PURPOSE: We investigated the direct labeling method of antibody with 99mTc and 188Re and examined the stability and function of these labeled compounds in in vitro and in vivo. MATERIALS AND METHODS: Disulfide bond of nonspecific human IgG was reduced to -SH group by 2-mercaptoethanol. Stannous ion was used to reduce 99mTc and 188Re. The stability of 99mTc-IgG and 188Re-IgG was estimated upto 24 hrs. Biodistribution was evaluated in abscess bearing rats at 4 and 24 hr post-injection of 99mTc or 188Re labeled IgG. RESULTS: The number of -SH group per reduced IgG molecule was 2.34. The labeling yield of 99mTc-IgG and 188Re-IgG were 90% and 95%, respectively. The stability of 99mTc-IgG at 1, 4, 6 and 24 hr was 91%, 83%, 78%, 7% and that of 188Re-IgG, high uptake was found on kidney, blood, stomach and abscess (9.42+/-0.68, 1.43+/-0.24, 0.86+/-0.18, 0.72+/-0.10 %ID/g, respectively). The uptakes at 24 hr were kidney, abscess, stomach, and blood in descending order. In case of 188Re-IgG, high uptake at 4 hr post injection appeared on kidney, blood, abscess and stomach (3.92+/-0.62, 1.32+/-0.08, 0.88+/-0.01, 0.26+/-0.06, respectively). The upatkes at 24 hr were kidney, abscess, blood abd stomach in descending order. The abscess to blood uptake ratio of 99mTc-IgG was 0.5 at 4 hr and 2.02 at 24 hr and that of 188Re-IgG was 0.67 and 1.29. CONCLUSION: 99mTc-IgG and 188Re-IgG and 188Re-IgG canbe labeled efficiently with direct labeling method. However, 99mTc-IgG and 188Re-IgG, labeled with direct method, was unstable. Further study in needed to enhance the stability of the antibody labeling.
Abscess ; Animals ; Humans ; Immunoglobulin G ; Kidney ; Mercaptoethanol ; Rats ; Stomach

PURPOSE: Radiolabeled CEA79.4 antibody has a possibility to be used in radioimmunoscintigraphy or radioimmunotheraphy of cancer. We investigated the in vitro properties and biodistribution of CEA79.4 antibody labeled with Re-188 or Tc-99m. MATERIALS AND METHODS: CEA79.4 was reduced by 2-mercaptoethanol to produce-SH reside, and was labeled with Re-188 or Tc-99m. For direct labeling of Tc-99m, methylene-diphosphonate was used as transchelating agent. CEA79.4 in 50 mM Acetate Buffered Saline (ABS, pH 5.3) was labeled with Re-188, using stannous tartrate as reducing agent. In order to measure immunoreactivity and the affinity constant of radiolabeled antibody, cell binding assay and Scatchard analysis using human colon cancer cells SNU-C4, were performed. Biodistribution study of labeled CEA79.4 was carried out at 1, 14 and 24 hr in ICR mice. RESULTS:. Labeling efficiencies of Tc-99m and Re-188 labeled antibodies were 92.4+/-5.9% and 84.7+/-4.6%, respectively. In vitro stability of Tc-99m-CEA79.4 in human serum was higher than Re-188-CEA79.4. Immunoreactivity and affinity constant of Tc-99m-CEA79.4 were 59.2% and 6.59x109 M-1, respectively, while those of Re-188-CEA79.4 were 41.6% and 4.2x109 M-1, respectively. After 24 hr of administrations of Re-188 and Tc-99m labeled antibody, the remaining antibody, the remaining antibodies in blood were 6.32 and 9.35% ID/g respectively. The biodistribution of each labeled antibody in other organs was similar because they did not accumulate in non-targeted organs. CONCLUSION: In vitro properties and biodistribution of Re-188-CEA79.4 were similar to those of Tc-99m-CEA79.4. It appears that Re-188-CEA79.4 can be used as a suitable agent for radioimmunotherapy.
Animals ; Antibodies ; Colonic Neoplasms ; Humans ; Hydrogen-Ion Concentration ; Mercaptoethanol ; Mice ; Mice, Inbred ICR ; Radioimmunodetection ; Radioimmunotherapy

P19, murine embryonal carcinoma (EC) cells can be induced to differentiate into neurons in the presence of retinoic acid (RA). To investigate neuronal differentiation of P19 cells in details, P19 aggregates were obtained in the presence or absence of RA, ascorbic acid (AA) and 2-mercaptoethanol (2-ME) in bacteriological Petri dishes. When the aggregates were transferred into the serum depleted medium, P19 cells exhibited dramatic morphological changes. Cells contained long and thin processes as detected in differentiated neurons. Western blot analysis showed that treatment of RA and AA induced expression of neuron-specific markers such as NCAM, NSE and Tuj1. Expression of GFAP was not detected, suggesting that P19 cells differentiate into neurons under our experimental condition. Immunocytochemical studies also revealed that treatment of RA and AA increased expression of NCAM and Tuj1. On the contrary, 2-ME was ineffective in the neuronal differentiation of P19 cells, which is consistent the results from the western blot analysis. These results suggest that differentiated P19 cells have similar characteristics to those of typically differentiated neurons. This study also suggests that P19 cells may provide useful tools to study neuronal differentiation in vitro.
Ascorbic Acid ; Blotting, Western ; Carcinoma, Embryonal ; Mercaptoethanol ; Neural Cell Adhesion Molecules ; Neurons* ; Tretinoin

OBJECTIVES: Brucellosis is a systemic disease with a wide spectrum of clinical manifestations. This study aimed to determine the seroprevalence of brucellosis in human immunodeficiency virus (HIV) infected patients in Hamadan Province in the west of Iran. METHODS: A total of 157 HIV-infected patients were screened through standard serological tests, including Wright’s test, Coombs’ Wright test, and 2-mercaptoethanol Brucella agglutination test (2ME test), blood cultures in Castaneda media, and CD4 counting. Data were analyzed using Stata version 11. RESULTS: Wright and Coombs’ Wright tests were carried out, and only 5 (3.2%) patients had positive serological results. However, all patients had negative 2ME results, and blood cultures were negative for Brucella spp. Moreover, patients with positive serology and a mean CD4 count of 355.8 ± 203.11 cells/μL had no clinical manifestations of brucellosis, and, and the other patients had a mean CD4 count of 335.55 ± 261.71 cells/μL. CONCLUSION: Results of this study showed that HIV infection is not a predisposing factor of acquiring brucellosis.
Agglutination Tests ; Brucella ; Brucellosis* ; Causality ; CD4 Lymphocyte Count ; HIV Infections ; HIV* ; Humans* ; Iran* ; Mercaptoethanol ; Seroepidemiologic Studies* ; Serologic Tests

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 micrometer CYS or 100 micrometer beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.
Animals ; Cysteamine ; Embryo Culture Techniques/*veterinary ; Embryo, Mammalian/*physiology ; Female ; Follicular Fluid ; Mercaptoethanol ; Nuclear Transfer Techniques/*veterinary ; Oocytes/*growth & development ; Sus scrofa/*physiology ; Time Factors

AIMTo establish model of differentiation of fetal liver stem cells induced by beta-ME + BHA into neural cells in vitro;

METHODSCD34+ cells from naturally aborted human fetal liver were isolated with MACS Kit, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After confluent more than 80%, the 5 passage cells were induced by 10(-3) mol/L beta-mercaptoethanol (beta-ME) and 2 x 10(-4) mol/L BHA for 24 hours, and washed with PBS, and then incubated in serum-free medium for 5 hours to 5 days. The characteristics of treated cells were assayed by immunocytochemistry staining analysis.

RESULTSCells treated by beta-ME+ BHA exhibited neuronal phenotype, and expressed neuronal specific proteins such as nestin, NeuN, TrnJ-1, and NF-M, which were not found in control cells. Statistic analysis showed that 81% cells were NeuN-positive, 75% cells TuJ-1-positive, 47% cells NF-M-positive, 90% cells NSE-positive.

CONCLUSIONbeta-ME + BHA can induce human fetal liver CD34+ cells to produce neuronal specific antigens and proteins in vitro and become neuronal cells. CD34+ cells from human fetal liver possess potentials of differentiation into neural cells.

Antigens, CD34 ; Butylated Hydroxyanisole ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Liver ; cytology ; embryology ; Mercaptoethanol ; pharmacology ; Neurons ; cytology

The effect of antioxidants on the in vitro life span of mouse keratinocytes was investigated in this work. It was found that the life span of the keratinocytes cultured in the medium supplemented with antioxidants was extended significantly. The most beneficial antioxidant used in this work was the mercaptoethanol, followed by the catalase and SOD. However, the growth rates of keratinocytes in vitro under all the experimental conditions still declined with the culture time. It was also found that the antioxidants added in the medium were also helpful to enhance the keratinocyte colony formation. In addition, the aging kinetics of the mouse epidermal keratinocytes in vitro were analyzed, and finally the aging rate constants corresponding to antioxidants used were calculated.
Animals ; Antioxidants ; pharmacology ; Catalase ; pharmacology ; Cell Division ; drug effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; Keratinocytes ; cytology ; drug effects ; Mercaptoethanol ; pharmacology ; Mice ; Superoxide Dismutase ; pharmacology

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.
Animals ; Benzimidazoles/chemistry ; Dogs/*physiology ; Epidermal Growth Factor/*pharmacology ; Estrus/*physiology ; Female ; Fluorescent Dyes/chemistry ; Meiosis/drug effects/physiology ; Mercaptoethanol/*pharmacology ; Microscopy, Ultraviolet/veterinary ; Oocytes/drug effects/growth&development/*physiology ; Ovary/drug effects/*physiology

Guanine aminohydrolase(GAH;EC 3. 5. 4. 3.) was partially purified 122-fold from rat cerebrum to a specific activity of 7.22 in its per mg protein with a recovery of 7.47% by fractionation with ammonium sulfate, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and isoelectric focusing(pH4-6). The molecular weight of partially purified rat cerebral guanine aminohydrolase was estimated to be 110,000. But, in the cerebral cytosol, a rather higher molecular weight form of the enzyme was identified. The activity of the higher molecular weight form of guanine aminohydrolase was increased by dialyzing the cytosol, and it was converted into the lower molecular weight form(M.W.110,000) by addition of 2-mercaptoethanol. The reaction velocity of partially purified guanine aminohydrolase of rat cerebrum disclosed a hyperbolic curve, with its KM being 6.0uM at pH 8.0. The preparation showed high substrate specificity:among the purine nucleotides, nucleosides and bases with amino group, only guanosine and guanine were deaminated by the enzyme, and the reaction rate of the enzyme displayed by guanosine was less than 10% of that by guanine. When observed under the equimolar concentration of the substrate, hypoxanthine as well as inosine inhibited the activity of the rat cerebral guanine aminohydrolase by 9.4 and 7.8%, respectively, while 5-aminoimidazole-4-carboxamide inhibited the activity of it by 38%. The activity was inhibited by p-hydroxymercuric benzoate as well. Complete loss of its activity was observed after 30 minutes incubation at 60 degrees C, suggesting the preparation was heat labile.
Ammonium Sulfate ; Animals ; Benzoates ; Cerebrum* ; Chromatography ; Chromatography, Gel ; Cytosol ; DEAE-Cellulose ; Durapatite ; Filtration ; Guanine Deaminase* ; Guanine* ; Guanosine ; Hot Temperature ; Hydrogen-Ion Concentration ; Hypoxanthine ; Inosine ; Mercaptoethanol ; Molecular Weight ; Nucleosides ; Purine Nucleotides ; Rats* ; Xanthine Oxidase

BACKGROUND: Nitric oxide is a short-lived effector molecule derived from L-arginine by the nitric oxide synthase(NOS). Nitric oxide plays a role in a number of physiologic and pathophysiologic functions including host defense, edema formation, and regulation of smooth muscle tone. Some kinds of cells including macrophage are known to produce large quantities of nitric oxide in response to inflammatory stimuli such as interleukin-1beta(IL-1beta), tumor necrosis factor-alpha(TNF-alpha), interferon-gamma(IFN-gamma) and lipopolysaccharide(LPS). Reactive oxygen species are also known to be important in the pathogenesis of acute cell and tissue injury such as acute lung injury model. METHODS: Using the RAW264.7 cells, we have examined the ability of oxidant hydrogen peroxide(H2O2) to stimulate nitric oxide production and inducible NOS mRNA expression. Also, we have examined the effects of NOS inhibitors and antioxidants on H2O2 induced nitric oxide production. RESULTS: Stimulation of RAW264.7 cells with combinations of 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, and 100 U/ml IFN-gamma or 100 U/ml IFN-gamma and 1 microg/ml LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite(NO2-) and nitrate(NO3-). Addition of 250 microM- 2 mM H2O2 to the cytokines significantly augmented the synthesis of NO2- and NO3-(p<0.05). When cells were incubated with increasing concentrations of H2O2 in the presence of IL-1beta, TNF-alpha and IFN-gamma at constant level, the synthesis of NO2- and NO3- was dose-dependently increased(p<0.05). 3. NG-nitro-L-arginine methyl ester(L-NAME), dose dependently, significantly inhibited the formation of NO2- and NO3- in cells stimulated with LPS, IFN-gamma and H2O2 at constant level(p<0.05). 4. Catalase significantly inhibited the H2 O2-induced augmentation of cytokine- induced NO2- and NO3- formation(p<0.05). But, boiled catalase did not produce a significant inhibition in comparison with the native enzyme. Another antioxidant 2-mercaptoethanol and orthophenanthroline dose-dependently suppressed NO2- and NO3- synthesis(p<0.05). Northern blotting demonstrated that H2O2 synergistically stimulated the cytokine-induced iNOS mRNA expression in RAW264.7. CONCLUSION: These results suggest that H2O2 contributes to inflammatory process by augmenting the iNOS expression and nitric oxide synthesis induced by cytokines.
Acute Lung Injury ; Antioxidants ; Arginine ; Blotting, Northern ; Catalase ; Cytokines ; Edema ; Hydrogen Peroxide* ; Hydrogen* ; Macrophages* ; Mercaptoethanol ; Muscle, Smooth ; Necrosis ; Nitric Oxide ; Nitric Oxide Synthase Type II* ; Nitroarginine ; Reactive Oxygen Species ; RNA, Messenger ; Tumor Necrosis Factor-alpha