Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 49
Filter
1.
Acta amaz ; 50(4): 335-338, out. - dez. 2020.
Article in English | LILACS | ID: biblio-1146378

ABSTRACT

Muitas árvores tropicais possuem dossel alto e folhas não facilmente acessíveis. O uso de tecido de um órgão mais acessível (câmbio) para extração de DNA pode ser uma alternativa para estudos moleculares. Nós adaptamos uma metodologia viável para extrair DNA genômico de tecido cambial coletado no campo para avaliação com PCR. Testamos três condições de armazenamento (dois tampões e sílica gel) e quatro períodos após a coleta. Utilizamos protocolos descritos anteriormente e os testamos em três espécies encontradas em florestas amazônicas e outros biomas: Anadenanthera peregrina var. peregrina, Cedrela fissilis e Ceiba speciosa. Nosso protocolo foi eficaz na obtenção de DNA adequado para sequenciamento e genotipagem de microssatélites. Recomendamos o uso de sílica para armazenamento de longo prazo e o tampão com ácido ascórbico para curto prazo. (AU)


Subject(s)
Ascorbic Acid , DNA , Dithiothreitol
2.
Article in English | WPRIM | ID: wpr-762436

ABSTRACT

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.


Subject(s)
Complement System Proteins , Dithiothreitol , Edetic Acid , Fluorescence , Humans , Immunoglobulin G , Transplant Recipients
3.
Article in English | WPRIM | ID: wpr-728620

ABSTRACT

Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.


Subject(s)
Acinar Cells , Animals , Antioxidants , Calcium , Catalase , Cell Membrane , Cytosol , Dithiothreitol , Extracellular Fluid , Hand , Hydrogen Peroxide , Hydrogen , Inositol 1,4,5-Trisphosphate , Ions , Manganese , Mice , Perfusion , Plasma Membrane Calcium-Transporting ATPases , Plasma , Reactive Oxygen Species
4.
IBJ-Iranian Biomedical Journal. 2017; 21 (4): 270-274
in English | IMEMR | ID: emr-189236

ABSTRACT

Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates [ADCs]. Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates


Methods: In the present study, we investigated the effect of various dithiothreitol [DTT] concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively


Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37[degree]C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively


Conclusion: Optimized site specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs


Subject(s)
Oxidation-Reduction , Pharmaceutical Preparations , Trastuzumab , Temperature , Sulfhydryl Compounds , Alkylation , Electrophoresis, Polyacrylamide Gel , Dithiothreitol
5.
Article in Korean | WPRIM | ID: wpr-34205

ABSTRACT

LW antigens are expressed in higher intensities in D-positive blood cells than D-negative cells, which can result in false identification of anti-D in pretransfusion testing. Although several cases of anti-LW have been reported abroad, to the best of our knowledge, none have been reported in Korea. Herein, we report a case of anti-LW in a 58 year-old RhD positive patient with non-Hodgkin's lymphoma with a positive direct Coombs test and a suspicion of the presence of passive anti-D antibodies because of a history of intravenous immunoglobulin administration. However, during a 5-month follow up, the antibody was confirmed as anti-LW on grounds that it showed weakened reaction in dithiothreitol treated cells and enforced reaction in cord O+ cells when compared to the results from antibody identification panel cells.


Subject(s)
Antibodies , Blood Cells , Coombs Test , Dithiothreitol , Follow-Up Studies , Humans , Immunoglobulins , Korea , Lymphoma, Non-Hodgkin , Sensitivity and Specificity
6.
Article in English | WPRIM | ID: wpr-165359

ABSTRACT

A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 µg/mL. The inter- and intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.


Subject(s)
Administration, Oral , Ascorbic Acid , Chromatography, High Pressure Liquid , Chromatography, Liquid , Dehydroascorbic Acid , Dithiothreitol , Humans , Plasma , Tablets , Volunteers
7.
Article in Korean | WPRIM | ID: wpr-215694

ABSTRACT

BACKGROUND: ABO antibody titration is important in cases such as ABO incompatible hemolytic disease of the fetus and newborn (HDFN), ABO incompatible bone marrow, or solid organ transplantation. This study was conducted in order to evaluate usability of ORTHO VISION (Ortho Clinical Diagnostics, Raritan, USA) designed automated ABO antibody titration equipment. METHODS: The isoagglutination titers were determined in 80 subjects (20 A, 20 B, 40 O (anti-A 20, anti-B 20)) using a conventional tube technique, including a 30 minute room temperature phase (CTT), Dithiothreitol treated manual column agglutination technique (MCAT), and automated column agglutination technique (ACAT) by ORTHO VISION. The concordance of titer was compared within one dilution step between the two methods. RESULTS: The isoagglutinin titers measured by the ACAT with anti-human globulin poly cassette (ACAT_Poly) and anti-human globulin IgG cassette (ACAT_IgG) were the highest and the isoagglutinin titer measured by the MCAT was also higher than that by the CTT. The isoagglutinin titer measured by the ACAT with reverse diluents cassette (ACAT_Reverse) was similar to that measured by the CTT. The concordance of anti-A and anti-B titers between CTT and ACAT_Reverse was 83% and 68%. The concordance of anti-A and anti-B titers between MCAT and ACAT_Poly was 100% and 83%. The concordance of anti-A and anti-B titers between MCAT and ACAT_IgG was 98% and 88%. CONCLUSION: Automated isoagglutinin titration using ACAT_Poly or ACAT_IgG without DTT showed reliable concordance with DTT treated MCAT, and it appears to be a possible replacement for the conventional MCAT method.


Subject(s)
ABO Blood-Group System , Agglutination , Automation , Bone Marrow , Dithiothreitol , Fetus , Humans , Immunoglobulin G , Infant, Newborn , Organ Transplantation , Transplants
8.
Article in Korean | WPRIM | ID: wpr-102158

ABSTRACT

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca2+]i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate (H2DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca2+ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca2+ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca2+-sensitive fluorescence dye fura-2, the [Ca2+]i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.


Subject(s)
Animals , Calcium , Calcium-Transporting ATPases , Central Nervous System Sensitization , Dithiothreitol , Fluoresceins , Fluorescence , Fura-2 , Humans , Membranes , Microscopy, Confocal , Neurons , Nociception , Perfusion , Rats , Reactive Oxygen Species , Substantia Gelatinosa , Thapsigargin , Tissue Donors
9.
Rio de Janeiro; s.n; 2011. 137 p. ilus, graf.
Thesis in Portuguese | LILACS | ID: lil-668610

ABSTRACT

Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, infecção fúngica oportunista com altas taxas de mortalidade afetando, principalmente, pacientes com neutropenia profunda e prolongada. Durante o processo de invasão e disseminação características desta infecção sistêmica, os conídios do fungo inalados e não eliminados pelas células do sistema imune inato diferenciam-se em hifas que, por sua vez, são angioinvasivas. Pouco se conhece sobre as moléculas da parede celular envolvidas na patogênese do A. fumigatus e/ou secretadas por este patógeno. Neste contexto, este trabalho procura ampliar o entendimento desta doença através do estudo de proteínas diferencialmente expressas na superfície de A. fumigatus durante a morfogênese. Foi utilizada uma abordagem proteômica e foram estudados extratos de superfície de células de A. fumigatus em diferentes estágios durante o processo de filamentação. Estas células foram denominadas, de acordo com o tempo de cultivo e a morfologia, como: TG6h (tubo germinativo), H12h ou H72h (hifas). As proteínas de superfície celular foram extraídas, a partir de células intactas, por tatamento brando com o agente redutor DTT (ditiotreitol). Observou-se que o perfil funcional das proteínas expressas por H12h e H72h foi similar, com exceç~çao de proteínas relacionadas à resposta ao estresse, enquanto o perfil para TG6h apresentou diferenças significativas para vários grupos funcionais de proteínas quando comparado às hifas. Desta forma, foram realizados experimentos de proteômica diferencial entre tubo germinativo (TG6h) e a hifa madura (H72h), pela técnica de DIGE (differential gel electrophoresis). Os resultados revelaram que entre as proteínas diferencialmente expressas, aquelas relacionadas às vias de biossíntese e outras denominadas multifuncionais encontram-se superexpressas em TG6h. Em relação às proteínas de resposta a estresse, observou-se que algumas HSPs eram mais expressas neste morfotipo...


Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA), a opportunistic a life-threatening disease for immunocompromised hosts, especially those with acute and prolonged neutropenia. During the invasion and dissemination, which occurs in this systemic infection, the A. fumigatus conidia, after its inhalation, germinates into angioinvasive hyphae in case the innate immune response fails in eliminate these cells. Little is known about the cell wall molecules and/or the secreted proteins involved on the A. fumigatus pathogenesis, at this context the present work aims to amplify the knowledge about the aspergillosis by studying the differentially surface proteins of A. fumigatus during the filamentation process. These cells were denominated according to their morphology and their growtn time as: TG6h (germ tubes), H12h and H72h (hyphae). The surface proteins were mildly extracted from intact cells using the reducing agent DTT (dithiothreitol). The functional profile of the H12h and H72h were similar except for the stress response proteins, while the TG6h presented significant differences for several functional groups. On this base, the DIGE (differential gel electrophoresis) was performed using the surface extracted proteins of the germ tubes (TG6h) and mature hyphae (H72h) cells. The results indicate that multiple functional proteins and proteins related to the biosynthesis pathways were overexpressed at TG6h. Some stress response proteins as the HSPs were overexpressed on this morphotype while the MnSOD, oxidative stress responsive protein, was most abundant at the hyphae. PhiA, an integrant protein of the cell wall, was the only protein with a secretion signal sequence. All other proteins identified on the cell surface lack an identifiable secretion sign, and are denominated atypical proteins. The plasma membrane integrity was verified after the mild extraction using DTT, and also the biotinylation of the cell extracted proteins...


Subject(s)
Aspergillus fumigatus/pathogenicity , Fungal Proteins/analysis , Dithiothreitol , Two-Dimensional Difference Gel Electrophoresis/methods , Hyphae/physiology , Membrane Proteins , Cell Wall , Proteome/analysis , Proteomics/methods
10.
Article in Korean | WPRIM | ID: wpr-200867

ABSTRACT

Several approaches have been introduced to detect allo-antibodies in the presence of warm auto-antibodies, and these methods include warm autoadsorption, cysteine-activated papain and dithiothreitol (ZZAP), and polyethylene glycol (PEG) and dilution of the patient's serum. Among them, the dilution technique is a simple and rapid method. During pretransfusion testing of a 33 year-old systemic lupus erythematosus (SLE) patient with warm auto-antibodies, antibody identification was done by the dilution technique with using serum diluted 1-in-8. The patient demonstrated an anti-Fy(b) pattern of reactivity in his sera. Contrary to our expectations, the phenotype of the erythrocytes was Fy(a+/b+) and the genotype, as assessed by performing PCR-restriction fragment length polymorphism (RFLP), was FY*A/FY*B. These results suggest that the antibody is an autoantibody showing anti-Fy(b) specificities. An antibody identification test using undiluted serum showed the same result when 40 days had passed. We report here on a case with auto-anti-Fy(b) proven by the dilution method in the presence of warm autoantibodies.


Subject(s)
Autoantibodies , Dithiothreitol , Erythrocytes , Genotype , Humans , Indicator Dilution Techniques , Lupus Erythematosus, Systemic , Papain , Phenotype , Polyethylene Glycols , Sensitivity and Specificity
11.
Article in Korean | WPRIM | ID: wpr-188568

ABSTRACT

BACKGROUND: The ABO isoagglutinin titer is useful for the evaluation and observation of ABO incompatible bone marrow transplantation or organ transplantation, yet the results can be different depending on the test methods. Measurement of isoagglutinin titer using the gel test has recently been reported, but this test is expensive. In this study, we investigated isoagglutinin titer distribution of normal individuals using the different tube hemagglutination technique to help select the best test method and to interpret the agglutinin titer. METHODS: Normal healthy individuals were selected from those patients who underwent a physical examination at Ajou University Hospital, during July 2009. Sixty healthy individuals, (10 men and 10 women per each ABO blood group) were recruited for the study. The immediate spin method (IS), the anti-human globulin method with dithiothreitol treatment (DTT-AHG) and the anti-human globulin method without DTT treatment (AHG) were performed simultaneously. The reciprocal of the highest serum dilution that showed macroscopic agglutination 1+ or more was regarded as the isoagglutinin titer. RESULTS: The isoagglutinin titer measured by the AHG was the highest in the all blood groups. In case of blood groups A and B, the isoagglutinin titer by the IS was higher than that by the DTT-AHG, but this was quite the reverse in the case of the blood group O. CONCLUSION: If it is not necessary to distinguish IgM antibody and IgG antibody, then it seems that the AHG is the best practical method of those three methods. It was more sensitive than the IS and more rapid and easier than the DTT-AHG.


Subject(s)
Agglutination , Blood Group Antigens , Bone Marrow Transplantation , Dithiothreitol , Female , Hemagglutination , Humans , Immunoglobulin G , Immunoglobulin M , Male , Organ Transplantation , Physical Examination , Transplants
12.
Article in Korean | WPRIM | ID: wpr-88366

ABSTRACT

BACKGROUND: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. OBJECTIVES: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. METHODS: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. RESULTS: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. CONCLUSION: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice.


Subject(s)
Acrylic Resins , Animals , Antibodies , Candida , Candida albicans , Cell Wall , Chromatography , Chromatography, High Pressure Liquid , Chromatography, Liquid , Chromatography, Reverse-Phase , Dithiothreitol , Endopeptidase K , Ethanolamines , Fungi , Glucan Endo-1,3-beta-D-Glucosidase , Immunization , Immunization, Passive , Mice , Multienzyme Complexes , Peptide Hydrolases , Phospholipases , Proteins , Survival Rate
13.
Pakistan Journal of Medical Sciences. 2008; 24 (3): 364-367
in English | IMEMR | ID: emr-89534

ABSTRACT

Currently, there are no well-defined biochemical markers for identification of myocardial ischemia in normolipidaemic AMI individuals. Biochemical markers such as CK-MB, cTnl and myoglobin, used in assessing cellular necrosis, are not suitable for assessing myocardial ischemia.The introduction of the Ischemia Modified Albumin [IMA] assay for the first time provides emergency physicians with an objective diagnostic study to determine the presence of myocardial ischemia completely within the control of the emergency department. The present study was planned to evaluate IMA concentration in myocardial infarction patients with normal lipid profile. The serum IMA levels were determined in 165 normolipademic MI patients and 165 age-sexes matched healthy volunteers served as control. The levels of IMA were determined by addition of known amount of Cobalt [II] to a serum specimen and measurement of the unbound cobalt [II] by colorimetric assay using dithiothreitol [DTT]. Lipid profile was also analyzed enzymatically in these subjects. The values were expressed as means +/- standard deviation [SD] and data from patients and control was compared using student's 't'-test. Serum IMA levels were significantly increased in Ml patients as compared to control [p<0.001]. Also total cholesterol, TC: HDL-C ratio, triglycerides, LDL-cholesterol, LDL-C: HDL-C ratio and TG: HDL-C ratio were higher in AMI subjects [pc0.001] and HDL-cholesterol were lower in MI patients [p<0.001]. IMA can be used as an alternative biochemical parameter to aid clinical diagnosis which is cost effective


Subject(s)
Humans , Male , Female , Serum Albumin , Ischemia , /blood , Cobalt , Dithiothreitol , Cholesterol , Cholesterol, LDL , Cholesterol, HDL , Triglycerides , Myocardial Infarction/diagnosis
14.
Article in English | WPRIM | ID: wpr-728385

ABSTRACT

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.


Subject(s)
Animals , Arachidonic Acid , Chimera , COS Cells , Dithionitrobenzoic Acid , Dithiothreitol , Membranes , Oxidants
15.
Mycobiology ; : 219-225, 2007.
Article in English | WPRIM | ID: wpr-729941

ABSTRACT

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and 28degrees C. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH (7~10) and temperature (30degrees C~70degrees C) profiles with the optimal for keratinase activity at pH 8 and 45degrees C. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.


Subject(s)
Amino Acid Sequence , Ammonium Sulfate , Animals , Aspergillus flavus , Aspergillus , Biomass , Chickens , Chromatography, Gel , Chromatography, Ion Exchange , Dithiothreitol , Electrophoresis, Polyacrylamide Gel , Feathers , Fungi , Fusarium , Hydrogen-Ion Concentration , Iodoacetic Acid , Meals , Molecular Weight , Phenylmethylsulfonyl Fluoride , Protease Inhibitors , Serine Proteases , Sodium Dodecyl Sulfate , Subtilisin , Subtilisins
16.
Article in English | WPRIM | ID: wpr-27252

ABSTRACT

BACKGROUND: The membrane permeability transition of mitochondria has been suggested to be involved in toxic and oxidative forms of cell injury. Mitochondrial dysfunction is considered to play a critical role in neurodegeneration in Parkinson's disease. Despite the suggestion that indole beta-carbolines may be neurotoxic, these compounds provide a protective effect against cytotoxicity of other neurotoxins. In addition, the effect of indole beta-carbolines on change in the mitochondrial membrane permeability due to reactive nitrogen species (RNS), which may lead to cell death, has not been clarified. METHODS: Differentiated PC12 cells were used as the experimental culture model for the investigation of neuronal cell injury, which occurs in Parkinson's disease. The effect of indole beta-carbolines (harmalol and harmine) on differentiated PC12 cells against toxicity of S-nitroso-N-acetyl-DL-penicillamine (SNAP) was determined by measuring the effect on the change in transmembrane potential, cytochrome c release, formation of ROS, GSH contents, caspase-3 activity and cell viability, and was compared to that of R-(-)-deprenyl. RESULTS: Specific inhibitors of caspases (z-LEHD.fmk, z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell death in PC12 cells due to SNAP. beta-Carbolines and R-(-)-deprenyl attenuated the SNAP-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 25-50 microM. The compounds inhibited the nuclear damage, decrease in mitochondrial transmembrane potential, cytochrome c release and formation of reactive oxygen species caused by SNAP in PC12 cells. beta-Carbolines and R-(-)-deprenyl attenuated the H2O2-induced cell death and depletion of GSH. CONCLUSIONS: The results suggest that indole beta-carbolines attenuate the SNAP-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability, which may be caused by free radicals. Indole beta-carbolines appear to exert a protective effect against the nitrogen species-mediated neuronal cell injury in Parkinson's disease comparable to R-(-)-deprenyl.


Subject(s)
Animals , Antioxidants , Carbolines , Caspase 3 , Caspases , Cell Death , Cell Survival , Cytochromes c , Dithiothreitol , Free Radicals , Melatonin , Membrane Potentials , Membranes , Mitochondria , Mitochondrial Membranes , Neurons , Neurotoxins , Nitrogen , Parkinson Disease , PC12 Cells , Permeability , Reactive Nitrogen Species , Reactive Oxygen Species
17.
Article in Korean | WPRIM | ID: wpr-16566

ABSTRACT

Hemolytic property is a specific feature of bacteria to obtain iron which is essential for its survival in host tissues. Therefore, it is thought to be one of several factors of virulence. The purpose of this study was to investigate the hemolytic properties of Prevotella nigrescens isolated from the teeth diagnosed as pulp necrosis and apical periodontitis under the presence of hemolysin inhibitors such as NaN3 and dithiothreitol, heat, various pH and cultural conditions. The results were as follows; 1. Clinically isolated P. nigrescens strains and standard P. nigrscens ATCC 33563 showed hemolytic activity. 2. P. nigrescens showed higher hemolytic activity against human erythrocytes than sheep or horse erythrocytes. 3. NaN3 and dithiothreitol (DTT) reduced the hemolytic activity of P. nigrescens in a dose dependent manner (p < 0.05). 4. Optimal pH for the maximum hemolytic activity of P. nigrescens was 4.0 and the hemolysin was stable under the 50degrees C, but the hemolytic activity was significantly decreased at 95degrees C. 5. P. nigrescens cultured in 10% CO2 condition showed higher hemolytic activity than the bacteria cultured in the anaerobic condition.


Subject(s)
Bacteria , Dental Pulp Necrosis , Dithiothreitol , Erythrocytes , Horses , Hot Temperature , Humans , Hydrogen-Ion Concentration , Iron , Periapical Periodontitis , Prevotella nigrescens , Prevotella , Sheep , Sodium Azide , Tooth , Virulence
18.
Braz. j. med. biol. res ; 37(5): 649-658, May 2004. ilus, tab, graf
Article in English | LILACS | ID: lil-357554

ABSTRACT

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 æl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 æl 70 percent perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100§C for 45 min. After cooling, 100 æl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (rý > 0.998), and the analytical recoveries were 73.2 percent for 6-TG, 119.1 percent for 6-MP and 97.4 percent for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3 percent, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15 percent) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.


Subject(s)
Humans , Child , Mercaptopurine , Chromatography, High Pressure Liquid , Erythrocytes , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Biomarkers , Dithiothreitol , Thioguanine
19.
Indian J Exp Biol ; 2004 Feb; 42(2): 143-8
Article in English | IMSEAR | ID: sea-60643

ABSTRACT

Effects of lead (Pb) and cadmium (Cd) both alone or in combination on the binding of LH and FSH on isolated granulosa cells were studied. Granulosa cells isolated from proestrous rats were incubated (in vitro) with lead acetate and/or cadmium acetate (0.03 microM of Pb or Cd) for 1 hr. LH binding was dropped to 84% in Pb treated cells, 72.5% in Cd treated cells and 74.8% in combined metal treated cells compared to control. FSH binding dropped to 85.5% in Pb treated cells, 71.16% in Cd treated cells and 72.5% in combined metal treated cells compared to control. Activity of 17beta Hydroxy Steroid Dehydrogenase (17betaHSDH), a key steroidogenic enzyme was reduced by 52% in Cd and 37% in combined metal exposed cells whereas Pb exposed cells showed 31% reduction in the enzyme activity. Pretreatment with SH groups protectants (glutathione [GSH], dithiothretol [DTT]) and zinc caused an ameriolation in enzyme activity whereas Zn pretreatment showed an increase in gonadotropin binding in metal exposed cells. These results suggest that both Pb and Cd can cause a reduction in LH and FSH binding, which significantly alters steroid production in vitro and exerts a direct influence on granulosa cell function.


Subject(s)
Animals , Cadmium/toxicity , Dithiothreitol/pharmacology , Drug Combinations , Female , Follicle Stimulating Hormone/metabolism , Glutathione/pharmacology , Granulosa Cells/drug effects , Hydroxysteroid Dehydrogenases/metabolism , Lead/toxicity , Luteinizing Hormone/metabolism , Proestrus/drug effects , Rats , Steroids/biosynthesis , Zinc/pharmacology
20.
Article in Korean | WPRIM | ID: wpr-652777

ABSTRACT

BACKGROUND AND OBJECTIVES: Postnasal drip is one of the most common symptoms of chronic rhinosinusitis (CRS), and is the main cause of chronic cough. To evaluate the effect of upper airway inflammation defined as CRS on lower bronchial airway, we compared the cytology of nasal secretion (NS) and bronchoalveolar lavage fluid (BALF) of normal controls and patients with chronic rhinosinusitis accompanying with and without chronic cough normal controls. MATERIALS AND METHOD: Ten patients of CRS with postnasal drip were selected. Five of them had chronic cough and the others not. Five normal controls were selected. NS collected using Juhn's tymanic tap and BALF collected through fiberoptic bronchoscopy were diluted with dithiothreitol and PBS. These samples were centrifused and then cytospin slide was prepared. The cytology of the slides were evaluated under light microscope after Wright stain. To examine neutrophil activity, nitroblue tetrazolium dye (NBT) test was performd. Statistical analysis was performed using Mann-Whitney Rank Sum W test. RESULTS: In NS, there were no significant differences in the cell populations among coughing, noncoughing, and the normal control group. NBT positivity of coughing (34.2%) and noncoughing (31.5%) groups showed significantly higher than those of controls (8.6%). In BALF of coughing group, the population of macrophages (78.0%) was significantly lower than noncoughing (86.6%) and control (92.8%) groups, and population of lymphocytes (20.8%) was significantly higher than noncoughing (12.6%) and the control (6.4%) groups. In BALF of noncoughing group, the population macrophages was lower and those of lymphocytes were higher than the control group. CONCLUSION: These results suggest that CRS enhances increased local immune responses and decreased phagocytic activity of the lower airway. And chronic cough in patients with CRS is thought to be dependent on individual tolerance to cough provocation, not on aspiration of postnasal drip of discharge.


Subject(s)
Bronchoalveolar Lavage Fluid , Bronchoalveolar Lavage , Bronchoscopy , Cough , Dithiothreitol , Humans , Inflammation , Lymphocytes , Macrophages , Neutrophils , Nitroblue Tetrazolium , Sinusitis
SELECTION OF CITATIONS
SEARCH DETAIL