 in the vehicle-control group and 913 ± 349 (s.e.) mm3 in the isobrucein B group (P=0.008, t-test). These results provide strong support for STAT3 targeting in antitumor drug discovery and suggest that quassinoids may have utility in such an approach.
50
Charuk, J. H. M., C. Guerin, and P. C. Holland. "Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures." Biochemical Journal 282, no. 2 (March 1, 1992): 399–407. http://dx.doi.org/10.1042/bj2820399.
Full textAbstract:
We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.