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1
Green, Kim Y., Aaron Mory, Mark H. Fogg, et al. "Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells." Journal of Virology 76, no. 17 (2002): 8582–95. http://dx.doi.org/10.1128/jvi.76.17.8582-8595.2002.
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ABSTRACT A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative “3A-like” protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.
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Carell, Thomas, Edward A. Wintner, Julius Rebek, and A. J. Sutherland. "A Solution-Phase Screening Procedure for the Isolation of Active Compounds from a Library of Molecules." Angewandte Chemie International Edition in English 33, no. 20 (1994): 2061–64. http://dx.doi.org/10.1002/anie.199420611.
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McIlwain, Henry. "Assay-guided isolation of naturally-occurring neuroactive substances." Psychological Medicine 15, no. 1 (1985): 15–26. http://dx.doi.org/10.1017/s0033291700020894.
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SynopsisWays in which chemical techniques could be applied to the understanding of neural systems, their functioning and their disorders were devised only gradually during the present century. In a particularly successful procedure, now termed assay-guided isolation, neural defects were made good by means of tissue-extracts and the restoration of function was established as an assay-system to guide the chemical separation and identification of the active tissue constituent. Thiamin was so isolated, using an experimental polyneuritis assay; subsequent instances among other metabolites, hormones, neurotransmitters and nerve growth factors are recounted. Procedures of assay-guided characterization ensured that links were retained between specific, sparsely-occurring substances and chosen aspects of their biological roles while their chemical nature was first explored and then established. The procedures discouraged the too-facile postulating of hypothetical molecules and contributed to the distinctiveness of neurochemistry as a subject within the neurosciences.
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Vakharia, Hema, Greg J. German, and Rajeev Misra. "Isolation and Characterization ofEscherichia coli tolC Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin." Journal of Bacteriology 183, no. 23 (2001): 6908–16. http://dx.doi.org/10.1128/jb.183.23.6908-6916.2001.
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ABSTRACT This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.
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Villa, Federico, Rodolfo Quarto, and Roberta Tasso. "Extracellular Vesicles as Natural, Safe and Efficient Drug Delivery Systems." Pharmaceutics 11, no. 11 (2019): 557. http://dx.doi.org/10.3390/pharmaceutics11110557.
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Extracellular vesicles (EVs) are particles naturally released from cells, delimited by a lipid bilayer, carrying functionally active biological molecules. In addition to their physiological role in cellular communication, the interest of the scientific community has recently turned to the use of EVs as vehicles for delivering therapeutic molecules. Several attempts are being made to ameliorate drug encapsulation and targeting, but these efforts are thwarted if the starting material does not meet stringent quality criteria. Here, we take a step back to the sources and isolation procedures that could guarantee significant improvements in the purification of EVs to be used as drug carriers, highlighting the advantages and shortcomings of each approach.
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Gu, Zhenhua, and Jia Feng. "Atropisomerism in Styrene: Synthesis, Stability, and Applications." SynOpen 05, no. 01 (2021): 68–85. http://dx.doi.org/10.1055/s-0040-1706028.
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AbstractAtropisomeric styrenes are a class of optically active compounds, the chirality of which results from restricted rotation of the C(vinyl)–C(aryl) single bond. In comparison with biaryl atropisomers, the less rigid skeleton of styrenes usually leads them to have lower rotational barriers. Although it has been overlooked for a long time, scientists have paid attention to this class of unique molecules in recent years and have developed many methods for the preparation of optically active atropisomeric styrenes. In this article, we review the development of the concept of atropisomeric styrenes, along with their isolation, asymmetric synthesis, and synthetic applications.1 Introduction2 The Concept of Styrene Atropisomerism3 Early Research: Separation of Optically Active Styrenes4 Synthesis of Optically Active Styrenes5 Stability of the Chirality of Atropisomeric Styrenes6 Outlook
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Li, L., and P. K. Moore. "An overview of the biological significance of endogenous gases: new roles for old molecules." Biochemical Society Transactions 35, no. 5 (2007): 1138–41. http://dx.doi.org/10.1042/bst0351138.
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Biologically active gases that occur naturally in the body include nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S). Each of these molecules is synthesized by enzymes which have been characterized biochemically and pharmacologically, and each acts, via well-established molecular targets, to effect physiological and/or pathophysiological functions within the body. Major biological roles that appear to be common to all three gases include the regulation of vascular homoeostasis and central nervous system function. It is becoming increasingly clear that both the synthesis and the biological activity of each gas are, to some extent, regulated by the presence of the others, and as such it is necessary to consider these molecules not in isolation but acting together to control cell function. Additional, more speculative candidates for gaseous cell signalling molecules include ammonia, acetaldehyde, sulfur dioxide and nitrous oxide. Whether such molecules also play a role in regulating body function remains to be determined.
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Fair, DS, DJ Revak, JG Hubbard, and A. Girolami. "Isolation and characterization of the factor X Friuli variant." Blood 73, no. 8 (1989): 2108–16. http://dx.doi.org/10.1182/blood.v73.8.2108.2108.
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Abstract Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two- dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195–424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.
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Fair, DS, DJ Revak, JG Hubbard, and A. Girolami. "Isolation and characterization of the factor X Friuli variant." Blood 73, no. 8 (1989): 2108–16. http://dx.doi.org/10.1182/blood.v73.8.2108.bloodjournal7382108.
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Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two- dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195–424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.
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Kwon, B. S., D. Kestler, E. Lee, M. Wakulchik, and J. D. Young. "Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes." Journal of Experimental Medicine 168, no. 5 (1988): 1839–54. http://dx.doi.org/10.1084/jem.168.5.1839.
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Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2-terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35-kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.
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Ohlendieck, K., ST Dhume, JS Partin, and WJ Lennarz. "The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor." Journal of Cell Biology 122, no. 4 (1993): 887–95. http://dx.doi.org/10.1083/jcb.122.4.887.
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The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.
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Sharifi, Mohammad, and Nirichan Kunchirman Bipinraj. "Isolation and Identification of Actinomycetes with Anticandida Activity from Mangrove Soil." Biosciences Biotechnology Research Asia 16, no. 3 (2019): 611–15. http://dx.doi.org/10.13005/bbra/2776.
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Candida albicans, a common human commensal, is the leading cause of nosocomial infections due to the emergence of drug resistance. The present study reports the isolation and identification of actinomycetes from mangrove soil and characterization of its antagonistic activity against drug resistant Candida species. Mangrove soils from Khargar, Navi Mumbai were screened for actinomycetes with anti-candida activity. In total, 20 actinomycetes culture were isolated from mangrove soil sample, amongst the culture designated as MB was found to inhibit all tested pathogenic Candida cultures. The isolate MB was identified using biochemical tests and 16S rRNA sequencing as Streptomyces viridocromogenes. MB culture showed maximum activity after incubation period of 48 to 72 h, pH of 6.2 and temperature of 30℃. Partially purified active molecule was found to be inactivated by heat treatment but resisted proteinase K, indicating the compound can be an antibiotic in nature. The study highlights the isolation of Streptomyces viridocromogenes with antagonistic activity against multidrug resistant Candida from mangrove soil. This culture is an ideal candidate for further characterization studies for anti-candida molecules.
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Ahuja, Ashok, Devinder Kaur, Mallubhotla Sharada, Arun Kumar, Krishan Avtar Suri, and Prabhu Dutt. "Glycowithanolides accumulation in in Vitro Shoot Cultures of Indian Ginseng (Withania somnifera Dunal)." Natural Product Communications 4, no. 4 (2009): 1934578X0900400. http://dx.doi.org/10.1177/1934578x0900400407.
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Phytochemical investigations of multiple shoot cultures of selected accessions AGB002 and AGB025 of Withania somnifera. established in vitro utilizing shoot tip apices cultured on Murashige and Skoog's medium supplemented with BAP (1 mg/L) have been carried out. This has lead to isolation of four glycowithanolides viz. Withanoside IV (WSG-3), Withanoside VI (WSG-3A), Physagulin D (WSG-P) and Withastraronolide (WSC-O). The structures of these have been confirmed on the basis of spectroscopic data. Multiple shoot cultures could be an alternative renewable resource for production of these biologically active molecules.
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Yao, Yuan, Dechao Jiao, Zhaonan Li, et al. "Roles of Bile-Derived Exosomes in Hepatobiliary Disease." BioMed Research International 2021 (January 13, 2021): 1–14. http://dx.doi.org/10.1155/2021/8743409.
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Exosomes are vesicles with a diameter of 30-150 nm produced by living cells and secreted into the extracellular matrix. Exosomes mediate cellular communication by carrying active molecules, such as nucleic acids, proteins, and liposomes. Although exosomes are found in various body fluids, little is known about bile-derived exosomes. This review is the first to summarize the methods of bile storage and isolation of biliary exosomes, highlighting the roles of bile-derived exosomes, especially exosomal noncoding RNAs, in physiological and disease states and discussing their potential clinical applications.
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Nikolic, Milan, and Sinisa Djordjevic. "Alkaloids in the pharmaceutical industry: Structure, isolation and application." Chemical Industry 57, no. 10 (2003): 471–78. http://dx.doi.org/10.2298/hemind0310471n.
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By the end of the 18th and the beginning of the 19th century a new era began in medicine, pharmaceutics and chemistry that was strongly connected with alkaloids and alkaloid drugs. Even before that it was known that certain drugs administered in limited doses were medicines, and toxic if taken in larger doses (opium, coke leaves, belladonna roots, monkshood tubers crocus or hemlock seeds). However, the identification, isolation and structural characterization of the active ingredients of the alkaloid drugs was only possible in the mid 20th century by the use of modern extraction equipment and instrumental methods (NMR, X-ray diffraction and others).In spite of continuing use over a long time, there is still great interest in investigating new drugs, potential raw materials for the pharmaceutical industry, as well as the more detailed investigation and definition of bio-active components and the indication of their activity range, and the partial synthesis of new alkaloid molecules based on natural alkaloids. The scope of these investigations, especially in the field of semi-synthesis is to make better use of the bio-active ingredients of alkaloid drugs, i.e. to improve the pharmacological effect (stronger and prolonged effect of the medicine, decreased toxicity and side effects), or to extend or change the applications. A combined classification of alkaloids was used, based on the chemical structure and origin, i.e. the source of their isolation to study alkaloid structure. For practical reasons, the following classification of alkaloids was used: ergot alkaloids, poppy alkaloids, tropanic alkaloids purine derivative alkaloids, carbon-cyclic alkaloids, and other alkaloids. The second part of this report presents a table of general procedures for alkaloid isolation from plant drugs (extraction by water non-miscible solvents, extraction by water-miscible solvents and extraction by diluted acid solutions). Also, methods for obtaining chelidonine and glaucine as hydrochloride bases and salts were presented in more details. Data from leading world pharmacopoeias (Ph. Eur. Ill/s 2000, DAB 1996, USP 23, JP XIII, BP 1993, Ph. Jug. IV) were used in the study of application of the pure alkaloids in pharmaceutical forms with predetermined doses. A comparative study of these data shows that a great number of preparations are produced worldwide based on alkaloids and alkaloids with modified structure. These medicines have found use in modern therapeutic practice in many countries. Most products are produced on the basis of caffeine, theophylline, ephedrine, atropine, scopolamine, reserpine and pilocarpine.
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Culotta, V. C., R. J. Wides, and B. Sollner-Webb. "Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors." Molecular and Cellular Biology 5, no. 7 (1985): 1582–90. http://dx.doi.org/10.1128/mcb.5.7.1582.
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RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.
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Culotta, V. C., R. J. Wides, and B. Sollner-Webb. "Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors." Molecular and Cellular Biology 5, no. 7 (1985): 1582–90. http://dx.doi.org/10.1128/mcb.5.7.1582-1590.1985.
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RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.
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Carson, C., M. A. Birkett, J. G. Logan, et al. "Novel use of stir bar sorptive extraction (SBSE) as a tool for isolation of oviposition site attractants for gravid Culex quinquefasciatus." Bulletin of Entomological Research 100, no. 1 (2009): 1–7. http://dx.doi.org/10.1017/s0007485309006701.
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AbstractMosquitoes such as Culex quinquefasciatus Say (Diptera: Culicidae) are important vectors of organisms that cause disease in humans. Research into the development of effective standardized odour baits for blood-fed females (oviposition attractants), to enable entomological monitoring of vector populations, is hampered by complex protocols for extraction of physiologically active volatile chemicals from natural breeding site water samples, which have produced inconsistent results. Air entrainment and solvent extraction are technically demanding methods and are impractical for use in resource poor environments where mosquito-borne disease is most prevalent. This study reports the first use of a simple, robust extraction technique, stir bar sorptive extraction (SBSE), to extract behaviourally active small lipophilic molecules (SLMs) present in water samples collected from Cx. quinquefasciatus breeding sites in Tanzania. Extracts from a pit latrine and from a cess pool breeding site attracted more gravid Cx. quinquefasciatus in pair choice bioassays than control extracts, and coupled gas chromatography-electroantennography (GC-EAG) allowed tentative identification of 15 electrophysiologically active chemicals, including the known oviposition attractant, skatole (3-methylindole). Here, we have demonstrated, using simple pair choice bioassays in controlled laboratory conditions, that SBSE is effective for the extraction of behaviourally and electrophysiologically active semiochemicals from mosquito breeding site waters. Further research is required to confirm that SBSE is an appropriate technique for use in field surveys in the search for oviposition cues for Cx. quinquefasciatus.
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Ortiz, Aurelio, Miriam Castro, and Estibaliz Sansinenea. "3,4-Dihydroisocoumarins, Interesting Natural Products: Isolation, Organic Syntheses and Biological Activities." Current Organic Synthesis 16, no. 1 (2019): 112–29. http://dx.doi.org/10.2174/1570179415666180924123439.
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Background:3,4-dihydroisocoumarins are an important small group belonging to the class of naturally occurring lactones isolated from different bacterial strains, molds, lichens, and plants. The structures of these natural compounds show various types of substitution in their basic skeleton and this variability influences deeply their biological activities. These lactones are structural subunits of several natural products and serve as useful intermediates in the synthesis of different heterocyclic molecules, which exhibit a wide range of biological activities, such as anti-inflammatory, antiplasmodial, antifungal, antimicrobial, antiangiogenic and antitumoral activities, among others. Their syntheses have attracted attention of many researchers reporting many synthetic strategies to achieve 3,4-dihydroisocoumarins and other related structures. </P><P> Objective: In this context, the isolation of these natural compounds from different sources, their syntheses and biological activities are reviewed, adding the most recent advances and related developments.Conclusion:This review aims to encourage further work on the isolation and synthesis of this class of natural products. It would be beneficial for synthetic as well as the medicinal chemists to design selective, optimized dihydroisocoumarin derivatives as potential drug candidates, since dihydroisocoumarin scaffolds have significant utility in the development of therapeutically relevant and biologically active compounds.
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Nicosia, Aldo, Alexander Mikov, Matteo Cammarata, et al. "The Anemonia viridis Venom: Coupling Biochemical Purification and RNA-Seq for Translational Research." Marine Drugs 16, no. 11 (2018): 407. http://dx.doi.org/10.3390/md16110407.
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Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis, which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation.
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Bishop-Hurley, Sharon L., Sarah A. Mounter, James Laskey, et al. "Phage-Displayed Peptides as Developmental Agonists for Phytophthora capsici Zoospores." Applied and Environmental Microbiology 68, no. 7 (2002): 3315–20. http://dx.doi.org/10.1128/aem.68.7.3315-3320.2002.
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ABSTRACT As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.
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Tanaka, Naonobu, and Yoshiki Kashiwada. "Phytochemical studies on traditional herbal medicines based on the ethnopharmacological information obtained by field studies." Journal of Natural Medicines 75, no. 4 (2021): 762–83. http://dx.doi.org/10.1007/s11418-021-01545-7.
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AbstractTraditional herbal medicines, which have been used in the matured traditional medical systems as well as those have been used in ethnic medical systems, are invaluable resources of drug seeds. Ethnobotanical and ethnopharmacological survey may provide useful information of these herbal medicines, which are valuable for searching new bioactive molecules. From this viewpoint, we have been performing the ethnobotanical and ethnopharmacological field studies in Yunnan Province and Guangxi Zhuang Autonomous Region, China, and Mongolia. Phytochemical studies on traditional herbal medicines were performed based on the information obtained by our ethnobotanical survey. Herbal medicines used in Uzbekistan and Bangladesh were also investigated on the basis of the ethnopharmacological information obtained from collaborative researchers in the respective regions. Some studies were carried out for searching active substance(s) based on bioassay-guided fractionation and isolation. Over 150 new molecules were isolated in these studies, and their various biological activities were also demonstrated. This review summarizes the results of phytochemical studies of those traditional herbal medicines as well as biological activities of the isolated molecules. Graphic abstract
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Kim, Ji Hoon, Eun Ju Jung, Yun Jung Lee, En Mei Gao, Ahmed Shah Syed, and Chul Young Kim. "Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography." Molecules 25, no. 13 (2020): 3077. http://dx.doi.org/10.3390/molecules25133077.
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A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane–acetonitrile–water (10:2:8, v/v), ethyl acetate–acetonitrile–water (10:2:8, v/v), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v). The lower phase of the n-hexane–acetonitrile–water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate–acetonitrile–water (10:2:8), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.
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Sutthirat, Natwara, Joseph W. Ziller, Jenny Y. Yang, and Zachary Thammavongsy. "Crystal structure of NiFe(CO)5[tris(pyridylmethyl)azaphosphatrane]: a synthetic mimic of the NiFe hydrogenase active site incorporating a pendant pyridine base." Acta Crystallographica Section E Crystallographic Communications 75, no. 4 (2019): 438–42. http://dx.doi.org/10.1107/s2056989019003256.
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The reaction of Ni(TPAP)(COD) {where TPAP = [(NC5H4)CH2]3P(NC2H4)3N} with Fe(CO)5 resulted in the isolation of the title heterobimetallic NiFe(TPAP)(CO)5 complex di-μ-carbonyl-tricarbonyl[2,8,9-tris(pyridin-2-ylmethyl)-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane]ironnickel, [FeNi(C24H30N7P)(CO)5]. Characterization of the complex by 1H and 31P NMR as well as IR spectroscopy are presented. The structure of NiFe(TPAP)(CO)5 reveals three terminally bound CO molecules on Fe0, two bridging CO molecules between Ni0 and Fe0, and TPAP coordinated to Ni0. The Ni—Fe bond length is 2.4828 (4) Å, similar to that of the reduced form of the active site of NiFe hydrogenase (∼2.5 Å). Additionally, a proximal pendant base from one of the non-coordinating pyridine groups of TPAP is also present. Although involvement of a pendant base has been cited in the mechanism of NiFe hydrogenase, this moiety has yet to be incorporated in a structurally characterized synthetic mimic with key structural motifs (terminally bound CO or CN ligands on Fe). Thus, the title complex NiFe(TPAP)(CO)5 is an unique synthetic model for NiFe hydrogenase. In the crystal, the complex molecules are linked by C—H...O hydrogen bonds, forming undulating layers parallel to (100). Within the layers, there are offset π–π [intercentroid distance = 3.2739 (5) Å] and C—H...π interactions present. The layers are linked by further C—H...π interactions, forming a supramolecular framework.
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Kim, Ji Hoon, Je-Seung Jeon, Jung Hoon Kim, et al. "Bioassay-Guided Isolation of Two Eudesmane Sesquiterpenes from Lindera strychnifolia Using Centrifugal Partition Chromatography." Molecules 26, no. 17 (2021): 5269. http://dx.doi.org/10.3390/molecules26175269.
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In this study, a centrifugal partition chromatography (CPC) separation was applied to identify antioxidant-responsive element (ARE) induction molecules from the crude extract of Lindera strychnifolia roots. CPC was operated with a two-phase solvent system composed of n-hexane-methanol-water (10:8.5:1.5, v/v/v) in dual mode (descending to ascending), which provided a high recovery rate (>95.5%) with high resolution. Then, ARE induction activity of obtained CPC fractions was examined in ARE-transfected HepG2 cells according to the weight ratios of the obtained fractions. The fraction exhibiting ARE-inducing activity was further purified by preparative HPLC that led to isolation of two eudesmane type sesquiterpenes as active compounds. The chemical structures were elucidated as linderolide U (1) and a new sesquiterpene named as linderolide V (2) by spectroscopic data. Further bioactivity test demonstrated that compounds 1 and 2 enhanced ARE activity by 22.4-fold and 7.6-fold, respectively, at 100 μM concentration while 5 μM of sulforaphane induced ARE activity 24.8-fold compared to the control.
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Adekenov, S. M. "Natural Sesquiterpene Lactones as Renewable Chemical Materials for New Medicinal Products." Eurasian Chemico-Technological Journal 15, no. 3 (2013): 163. http://dx.doi.org/10.18321/ectj220.
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<p>Literature data and own research results on the technology for isolating natural sesquiterpene lactones such as arglabin, alantolactone, artemisinin, grosheimin, isoalantolactone, parthenolide, santonin and potential possibilities of their use as renewable material for obtaining new compounds as well as biologically active derivatives are generalized in this review. Sesquiterpene lactones from plants are promising sources for the development and practical application of new original medical products possessing antitumor, anti-inflammatory, antimalarial, antiulcer, antiviral and immune-stimulating action. The technology for isolating sesquiterpene lactones is based on the extraction of raw plant material by different organic solvents with the subsequent chromatographic purification. The effective and environmentally safe technology for isolation and purification of sesquiterpene lactone arglabin from <em>Artemisia glabella</em> Kar. et Kir. by the СО<sub>2</sub>-extraction method is developed. Thereat, it was experimentally determined that the method for isolating arglabin from CO<sub>2</sub> extract of <em>Artemisia glabella</em> Kar. et Kir. using centrifugal partition chromatography is effective for preparative isolation of the active substance and its manufacturing application. It is practically important to obtain water-soluble derivatives of biologically active sesquiterpene lactones and also to use the nanotechnology achievements for directed transportation of a molecule of the medicine in the human body thereby reducing toxicity of an active component. Promising direction is chemical modification of molecules in sesquiterpene lactones which are renewable material for obtaining new derivatives, thanks to which it becomes possible to solve two problems at the same time. Firstly, these researches help to obtain derivatives with higher biological activity or improved physical and chemical properties. Secondly, these researches enable us to disclose the mechanism of action of different medicines within the framework of “structure-activity” correlation. The article presents the literature data and own results on chemical modification of sesquiterpene lactones of alantolactone, arglabin, artemisinin, grosheimin, isoalantolactone, parthenolide and santonin. Various reactions on functional groups of these molecules were used to obtain a number of new derivatives of sesquiterpene lactones containing haloid-, pyrazole-, triazole-, amino-, dialkylamino-, hydroxy-, dialkyl phosphonate- and cyclopropane groups, which have shown high physiological activity.</p>
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Domenis, Rossana, Rossella Zanutel, Federica Caponnetto, et al. "Characterization of the Proinflammatory Profile of Synovial Fluid-Derived Exosomes of Patients with Osteoarthritis." Mediators of Inflammation 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/4814987.
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The purpose of this study is to characterize synovial fluid- (SF-) derived exosomes of patients with gonarthrosis comparing two methods of isolation and to investigate their immune regulatory properties. Extracellular vesicles (EVs) have been isolated from inflamed SF by polymer precipitation method and quantified by Exocet kit and by nanoparticle tracking analysis. Vesicles expressed all the specific exosomal markers by immunoblot and FACS. After isolation with Exoquick, a relevant contamination by immune complexes was detected, which required further magnetic bead-based purification to remove. SF-derived exosomes significantly stimulated the release of several inflammatory cytokines and chemokines and metalloproteinases by M1 macrophages but did not influence the expression of CD80 and CD86 costimulatory molecules. In conclusion, we characterized purified exosomes isolated from inflamed SF and demonstrate that purified exosomes are functionally active in their ability to stimulate the release of proinflammatory factors from M1 macrophages. Our data indicate that SF-derived exosomes from gonarthrosis patients play a role in disease progression.
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Kuzmenkov, Alexey I., Maria Y. Sachkova, Sergey I. Kovalchuk, Eugene V. Grishin, and Alexander A. Vassilevski. "Lachesana tarabaevi, an expert in membrane-active toxins." Biochemical Journal 473, no. 16 (2016): 2495–506. http://dx.doi.org/10.1042/bcj20160436.
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In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18–79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present ‘idealized’ amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61–79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.
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Wainer, I. W., M. Crabos, and J. F. Cloix. "Rapid large-scale isolation of biologically active molecules using reversed-phase “flash” chromatography: Initial purification of endogenous Na+, K+-ATPase inhibitors from human urine." Journal of Chromatography B: Biomedical Sciences and Applications 338 (January 1985): 417–21. http://dx.doi.org/10.1016/0378-4347(85)80114-6.
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Buee, M., M. Rossignol, A. Jauneau, R. Ranjeva, and G. Bécard. "The Pre-Symbiotic Growth of Arbuscular Mycorrhizal Fungi Is Induced by a Branching Factor Partially Purified from Plant Root Exudates." Molecular Plant-Microbe Interactions® 13, no. 6 (2000): 693–98. http://dx.doi.org/10.1094/mpmi.2000.13.6.693.
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Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.
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Tava, Valeria, Anna Prigitano, Paolo Cortesi, Maria Carmela Esposto, and Matias Pasquali. "Fusarium musae from Diseased Bananas and Human Patients: Susceptibility to Fungicides Used in Clinical and Agricultural Settings." Journal of Fungi 7, no. 9 (2021): 784. http://dx.doi.org/10.3390/jof7090784.
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Fusarium musae belongs to the Fusarium fujikuroi species complex. It causes crown rot disease in banana but also keratitis and skin infections as well as systemic infections in immunocompromised patients. Antifungal treatments in clinical and agricultural settings rely mostly on molecules belonging to the azole class. Given the potential risk of pathogen spread from food to clinical settings, the goal of the work was to define the level of susceptibility to different azoles of a worldwide population of F. musae. Eight fungicides used in agriculture and five antifungals used in clinical settings (4 azoles and amphotericin B) were tested using the CLSI (Clinical and Laboratory Standards Institute) protocol methodology on 19 F. musae strains collected from both infected patients and bananas. The level of susceptibility to the different active molecules was not dependent on the source of isolation with the exception of fenbuconazole and difenoconazole which had a higher efficiency on banana-isolated strains. Minimal inhibitory concentrations (MICs) of the different molecules ranged from 0.12–0.25 mg/L for prochloraz to more than 16 mg/L for tetraconazole and fenbuconazole. Compared to the F. verticillioides, F. musae MICs were higher suggesting the importance of monitoring the potential future spread of this species also in clinical settings.
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Jaffe, Aron B., Pontus Aspenström, and Alan Hall. "Human CNK1 Acts as a Scaffold Protein, Linking Rho and Ras Signal Transduction Pathways." Molecular and Cellular Biology 24, no. 4 (2004): 1736–46. http://dx.doi.org/10.1128/mcb.24.4.1736-1746.2004.
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ABSTRACT Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.
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Hamimed, Souad, Nadji Boulebda, Hocine Laouer, and Abdelmalik Belkhiri. "Bioactivity-guided isolation of alkamides from a cytotoxic fraction of the ethyl acetate extract of Anacyclus pyrethrum (L.) DC. roots." Current Issues in Pharmacy and Medical Sciences 31, no. 4 (2018): 180–85. http://dx.doi.org/10.1515/cipms-2018-0033.
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Abstract Introduction. The alcohol extract of Pellitory (Anacyclus pyrethrum) roots has been previously shown to exert anticancer activities on the Human Colorectal Cancer Cell Line (HCT) by targeting apoptosis, metastasis and cell cycle arrest. However, the nature of the cytotoxic molecules associated with this activity remains unexplored. Aims. This study aims to reinvestigate Pellitory root extract as regard to its cytotoxic activity and to proceed to a bioguided fractionation to explore its active fraction and to give new insight in their phytochemical constituents. Methods. Powdered roots were subjected to repeated extraction with Petroleum ether (Pe), Chloroform (Ch), Ethyl acetate (Ea) and Methanol (Me). Pellitory extracts were then screened for cytotoxic activity using the Brine Shrimp Lethality (BSL) bioassay. Results. Ea extract exhibited a marked cytotoxic activity, with LC50 of 249.26 μg/mL in the BSL bioassay. The remaining extracts (Pe,Ch,Me) treated groups exhibited no or low mortality in the range of tested concentrations (1-1000 µg/mL). BSL assay-guided chromatographic fractionation of Ea active Extract revealed a highly cytotoxic fraction (F11) with LC50 of 42.5 µg/mL. Multistep purifications of the active F11 fraction afforded four alkamides, namely N-isobutyldeca-2,4-dienamide or Pellitorine (I), N-propyldodeca- -2,8-dienamide (II), N-isobutyltetradeca-2,4-dienamide (III) and N-propylnona-2,5- -dienamide (IV). Conclusions. This study suggests that cytotoxic activity is localized mainly in the ethyl acetate extract (Ea) of pellitory roots. BSL assay fractionation of this active extract leads to the isolation of four alkamides, including pellitorine (I). While this isobutyl alkamide has previously shown strong cytotoxic activities against human cancer cell lines, the other compounds (II to IV) were not previously reported as cytotoxic. Subsequently, the isolated alkamides will be considered in future study as candidates for in depth in-vitro evaluation of their cytotoxicity against cancer and normal cell lines. Finally, through this study, BSL assay demonstrate again its usefulness as bench-top assay in exploring plant extracts for cytotoxic compounds.
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Lakshmi, Vijai, and Sheela Ghosal. "In vitro and in vivo Antiamoebic Potential of Actinopyga lecanora (Jaeger)." Bangladesh Pharmaceutical Journal 18, no. 2 (2015): 118–20. http://dx.doi.org/10.3329/bpj.v18i2.24308.
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Human amoebiasis, due to Entamoeba histolytica infection, is mainly associated with morbidity thus affecting the quality of life and pace of development in the countries with warm climatic conditions. So far, the available drugs provide only symptomatic relief and they are not devoid of side effects. This leads to obtain novel molecules from natural sources having antiamoebic activity. The methanol extract of Actinopyga lecanora (Jaeger) displayed antiamoebic activity. It showed MIC 125 ?g/ml in our in-vitro studies, but when it was tested in rats, it revealed 88% inhibition of trophozoites at the dose of 900 mg/kg body weight against Entamoeba histolytica. Further work is in progress for the isolation and characterization of active molecules.Bangladesh Pharmaceutical Journal 18(2): 118-120, 2015
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Gensure, Robert C., Bhaskar Ponugoti, Yasemin Gunes, et al. "Identification and Characterization of Two Parathyroid Hormone-Like Molecules in Zebrafish." Endocrinology 145, no. 4 (2004): 1634–39. http://dx.doi.org/10.1210/en.2003-0964.
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Abstract Zebrafish (Danio rerio) have receptors homologous to the human PTH (hPTH)/PTHrP receptor (PTH1R) and PTH-2 receptor (PTH2R) and an additional receptor (PTH3R) with high homology to the PTH1R. To find natural ligands for zPTH1R and zPTH3R, we searched the zebrafish genomic database and discovered two distinct regions that, when translated (zPTH1 and zPTH2), showed high homology to hPTH. Isolation of cDNAs and determination of the intron/exon boundaries revealed genomic structures which were similar to known PTHs. Peptides consisting of the first 34 amino acids after the pre- and prosequences of the zebrafish PTHs (zPTHs) were synthesized and were shown to be fully active at the hPTH1R. zPTH2(1–34) was, however, approximately 30-fold less potent at the zPTH1R than hPTH(1–34), hPTHrP(1–36), and zPTH1(1–34). When tested with zPTH3R, zPTH1(1–34) and hPTHrP(1–36) showed similar potencies, whereas the potency of zPTH2(1–34) was moderately (3-fold) reduced. To determine whether other fishes have multiple PTHs, we searched the genomic database of the Japanese pufferfish (Takifugu rubripes) and identified zPTH1 and zPTH2 homologs. Phylogenetic analysis showed that PTHs from zebrafish and pufferfish are more closely related to each other than to known mammalian PTH homologs or to PTHrP and tuberoinfundibular peptide of 39 residues. This is consistent with evolution of two teleost PTH-like peptides occurring after the evolutionary divergence between fishes and mammals. Overall, the PTH system appears more complex in fishes than in mammals, providing evidence of continued evolution in nontetrapod species. The availability of multiple forms of fish PTH and their receptors provide additional tools for PTH ligand/receptor structure-function studies.
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Yan, Litao, Jin Chu, Mingshu Li, et al. "Pharmacological Properties of the Medical Maggot: A Novel Therapy Overview." Evidence-Based Complementary and Alternative Medicine 2018 (2018): 1–11. http://dx.doi.org/10.1155/2018/4934890.
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In the last decade, maggot has been hailed as the miraculous “medicinal maggot” for its diverse properties, including antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities. The fact that maggots show so many beneficial properties has increased the interest in these tiny larvae dramatically. Whilst there is relatively abundant clinical evidence to demonstrate the success of maggots as debridement agents, not so much emphasis has been placed on the basic science evidence, which was a combination of physical and biochemical actions. This review differs from those earlier works in that it is undertaken to provide an update of the latest scientific basis published on maggot, particularly active ingredients within maggot excretions/secretions (ES). Further investigations should focus on the isolation, identification, recombination, transgenosis, and mass production of the beneficial molecules within maggots.
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Stierle, Andrea A., and Donald B. Stierle. "Bioactive Secondary Metabolites from Acid Mine Waste Extremophiles." Natural Product Communications 9, no. 7 (2014): 1934578X1400900. http://dx.doi.org/10.1177/1934578x1400900738.
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The extremophilic microbes of the Berkeley Pit Lake are a valuable source of new and interesting secondary metabolites. It is of particular interest that these acidophilic microbes produce small molecule inhibitors of pathways associated with low pH and high Eh. These same small molecules also inhibit molecular pathways induced by reactive oxygen species (ROS) and inflammation in mammalian cells. Low pH is a hallmark of inflammation and high Eh is one of ROS, so the suitability of this collection as a source of bioactive metabolites is actually quite biorational. Compound isolation was guided by inhibition of caspase-1 and matrix metalloproteinase-3, and active compounds were sent to the National Cancer Institute-Developmental Therapeutics Program and Memorial Sloan Kettering Cancer center for evaluation as either antiproliferative or cytotoxic agents.
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Khurelbaatar, Tsendsuren, Alexander Gliserin, Je-Hoi Mun, Jaeuk Heo, Yunman Lee, and Dong-Eon Kim. "Realization of a Continuously Phase-Locked Few-Cycle Deep-UV/XUV Pump-Probe Beamline with Attosecond Precision for Ultrafast Spectroscopy." Applied Sciences 11, no. 15 (2021): 6840. http://dx.doi.org/10.3390/app11156840.
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Chemical and physical processes in molecules can be controlled through the manipulation of quantum interferences between rotational, vibrational, and electronic degrees of freedom. Most of the past efforts have been focused on the control of nuclear dynamics. Even though electronic coherence and its coupling to nuclear degrees of freedom may profoundly affect the outcome of these processes, electron dynamics have received less attention. Proper investigation of electron dynamics in materials demands ultrafast sources in the visible, ultraviolet (UV), and extreme ultraviolet (XUV) spectral region. For this purpose, a few-cycle deep-UV and XUV beamlines have been constructed for studying ultrafast electron dynamics in molecules. To ensure the required high temporal resolution on the attosecond time scale, vibration isolation from environmental mechanical noise and active stabilization have been implemented to achieve attosecond timing control between pump and probe pulses with excellent stability. This is achieved with an actively phase-stabilized double-layer Mach-Zehnder interferometer system capable of continuous time-delay scans over a range of 200 fs with a root-mean-square timing jitter of only 13 as over a few seconds and ~80 as of peak-to-peak drift over several hours.
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van den Heuvel, Marianne J., Barbara J. Jefferson, and Robert M. Jacobs. "Isolation of a Bovine Plasma Fibronectin-Containing Complex Which Inhibits the Expression of Bovine Leukemia Virus p24." Journal of Virology 79, no. 13 (2005): 8164–70. http://dx.doi.org/10.1128/jvi.79.13.8164-8170.2005.
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ABSTRACT Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the “plasma blocking factor” (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65°C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.
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Stephen, Cayman, Abdelfatteh El Omri, and Lukasz M. Ciesla. "Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices." ADMET and DMPK 6, no. 3 (2018): 200–214. http://dx.doi.org/10.5599/admet.535.
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Secondary plant metabolites are evolutionary-designed molecules that interact with multiple biological targets in human organisms. Identification of pharmacologically active phytochemicals is usually a time consuming and costly process. Cellular membrane affinity chromatography (CMAC) allows the detection of secondary metabolites present in complex natural matrices, e.g. plant extracts and their interactions with the immobilized fully-functional transmembrane proteins. After the isolation process of the binding compounds, CMAC columns can be used to study the binding process between the potential new ligands and the immobilized transmembrane protein target. The following parameters can be determined using CMAC columns: binding affinity (Kd), association rate constant (kon), dissociation rate constant (koff) and the equilibrium constant for complex formation (K). This review summarizes the preparation steps and the use of CMAC columns in the drug discovery process of new potential drug leads present in complex natural matrices.
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YOUSAFZAI, Faridoon K., Martin BUCK, and Barry E. SMITH. "Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine." Biochemical Journal 318, no. 1 (1996): 111–18. http://dx.doi.org/10.1042/bj3180111.
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Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 of Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, α-Cys-89 and β-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal α2β2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chromatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron–molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules.
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Calderón, C., Z. H. Huang, D. A. Gage, E. M. Sotomayor, and D. M. Lopez. "Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine." Journal of Experimental Medicine 180, no. 3 (1994): 945–58. http://dx.doi.org/10.1084/jem.180.3.945.
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Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a decreased capacity to kill tumor targets. This effect is due to an impaired ability to produce nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor that inhibits both NO production/release and cytotoxicity of LPS-activated peritoneal exudate macrophages (PEM). However, other complex macrophage functions such as phagocytosis, superoxide production, mitochondrial dehydrogenase activity, and synthesis of proteins were not reduced by this factor. The NO inhibitor has been found to be lipid in nature. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was unambiguously characterized as phosphatidyl serine (PS) by fast atom bombardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activated PEM pretreated with PS. The ubiquity of PS in the inner leaflet of biological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function that contributes to the protection against developing neoplasms.
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Zhang, Xiao-Yue, Yi-Han Liu, Da-Zhi Liu, Jia-Yang Xu, and Qiang Zhang. "Insulin-Mimic Components in Acer truncatum Leaves: Bio-Guided Isolation, Annual Variance Profiling and Regulating Pathway Investigated by Omics." Pharmaceuticals 14, no. 7 (2021): 662. http://dx.doi.org/10.3390/ph14070662.
Full textAbstract:
Insulin mimic can promote transporting glucose to muscle tissue and accelerate glucose consumption. It is commonly occurring in many functional foods or traditional medicines. Anti-diabetes molecules from food sources are highly safe and suitable for long-term use to prevent early diabetes. The leaves of Acer truncatum was found glucose uptake promotion in our phenotypic screening. However, its bioactive components and mechanism are still unclear. We collected leaves from trees of different ages (2, 3, 4, 7 and 11 years old) and profiled the ingredients by LC-MS/MS. The essential active component (myricitrin) was acquired following bio-guide on a whole organism Zebrafish (Danio rerio). Its content in the leaves was not affected by tree ages. Therefore, myricitrin can serve as a quality mark for functional foods derived from A. truncatum leaves. The transcriptomic and metabolomic analysis in Zebrafish explored the differentially expressed genes and metabolites. Based on joint-pathway enrichment and qRT-PCR verification, the critical bioactive component myricitrin was found to affect toll-like receptors signaling pathways to regulate glucose uptake. Our findings disclosed a bioactive marker (myricitrin) in A. truncatum leaves and explored its regulation mechanism, which rationalized the anti-diabetes function of the herbal food.
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Said Hassane, Charifat, Mireille Fouillaud, Géraldine Le Goff, et al. "Microorganisms Associated with the Marine Sponge Scopalina hapalia: A Reservoir of Bioactive Molecules to Slow Down the Aging Process." Microorganisms 8, no. 9 (2020): 1262. http://dx.doi.org/10.3390/microorganisms8091262.
Full textAbstract:
Aging research aims at developing interventions that delay normal aging processes and some related pathologies. Recently, many compounds and extracts from natural products have been shown to delay aging and/or extend lifespan. Marine sponges and their associated microorganisms have been found to produce a wide variety of bioactive secondary metabolites; however, those from the Southwest of the Indian Ocean are much less studied, especially regarding anti-aging activities. In this study, the microbial diversity of the marine sponge Scopalina hapalia was investigated by metagenomic analysis. Twenty-six bacterial and two archaeal phyla were recovered from the sponge, of which the Proteobacteria phylum was the most abundant. In addition, thirty isolates from S. hapalia were selected and cultivated for identification and secondary metabolites production. The selected isolates were affiliated to the genera Bacillus, Micromonospora, Rhodoccocus, Salinispora, Aspergillus, Chaetomium, Nigrospora and unidentified genera related to the family Thermoactinomycetaceae. Crude extracts from selected microbial cultures were found to be active against seven targets i.e., elastase, tyrosinase, catalase, sirtuin 1, Cyclin-dependent kinase 7 (CDK7), Fyn kinase and proteasome. These results highlight the potential of microorganisms associated with a marine sponge from Mayotte to produce anti-aging compounds. Future work will focus on the isolation and the characterization of bioactive molecules.
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Chen, Liang, Dimitra L. Capone, and David W. Jeffery. "Analysis of Potent Odour-Active Volatile Thiols in Foods and Beverages with a Focus on Wine." Molecules 24, no. 13 (2019): 2472. http://dx.doi.org/10.3390/molecules24132472.
Full textAbstract:
Certain volatile thiols are some of the most potent odour-active molecules that are found in nature. Thiols play significant roles in the aroma qualities of a range of foods and beverages, including wine, with extremely low odour detection thresholds (nanogram per litre range). A fundamental understanding of their formation, fate, and impact essentially depends on the development of suitable analytical methods. The analysis of volatile thiols in foods and beverages is a challenging task when considering (1) the complexity of food and beverage matrices and (2) that thiols are highly reactive, low molecular-weight volatiles that are generally present at trace to ultra-trace concentrations. For the past three decades, the analytical evaluation of volatile thiols has been intensively performed in various foods and beverages, and many novel techniques related to derivatisation, isolation, separation, and detection have been developed, particularly by wine researchers. This review aims to provide an up-to-date overview of the major analytical methodologies that are proposed for potent volatile thiol analysis in wine, foods, and other beverages. The analytical challenges for thiol analysis in foods and beverages are outlined, and the main analytical methods and recent advances in methodology are summarised and evaluated for their strengths and limitations. The key analytical aspects reviewed include derivatisation and sample preparation techniques, chromatographic separation, mass spectrometric detection, matrix effects, and quantitative analysis. In addition, future perspectives on volatile thiol research are also suggested.
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Upadhyay, Harish C. "Medicinal Chemistry of Alternative Therapeutics: Novelty and Hopes with Genus Ammannia." Current Topics in Medicinal Chemistry 19, no. 10 (2019): 784–94. http://dx.doi.org/10.2174/1568026619666190412101047.
Full textAbstract:
The plants have formed the basis of folklore remedy since the beginning of human civilization. The cumulative human endeavor and experience over a period of thousands of years developed into well to organize traditional medicine systems viz. Ayurvedic, Unani, Chinese amongst others. Across the world, traditional medicine is either the mainstay of health care or serves as a complement to modern drugs. In view of worldwide use of traditional medicines, World Health Organization launched ‘WHO-Traditional Medicine Strategy 2014-2023’ for the development of strong policies regarding knowledge-base, safety, quality-control and effectiveness of traditional/alternative therapeutics for national health systems. Besides their use in traditional medicine, plants have always been a good source of modern drug/pharmacologically active molecules. More than half of the modern pharmaceuticals are either plant isolates or their derivatives. The plant-based drugs are not only effective, but have better compatibility with human biological systems because of more biologically relevant chemistry, hence lesser side effects. Some of the species of genus Ammannia (Lythraceae) have been reported for their magical medicinal values. Many herbal formulations containing Ammannia spp. have been patented for treatment of serious diseases/disorders like cancer, spinal disease, human female infertility, chronic tonsillitis, pelvic inflammatory disease, treatment of bladder stones, urinary tract infections, dermatitis etc. The uses of Ammannia spp. in traditional medicine have been further verified by the biological activities of their extracts as well as isolation of bioactive phytomolecules. The current review provides details about Ammannia spp.; its use in folklore remedy, herbal formulations, biological activities of extracts, isolation of bioactive phytomolecules and SAR study of semi-synthetic derivatives to analyze the possibility of new drug molecules of plant origin.
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Saadouli, Ilhem, Imène Zendah El Euch, Emna Trabelsi, et al. "Isolation, Characterization and Chemical Synthesis of Large Spectrum Antimicrobial Cyclic Dipeptide (l-leu-l-pro) from Streptomyces misionensis V16R3Y1 Bacteria Extracts. A Novel 1H NMR Metabolomic Approach." Antibiotics 9, no. 5 (2020): 270. http://dx.doi.org/10.3390/antibiotics9050270.
Full textAbstract:
Streptomyces is the most frequently described genus of Actinomycetes, a producer of biologically active secondary metabolites. Indeed, the Streptomyces species produces about 70% of antibiotics and 60% of antifungal molecules used in agriculture. Our study was carried out with the goal of isolating and identifying antimicrobial secondary metabolites from Streptomyces misionensis V16R3Y1 isolated from the date palm rhizosphere (southern Tunisia). This strain presented a broad range of antifungal activity against Fusarium oxysporum, Aspergillus flavus, Penicillium expansum, Aspergillus niger, Candida albicans, Candida metapsilosis, and Candida parapsilosis and antibacterial activity against human pathogenic bacteria, including Escherichia fergusonii, Staphylococcus aureus, Salmonella enterica, Enterococcus faecalis, Bacillus cereus and Pseudomonas aeruginosa. The purification procedure entailed ethyl acetate extract, silica gel column, and thin layer chromatography. Based on 1H NMR metabolomic procedure application, also supported by the GC-MS analysis, cyclic dipeptide (l-Leucyl-l-Proline) was identified as the major compound in the bioactive fraction. In order to confirm the identity of the active compound and to have a large quantity thereof, a chemical synthesis of the cyclic dipeptide was performed. The synthetic compound was obtained with a very good yield (50%) and presented almost the same effect compared to the extracted fraction. This study indicates for the first time that Streptomyces misionensis V16R3Y1 exhibits a broad spectrum of antimicrobial activities, produced cyclic dipeptide (l-Leucyl-l-Proline) and might have potential use as a natural agent for pharmaceutical and agri-food applications.
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Yang, Ning Ning, Qing Yun Ma, Fan Dong Kong, et al. "Napthrene Compounds from Mycelial Fermentation Products of Marasmius berteroi." Molecules 25, no. 17 (2020): 3898. http://dx.doi.org/10.3390/molecules25173898.
Full textAbstract:
The metabolites of the genus Marasmius are diverse, showing good research prospects for finding new bioactive molecules. In order to explore the active metabolites of the fungi Marasmius berteroi, the deep chemical investigation on the bioactive compounds from its cultures was undertaken, which led to the isolation of three new naphthalene compounds dipolynaphthalenes A–B (1,2) and naphthone C (3), as well as 12 known compounds (4–15). Compounds 1, 2, and 4 are dimeric naphthalene compounds. Their structures were elucidated by MS, 1D and 2D NMR spectroscopic data, as well as ECD calculations. Compounds 2–4 and 7 exhibited acetylcholinesterase (AChE) inhibitory activities at the concentration of 50 μg/mL with inhibition ratios of 42.74%, 44.63%, 39.50% and 51.49%, respectively. Compounds 5 and 7,8 showed weak inhibitory activities towards two tumor cell lines, with IC50 of 0.10, 0.076 and 0.058 mM (K562) and 0.13, 0.18, and 0.15 mM (SGC-7901), respectively.
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Luparello, Claudio. "Marine Animal-Derived Compounds and Autophagy Modulation in Breast Cancer Cells." Foundations 1, no. 1 (2021): 3–20. http://dx.doi.org/10.3390/foundations1010002.
Full textAbstract:
It is known that in breast cancer biology, autophagy mainly plays a cytoprotective and anti-apoptotic role in vitro, being conceivably responsible for cell resistance to drug exposure and a higher metastatic attitude in vivo. Thus, the development of novel autophagy-targeting agents represents a valuable strategy to improve the efficacy of anticancer interventions. It is widely acknowledged that the enormous biodiversity of marine organisms represents a highly promising reserve for the isolation of bioactive primary and secondary metabolites targeting one or several specific molecular pathways and displaying active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of marine animal-derived extracts or isolated compounds which exert a modulatory effect on the autophagic process in breast cancer cells and list them with respect to the taxonomical hierarchy of the producing species. Where available, the molecular and biochemical aspects associated with the molecules or extracts under discussion will be also summarized.
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Abdelhameed, Reda F. A., Enas E. Eltamany, Dina M. Hal, et al. "New Cytotoxic Cerebrosides from the Red Sea Cucumber Holothuria spinifera Supported by In-Silico Studies." Marine Drugs 18, no. 8 (2020): 405. http://dx.doi.org/10.3390/md18080405.
Full textAbstract:
Bioactivity-guided fractionation of a methanolic extract of the Red Sea cucumber Holothuria spinifera and LC-HRESIMS-assisted dereplication resulted in the isolation of four compounds, three new cerebrosides, spiniferosides A (1), B (2), and C (3), and cholesterol sulfate (4). The chemical structures of the isolated compounds were established on the basis of their 1D NMR and HRMS spectral data. Metabolic profiling of the H. spinifera extract indicated the presence of diverse secondary metabolites, mostly hydroxy fatty acids, diterpenes, triterpenes, and cerebrosides. The isolated compounds were tested for their in vitro cytotoxicities against the breast adenocarcinoma MCF-7 cell line. Compounds 1, 2, 3, and 4 displayed promising cytotoxic activities against MCF-7 cells, with IC50 values of 13.83, 8.13, 8.27, and 35.56 µM, respectively, compared to that of the standard drug doxorubicin (IC50 8.64 µM). Additionally, docking studies were performed for compounds 1, 2, 3, and 4 to elucidate their binding interactions with the active site of the SET protein, an inhibitor of protein phosphatase 2A (PP2A), which could explain their cytotoxic activity. This study highlights the important role of these metabolites in the defense mechanism of the sea cucumber against fouling organisms and the potential uses of these active molecules in the design of new anticancer agents.