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Fractions Isolated (fraction + isolated)
Selected AbstractsNeurotoxic Effects of Three Fractions Isolated from Tityus serrulatus Scorpion VenomBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2000Ana Leonor A. Nencioni Scorpion venoms contain low molecular weight basic polypeptides, neurotoxins, that are the principal toxic agents. These toxins act on ion channels, promoting a derangement that may result in an abnormal release of neurotransmitters. In the present study we investigated some of the effects of the F, H and J fractions isolated from Tityus serrulatus scorpion venom on the central nervous system of rodents. The venom was partially purified by gel filtration chromatography. The neurotoxic effect of these fractions was studied on convulsive activity after intravenous injection, and on electrographic activity and neuronal integrity of rat hippocampus when injected directly into this brain area. The results showed that intravenous injection of the F and H fractions induced convulsions, and intrahippocampal injection caused electrographic seizures in rats and neuronal damage in specific hippocampal areas. Fraction J injected intravenously reduced the general activity of mice in the open field but induced no changes when injected into the brain. These results suggest that scorpion toxins are able to act directly on the central nervous system promoting behavioural, electrographic and histological modifications. [source] Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilumJOURNAL OF FISH DISEASES, Issue 12 2008E Högfors Abstract Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25,33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti- F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction. [source] Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fractionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Huei-Hsuan Cheng Abstract A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for ,3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal. © 2006 Wiley-Liss, Inc. [source] Protein fraction isolated from epididymal fluid re-associates sperm in vitro: Possible role of serpins in rat rosettes assemblyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2010María A. Monclus Abstract In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an ,-1 antitrypsin and a new protein with an ,-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm,proteases interaction was proposed. Mol. Reprod. Dev. 77: 410,419, 2010. © 2010 Wiley-Liss, Inc. [source] Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patientsACTA OPHTHALMOLOGICA, Issue 8 2009Nils Eide Abstract. Purpose:, Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells. Methods:, Mononuclear cell (MNC) fractions isolated from BM and PB in uveal melanoma patients were examined for tumour cells using our IMS method. Sheep-anti-mouse IgG antibody-coated super paramagnetic particles were conjugated to an anti-melanoma antibody. Microscopy of the magnetic fraction isolated from MNCs was performed to identify and count the number of bead-rosetted cells. The finding of at least two rosettes with coated beads in a 20-,l fraction of a sample was registered as a positive test. The melanocytic nature of the tumour cells was ascertained with a double labelling procedure using fluorescent microparticles. Results:, Using IMS in a study of 328 patients, tumour cells were at initial diagnosis found in BM and PB in 29.9% and 1.6% of cases, respectively. In positive samples, a median of 56 tumour cells (range 2,500) were identified. The captured cells were documented to be of melanocytic origin by the simultaneous binding of fluorescent beads coated with another melanoma-associated antibody. Conclusions:, The IMS method was sensitive and efficient in the detection of occult melanoma tumour cells in BM. The validity of the immunomagnetic technique was strengthened by verifying the melanocytic characteristics of the isolated cells. The IMS procedure identifies intact, vital tumour cells, permitting further molecular characterization, an advantage which makes this method attractive for extended use. The clinical relevance of the findings will be further investigated in follow-up studies with repeated sampling and characterization of the isolated tumour cells. [source] Quantitative solid-state 13C NMR spectroscopy of organic matter fractions in lowland rice soilsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 2 2004R. J. Smernik Summary Spin counting on solid-state 13C cross-polarization (CP) nuclear magnetic resonance (NMR) spectra of two humic fractions isolated from tropical lowland soils showed that only 32,81% of potential 13C NMR signal was detected. The observability of 13C NMR signal (Cobs) was higher in the mobile humic acid (MHA) than in the calcium humate (CaHA) fraction, and increased with increasing intensity of irrigated rice cropping. NMR observability appeared to be related to the nature of the organic carbon, with phenol- and methoxyl-rich samples having the higher values of Cobs. The Bloch decay (BD) technique provided more quantitatively reliable 13C NMR spectra, as evidenced by values of Cobs in the range 91,100% for seven of the eight humic fractions studied. The BD spectra contained considerably more aryl and carbonyl signal, and less O,alkyl and alkyl signal, with the greatest differences between CP and BD spectra observed for the samples with low Cobs(CP). The causes of low CP observability were investigated using the spectral editing technique RESTORE ( REstoration of Spectra via TCH and T One Rho (T1,H) Editing). Rapid T1,H relaxation was found to be primarily responsible for the under-representation of carbonyl carbon, whereas inefficient cross-polarization was primarily responsible for the under-representation of aryl carbon in CP spectra. Proton NMR relaxation rates T1H and T1,H were found to correlate with other NMR properties and also with cropping management. Non-uniform rates of T1H relaxation in two of the CaHA fractions enabled the generation of proton spin relaxation editing subspectra. [source] Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2001Edward D. Karoly Abstract The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5, flanking region, 1607 bases of coding region, and 309 bases of 3, flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-,-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45°C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:300,308, 2001 [source] Cerebral vascular accumulation of Dutch-type A,42, but not wild-type A,42, in hereditary cerebral hemorrhage with amyloidosis, Dutch typeJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Kazuchika Nishitsuji Abstract Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), is an autosomal dominant disorder caused by the Dutch mutation (E693Q) in the ,-amyloid precursor protein. This mutation produces an aberrant amyloid , (A,) species (A,E22Q) and causes severe meningocortical vascular A, deposition. We analyzed the A, composition of the vascular amyloid in the brains of HCHWA-D patients. Immunohistochemistry demonstrated that the vascular amyloid contained both A,40 and A,42, with a high A,40/A,42 ratio. In Western blotting of cerebral microvessel fractions isolated from the brains, both wild-type and Dutch-type A,40 were observed as major species. Reverse-phase HPLC-mass spectrometric analysis of the fractions revealed both wild-type and Dutch-type A,38 as the other main components of the vascular amyloid. Moreover, we detected peaks corresponding to Dutch-type A,42 but not to wild-type A,42. These results suggest a pathogenic role for the mutant A,42 in addition to the mutant A,40 in the cerebral amyloid angiopathy of HCHWA-D. © 2007 Wiley-Liss, Inc. [source] Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fractionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Huei-Hsuan Cheng Abstract A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for ,3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal. © 2006 Wiley-Liss, Inc. [source] A comparison of two methods for the isolation of free and occluded particulate organic matterJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 5 2005Angelika Kölbl Abstract Various methods exist for the isolation of particulate organic matter (POM), one of the soil-organic-matter (SOM) fractions reacting most sensitive on land-use or soil-management changes. A combination of density separation and ultrasonic treatment allows to isolate two types of POM: (1) free POM and (2) POM occluded in soil aggregates. POM fractions are closely linked to their biochemical function for the formation and stabilization of aggregates, therefore methods using different aggregate sizes may result in different POM fractions isolated. We evaluated two physical fractionation procedures to reveal whether they yield different POM fractions with respect to amount and composition, using grassland and arable soils with sandy-loam to sandy,clay-loam texture and thus low macroaggregate stability. Method I used air-dried aggregates of <2.0 mm size and a low-energy sonication for aggregate disruption, method II used field-moist aggregates <6.3 mm and a high-energy,sonication procedure for aggregate disruption. POM fractions were analyzed by elemental analysis (C, N) and CPMAS 13C-NMR spectroscopy. With both methods, about similar proportions of the SOM are isolated as free or occluded POM, respectively. The free- and occluded-POM fractions obtained with method I are also rather similar in C and N concentration and composition as shown by 13C-NMR spectroscopy. Method II isolates a free- and occluded-POM fraction with significantly different C and N concentrations. NMR spectra revealed significant differences in the chemical composition of both fractions from method II, with the occluded POM having lower amounts of O-alkyl C and higher amounts of aryl C and alkyl C than the free POM. Due to the use of larger, field-moist aggregates with minimized sample pretreatment, two distinctly different POM fractions are isolated with method II, likely to be more closely linked to their biochemical function for the formation and stabilization of aggregates. High-energy sonication as in method II also disrupts small microaggregates <63 µm and releases fine intraaggregate POM. This fraction seems to be a significant component of occluded POM, that allows a differentiation between free and occluded POM in sandy soils with significant microaggregation. It can be concluded, that microaggregation in arable soils with sandy texture is responsible for the storage of a more degraded occluded POM, that conversely supports the stabilization of fine microaggregates. Ein Vergleich zweier Methoden zur Isolierung von freier und okkludierter partikulärer organischer Substanz Partikuläres organisches Material (POM) wird im Hinblick auf die Landnutzung als sensitive Fraktion der organischen Bodensubstanz (SOM) angesehen, aber die unterschiedlichen Methoden seiner Isolierung erschweren den Vergleich zwischen verschiedenen Studien. Wir haben zwei physikalische Fraktionierungsmethoden ausgewertet, um zu zeigen, ob sie im Hinblick auf Menge und Zusammensetzung zu unterschiedlichen POM-Fraktionen führen. Hierfür wurden Proben von Grünland- und Ackerböden verwendet. Für Methode I wurden luftgetrocknete Aggregate der Größe <2 mm verwendet, zu deren Zerstörung eine Ultraschallbehandlung mit geringem Energieeintrag eingesetzt wurde. Für Methode II wurden feldfeuchte Aggregate der Größe <6.3 mm und eine Ultraschallbehandlung mit vergleichsweise hoher Energie zur Aggregatzerstörung herangezogen. Mit beiden Methoden konnten zwei POM-Gruppen gewonnen werden: (1) freies POM und (2) in Bodenaggregaten eingeschlossenes POM. Die POM-Fraktionen wurden mittels Elementanalyse (C, N) und CPMAS- 13C-NMR,Spektroskopie untersucht. Methode I zeigte im Hinblick auf Menge und Zusammensetzung nur sehr geringe Unterschiede zwischen freien und okkludierten POM-Fraktionen. Methode II isolierte freie und okkludierte POM-Fraktionen mit signifikant unterschiedlichen C- und N-Konzentrationen. Auch die NMR-Spektren zeigten Unterschiede in der chemischen Zusammensetzung der mit Methode II gewonnenen Fraktionen, die sich in signifikant geringeren O-Alkyl-C-Gehalten bei höheren Aryl-C- und Alkyl-C-Gehalten des okkludierten POM nachweisen ließen. Die Verwendung von größeren, feldfeuchten Aggregaten und die Minimierung der Probenvorbehandlung führt zu einer besseren Differenzierung beider POM-Fraktionen, die wahrscheinlich ihre biologische Funktion besser widerspiegelt. Zusätzlich führt eine Ultraschall-Behandlung mit hohem Energieeintrag zur Zerstörung von kleinen Mikroaggregaten <63 µm und damit zur Freisetzung von feinem Intraaggregat-POM. Diese Fraktion scheint in sandigen Böden mit niedriger Makroaggregat-Stabilität aussagekräftiger zwischen freier und okkludierter POM unterscheiden zu können. Folglich ist eine an das Bodenmaterial angepasste Probenvorbehandlung und Fraktionierungsmethode entscheidend, um eine präzise Charakterisierung der POM-Fraktionen zu gewährleisten. [source] Opsonization of late apoptotic cells by systemic lupus erythematosus autoantibodies inhibits their uptake via an Fc, receptor,dependent mechanismARTHRITIS & RHEUMATISM, Issue 10 2007Esther Reefman Objective Decreased clearance of apoptotic cells is suggested to be a major pathogenic factor in systemic lupus erythematosus (SLE). The aim of this study was to investigate whether the binding of SLE autoantibodies to apoptotic cells influences the phagocytosis of these cells by macrophages. Methods Apoptosis was induced in a human T cell line (Jurkat) and a keratinocyte cell line (HaCaT) by ultraviolet B irradiation. Binding of purified IgG from 26 SLE patients and 15 healthy controls to apoptotic cells was assessed by flow cytometry and Western blotting. Phagocytosis of IgG-opsonized apoptotic cells by monocyte-derived macrophages was assessed by light microscopy. Similar experiments were performed with a monoclonal antibody against SSA/Ro and IgG fractions from 5 patients with Sjögren's syndrome (SS) and 5 patients with rheumatoid arthritis (RA). Results IgG fractions from all 26 SLE patients bound to late apoptotic, but not early apoptotic, cells. IgG fractions isolated from SLE patients with different autoantibody profiles showed comparable levels of binding. IgG fractions from healthy controls did not bind. Opsonization of apoptotic cells with IgG fractions from SLE patients resulted in a significant inhibition of phagocytosis as compared with healthy control IgG fractions. A monoclonal antibody directed against SSA/Ro and IgG isolated from 5 antinuclear antibody (ANA),positive patients with SS were also able to elicit these effects, whereas IgG from 5 ANA-negative patients with RA did not. The inhibitory effect of patient IgG was abolished by blocking either the Fc, receptors (Fc,R) or the constant region of IgG, using a specific Fc-blocking peptide. Conclusion Autoantibodies from SLE patients are able to opsonize apoptotic cells and inhibit their uptake by macrophages via an Fc,R-dependent mechanism. [source] Neurotoxic Effects of Three Fractions Isolated from Tityus serrulatus Scorpion VenomBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2000Ana Leonor A. Nencioni Scorpion venoms contain low molecular weight basic polypeptides, neurotoxins, that are the principal toxic agents. These toxins act on ion channels, promoting a derangement that may result in an abnormal release of neurotransmitters. In the present study we investigated some of the effects of the F, H and J fractions isolated from Tityus serrulatus scorpion venom on the central nervous system of rodents. The venom was partially purified by gel filtration chromatography. The neurotoxic effect of these fractions was studied on convulsive activity after intravenous injection, and on electrographic activity and neuronal integrity of rat hippocampus when injected directly into this brain area. The results showed that intravenous injection of the F and H fractions induced convulsions, and intrahippocampal injection caused electrographic seizures in rats and neuronal damage in specific hippocampal areas. Fraction J injected intravenously reduced the general activity of mice in the open field but induced no changes when injected into the brain. These results suggest that scorpion toxins are able to act directly on the central nervous system promoting behavioural, electrographic and histological modifications. [source] Preparative isolation and purification of alkannin/shikonin derivatives from natural products by high-speed counter-current chromatography,BIOMEDICAL CHROMATOGRAPHY, Issue 2 2009Andreana N. Assimopoulou Abstract Alkannin and shikonin (A/S) and their derivatives have been found in the roots of several Boraginaceous species and are also produced through plant tissue cultures. The chiral compounds A/S are potent pharmaceutical substances with a wide spectrum of biological and pharmacological activities like wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant activity. High-speed counter-current chromatography (HSCCC) was applied for the first time to the separation, preparative isolation and purification of A/S and their esters from extracts of Alkanna tinctoria roots, as well as commercial samples. The constituents of HSCCC fractions and their purity were determined by high-performance liquid chromatography,diode array detection,mass spectrometry (HPLC-DAD-MS), since DAD cannot detect oligomeric A/S derivatives that are present in most of the samples containing the respective monomeric derivatives. The purity of HSCCC fractions was compared with the one of fractions isolated by column chromatography (CC) using as stationary phases silica gel and Sephadex LH-20. As shown, the purity of monomeric alkannin/shikonin was greater by HSCCC than CC separation of commercial A/S samples. Copyright © 2008 John Wiley & Sons, Ltd. [source] Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patientsACTA OPHTHALMOLOGICA, Issue 8 2009Nils Eide Abstract. Purpose:, Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells. Methods:, Mononuclear cell (MNC) fractions isolated from BM and PB in uveal melanoma patients were examined for tumour cells using our IMS method. Sheep-anti-mouse IgG antibody-coated super paramagnetic particles were conjugated to an anti-melanoma antibody. Microscopy of the magnetic fraction isolated from MNCs was performed to identify and count the number of bead-rosetted cells. The finding of at least two rosettes with coated beads in a 20-,l fraction of a sample was registered as a positive test. The melanocytic nature of the tumour cells was ascertained with a double labelling procedure using fluorescent microparticles. Results:, Using IMS in a study of 328 patients, tumour cells were at initial diagnosis found in BM and PB in 29.9% and 1.6% of cases, respectively. In positive samples, a median of 56 tumour cells (range 2,500) were identified. The captured cells were documented to be of melanocytic origin by the simultaneous binding of fluorescent beads coated with another melanoma-associated antibody. Conclusions:, The IMS method was sensitive and efficient in the detection of occult melanoma tumour cells in BM. The validity of the immunomagnetic technique was strengthened by verifying the melanocytic characteristics of the isolated cells. The IMS procedure identifies intact, vital tumour cells, permitting further molecular characterization, an advantage which makes this method attractive for extended use. The clinical relevance of the findings will be further investigated in follow-up studies with repeated sampling and characterization of the isolated tumour cells. [source] | |||||||||||||||||||