Academic literature on the topic 'Isolation ofbiologically active molecules'

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.

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.

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.

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.

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

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.

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.

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.

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.

У оквиру ове докторске дисертације изведено јеиспитивање различитих екстракционих поступака заизоловање апигенина из цвета камилице, као и евалуацијабиолошке активности добијених екстраката. Полазнибиљни материјал сачињавале су две групе латицакамилице: ферментисане и неферментисане (нативне).Екстракција ферментисаних цветова је извођена применомултразвучне екстракције користећи етанол као екстрагенс,а добијени екстракти су се одликовали изузетно високимсадржајем апигенина. Оптимизација екстракције је билаизведена применом методе одзивне површине. Применомелектрон-спин резонанце испитана је антирадикалскаактивност екстраката. Додатно, фармаколошка вредностдобијених екстраката је потврђена и одређивањем њиховогантимикробног и антипролиферативног потенцијала.Нативни цветови камилице су екстраховани применомразличитих екстаркционих техника: микроталасне,ултразвучне, Soxhlet екстракције као и екстракцијесубкритичном водом. Eкстрaкција водом у субкритичномстању се показала супериорнијом у односу на све осталетехнике у погледу садржаја укупних фенола и флавоноида.У циљу добијања екстраката са максималним садржајемапигенина изведена је оптимизација овог екстракционогпроцеса. Изоловање чистог апигенина је изведено изекстракта добијеног под оптималним екстракцијомусловима (однос дрога:растварач 1:30, брзина мешања 3 Hz,притисак 45 bar, температура 115&ordm;C, време 30 мин,концентрација модификатора 0,001 М) применом поступкаколонске хроматографије на стубу полиамида. Хемијскипрофил као и садржај појединачних полифенолнихкомпонената у екстрактима добијеним на различитимпритисцима, температурама и уз присуство модификатораразличитих концентрација одређен је применом UHPLCDAD-HESI-MS/MS. У свим анализираним екстрактимадетектован је велики број полифенолних компонената, докје апигенин у свима био доминантно једињење. Садржајапигенина у екстракту добијеном под оптималнимекстракционим условима је износио 1.700,34 mg/kg.Применом седам различитих тестова извршена јеевалуација антиоксидативног и антирадикалскогпотенцијала екстраката. Антимикробни потенцијалекстраката је одређен за осам различитих микробнихлинија. in vitro тестовима испитана је способностинхибиције &alpha;-амилазе, &alpha;-глукозидазе и тирозиназе.Деловањем на раст три хистолошки различите ћелијскелиније, испитана је антипролиферативна активностекстраката добијених субкритичном водом.Антимотилитетна активност обе групе екстраката(ферментисаних и неферментисаних цветова) одређена је уin vitro условима.<br>U okviru ove doktorske disertacije izvedeno jeispitivanje različitih ekstrakcionih postupaka zaizolovanje apigenina iz cveta kamilice, kao i evaluacijabiološke aktivnosti dobijenih ekstrakata. Polaznibiljni materijal sačinjavale su dve grupe laticakamilice: fermentisane i nefermentisane (nativne).Ekstrakcija fermentisanih cvetova je izvođena primenomultrazvučne ekstrakcije koristeći etanol kao ekstragens,a dobijeni ekstrakti su se odlikovali izuzetno visokimsadržajem apigenina. Optimizacija ekstrakcije je bilaizvedena primenom metode odzivne površine. Primenomelektron-spin rezonance ispitana je antiradikalskaaktivnost ekstrakata. Dodatno, farmakološka vrednostdobijenih ekstrakata je potvrđena i određivanjem njihovogantimikrobnog i antiproliferativnog potencijala.Nativni cvetovi kamilice su ekstrahovani primenomrazličitih ekstarkcionih tehnika: mikrotalasne,ultrazvučne, Soxhlet ekstrakcije kao i ekstrakcijesubkritičnom vodom. Ekstrakcija vodom u subkritičnomstanju se pokazala superiornijom u odnosu na sve ostaletehnike u pogledu sadržaja ukupnih fenola i flavonoida.U cilju dobijanja ekstrakata sa maksimalnim sadržajemapigenina izvedena je optimizacija ovog ekstrakcionogprocesa. Izolovanje čistog apigenina je izvedeno izekstrakta dobijenog pod optimalnim ekstrakcijomuslovima (odnos droga:rastvarač 1:30, brzina mešanja 3 Hz,pritisak 45 bar, temperatura 115&ordm;C, vreme 30 min,koncentracija modifikatora 0,001 M) primenom postupkakolonske hromatografije na stubu poliamida. Hemijskiprofil kao i sadržaj pojedinačnih polifenolnihkomponenata u ekstraktima dobijenim na različitimpritiscima, temperaturama i uz prisustvo modifikatorarazličitih koncentracija određen je primenom UHPLCDAD-HESI-MS/MS. U svim analiziranim ekstraktimadetektovan je veliki broj polifenolnih komponenata, dokje apigenin u svima bio dominantno jedinjenje. Sadržajapigenina u ekstraktu dobijenom pod optimalnimekstrakcionim uslovima je iznosio 1.700,34 mg/kg.Primenom sedam različitih testova izvršena jeevaluacija antioksidativnog i antiradikalskogpotencijala ekstrakata. Antimikrobni potencijalekstrakata je određen za osam različitih mikrobnihlinija. in vitro testovima ispitana je sposobnostinhibicije &alpha;-amilaze, &alpha;-glukozidaze i tirozinaze.Delovanjem na rast tri histološki različite ćelijskelinije, ispitana je antiproliferativna aktivnostekstrakata dobijenih subkritičnom vodom.Antimotilitetna aktivnost obe grupe ekstrakata(fermentisanih i nefermentisanih cvetova) određena je uin vitro uslovima.<br>In the frame of this thesis different extraction approaches forapigenin isolation from chamomile ligulate flowers wereexamined and biological activity of obtained extracts wasevaluated. Starting plant samples included fermented andnonfermented (native) flowers.Extraction of fermented flowers was performed by usingultrasound-assisted extraction with ethanol. The concentrationof apigenin was high in obtained extracts. Optimization of theextraction procedures was performed by response surfacemethodology. Antiradical activity of observed extracts wasexamined by electron-spin resonance spectroscopy.Furthermore, pharmacological potential of obtained extractswas confirmed by determining their antimicrobial andantiproliferative activity.Native chamomile flowers were extracted by differentextraction techniques: microwave, ultrasound, Soxhlet andsubcritical water extraction. Subcritical water extractionshowed to be superior in comparison to other applied techniquesin respect to total phenols and flavonoids content. Optimizationof the subcritical water extraction was directed to maximizationof apigenin content. Isolation of pure apigenin from extractsobtained under optimal extraction conditions (sample-tosolventratio 1:30, agitation rate 3 Hz, temperature 115&ordm;C,pressure 45 bar, extraction time 30 min) was performed bypreparative chromatography. Chemical profiles and content ofindividual polyphenolic components in extracts obtained atdifferent pressures, temperatures, and with differentconcentrations of a modifier was determined by UHPLC-DADHESI-MS/MS. In all analyzed extracts the great number ofpolyphenolic components was detected while apigenin was thedominant compound in all extracts. Content of apigenin in theextract obtained under optimal extraction condition was1,700.34 mg/kg. Antioxidant and antiradical potential ofextracts was evaluated according to different mechanisms.Antimicrobial potential of extracts was determined against eightdifferent microbial strains. Ability of extracts to inhibit &alpha;-amylase, &alpha;-glucosidase and tyrosinase was determined by invitro assays. Antiproliferative activity of subcritical waterextracts was defined by testing their influence on the growth ofthree histologically different cell lines.Anti-intestinal motility activity of both group of extracts (nativeand fermented) was determined by in vivo experiments.

Our research group was the first one to microfabricate and demonstrate nano-channels in silicon membranes (1, 2). We employed nano-channeled chips to provide immuno-isolation for cell transplantation towards the treatment of diabetes (3), for biomolecular separation (4), and for the controlled passive and active release of drug molecules from implanted capsules (5). We showed that the constraints placed upon molecular agitation in nano-channels affected their concentration-driven transport kinetics (6, 7). A zero-order passive release of biological molecules was achieved, by the rational tailoring of nano-channels dimensions. This achievement allowed releasing of a constant amount of drugs over a long period of time. However, the development and optimization of many drug therapies require long-term drug delivery with controlled but variable dosage using miniaturized systems (8). Moreover, application such as drug release from implanted devices requires tight operational control, of regulatory agency caliber. We have engaged in the development and characterization of elecroosmotic nano-channels membranes, and present our results in this communication. These include the influence of the drug release rate on nanochannel size, membrane configuration, and applied voltage.