Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.
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PMID:Apoptosis of mouse embryonic stem cells induced by single cell suspension. 1079 19

Nasopharyngeal carcinoma (NPC) is universally associated with EBV infection. We have shown that the phosphonated nucleoside analog, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (HPMPC) strongly inhibits growth of NPC xenografts in nude mice by causing apoptosis (J. Neyts et al., Cancer Res., 58, 384-388, 1998). We, therefore, tested two additional members of this drug family that have different degrees of antiviral activity, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-2-(R)-(phosphonomethoxy)propyladenine (PMPA). Intratumoral injection of PMEA (75 microl of 2% solution) in C15 NPC xenografts, which are latently infected with EBV, slowed tumor growth moderately, whereas PMPA (75 microl of 2% solution) slowed tumor growth only marginally. Compared with the previous results showing complete regression of tumor, PMEA had less antitumoral effect than HPMPC, and PMPA had the least. After 4 weeks of preventive treatment, tumors formed in 12.5, 50, and 100% of mice treated with HPMPC, PMEA, and PMPA, respectively, in contrast to the development of tumors in all of the PBS-treated control mice. We also investigated the effect of each drug on the EBV-positive epithelial cell line NPC-KT in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed inhibition of growth of NPC-KT cells by HPMPC and PMEA, but not by PMPA, which correlates with the results observed in tumor xenografts. Growth inhibition was attributable to induction of apoptosis in NPC-KT cells as indicated by a DNA fragmentation assay. Cleavage of poly(ADP-ribose) polymerase after treatment of NPC-KT cells with HPMPC was observed, which suggested that the apoptosis may be mediated by caspase(s). The apoptotic effects of the drugs are independent of any effects on EBV DNA polymerase, which is not expressed in these latently infected NPCs. These results suggest that HPMPC as well as PMEA could provide an adjunctive treatment for NPC.
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PMID:Prevention and inhibition of nasopharyngeal carcinoma growth by antiviral phosphonated nucleoside analogs. 1169 6

Based on the recently highlighted potential of nucleophilic heteroaromatic ortho-radiofluorinations in the preparation of fluorine-18-labeled radiotracers and radiopharmaceuticals for PET, a [(18)F]fluoropyridine-based bromoacetamide reagent has been prepared and used in prosthetic group introduction for the labeling of oligonucleotides. [(18)F]FPyBrA (2-bromo-N-[3-(2-[(18)F]fluoropyridin-3-yloxy)propyl]acetamide) was designed as a radiochemically feasible reagent, its pyridinyl moiety both carrying the radioactive halogen (fluorine-18) and allowing its efficient incorporation via a nucleophilic heteroaromatic substitution, and its 2-bromoacetamide function, ensuring the efficient alkylation of a phosphorothioate monoester group born at the 3'- or 5'-end of single-stranded oligonucleotides. [(18)F]FPyBrA (HPLC-purified) was efficiently prepared in 18-20% non-decay-corrected yield (based on starting [(18)F]fluoride) using a three-step radiochemical pathway in 80-85 min. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination as the fluorine-18 incorporation-step (70-85% radiochemical yield) and uses [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as precursor for labeling, followed by (2) rapid and quantitative TFA-removal of the N-Boc-protective group and (3) condensation with 2-bromoacetyl bromide (45-65% radiochemical yield). Typically, 3.3-3.7 GBq (90-100 mCi) of HPLC-purified [(18)F]FPyBrA could be obtained in 80-85 min, starting from 18.5 GBq (500 mCi) of a cyclotron production batch of [(18)F]fluoride. [(18)F]FPyBrA was regioselectively conjugated with 9-mer and 18-mer single-stranded oligonucleotides, provided with a phosphorothioate monoester group at their 3'-end. Both natural phosphodiester DNAs and in vivo-stable 2'-methoxy and -fluoro-modified RNAs were used. Conjugation uses optimized, short-time reaction conditions (MeOH/0.1 M PBS pH 7.4, 15 min, 120 degrees C), both compatible with the chemical stability of the oligonucleotides (ONs) and the half-life of fluorine-18. Conjugated [(18)F]ONs were finally purified by RP-HPLC and desalted using a Sephadex NAP-10 column. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the oligonucleotide, and the HPLC purification and formulation lasted 140-160 min. [(18)F]FPyBrA represents a valuable alternative to the already reported N-(4-[(18)F]fluorobenzyl)-2-bromoacetamide for the design and development of oligonucleotide-based radiopharmaceuticals for PET.
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PMID:Design and synthesis of a new [18F]fluoropyridine-based haloacetamide reagent for the labeling of oligonucleotides: 2-bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide. 1514 90

A stable suspension of carbon nanotubes (CNT) was obtained by dispersing the CNT in a solution of surfactant, such as cetyltrimethylammonium bromide (CTAB, a cationic surfactant). CNT (dispersed in the solution of 0.1% CTAB) has promotion effects on the direct electron transfer of glucose oxidase (GOx), which was immobilized onto the surface of CNT. The direct electron transfer rate of GOx was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of GOx, with a midpoint potential of about -0.466 V (vs SCE (saturated calomel electrode)) in the phosphate buffer solution (PBS, pH 6.9). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (ks) and the value of midpoint potential (E1/2) were estimated. The dependence of E1/2 on solution pH indicated that the direct electron transfer reaction of GOx is a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOx retained its bioelectrocatalytic activity for the oxidation of glucose, suggesting that the electrode may find use in biosensors (for example, it may be used as a bioanode in biofuel cells). The method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.
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PMID:Direct electron transfer of glucose oxidase promoted by carbon nanotubes. 1530 51

To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase. The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. An animal study was carried out in which BALB/C nude mice bearing yCDglyTK gene-modified tumors were treated with prodrugs and radiation. Our results revealed that the yCDglyTK gene could be expressed in CNE-2 cells in vitro. In MTT analysis, at the transfection rate of 10%, 66% cells were killed. The synergistic effect of CD and TK showed 91% of yCDglyTK-transfected cells were killed with the treatment of 5-fluorocytosine (5-FC) alone, 60% killed with ganciclovir (GCV) alone, and 75% killed with 5-FC and GCV together. In vivo, the tumor volume in all of the four prodrugs and/or radiation-treated groups were significantly different from that in the PBS-controlled group (P<.01); also yCDglyTK+prodrug+radiation group was different from the other three groups (P<.05). Our findings suggested there was a synergistic antitumor effect when combining suicide gene therapy and radiation, and yCDglyTK has potent antitumor efficacy and may be a candidate suicide gene for cancer therapy.
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PMID:A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma. 1549 80

Minocycline, a tetracycline derivative, disrupts inflammatory processes within the CNS and reduces demyelination in experimental autoimmune encephalomyelitis. Several recent studies indicate that components of the inflammatory response to demyelination may be beneficial for the regenerative process of remyelination. In this study we examined the effects of minocycline on remyelination independent of its effects in limiting immune-mediated white matter damage using a toxin model of demyelination. Demyelinating lesions were induced by injection of ethidium bromide into caudal cerebellar peduncles of adult rats. Minocycline or PBS was administered by twice daily injections from day 1 prior to lesion-induction to post lesion day 3. Remyelination was assessed, blinded to grouping, using standard morphological criteria. The microglia activation within the lesion was assessed by examining the expression of OX-42 and major histocompatibility class II immunoreactivity. The oligodendrocyte progenitor cell (OPC) response was quantified by in situ hybridization using probes for OPC-expressed mRNAs, platelet-derived growth factor receptor-alpha and Olig-1. Minocycline treatment strongly inhibited microglia/macrophage activation at day 1 and day 3 post-lesion induction, and suppressed the OPC response to demyelination. We also found a significant decrease in the extent of oligodendrocyte but not Schwann cell remyelination in the minocycline-treated animals as compared with controls at 3 weeks post-lesion induction. These results indicate that microglia/macrophage activation is an important process for remyelination and further support the concept that suppression of inflammatory response may impair remyelination.
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PMID:Minocycline-mediated inhibition of microglia activation impairs oligodendrocyte progenitor cell responses and remyelination in a non-immune model of demyelination. 1558 38

Flavonoids, which are main constituents of herbal medicines, have been reported to inhibit the growth of Helicobacter pylori (HP). Therefore, to evaluate the anti-HP activity of some flavonoids (flavanols, flavones, flavonols and isoflavonoids), their effects on the growth and vacuolation of HP as well as the infective properties of HP against HeLa cells were investigated. Catechins, quercetin and naringenin weakly inhibited the growth of HP, but all tested compounds did not inhibit HP infection into KATO III cells and HP urease activity. Quercetin and naringenin inhibited HP VacA vacuolation in HeLa cells with IC (50) values of 0.046 and 0.36 mM, respectively. Quercetin also inhibited procaspase-3 activation to caspase-3 in HeLa cells induced by HP VacA toxin, which may induce cell death via the proteolytic activation of a cascade of caspases. However, quercetin did not affect Bax and Bcl-2 protein levels. Based on these findings, quercetin may improve gastric cell death by inhibiting apoptotic signaling by HP VacA toxin. Abbreviations. HP: Helicobacter pyloriBSA:bovine serum albumin ESL:enhanced chemiluminescence MIC:minimum inhibitory concentration MTT:methylthiazolyldiphenyl-tetrazolium bromide PBS:phosphate-buffered saline VacA:Vacuolating cytotoxin.
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PMID:In vitro inhibitory effect of flavonoids on growth, infection and vacuolation of Helicobacter pylori. 1577 May 37

The DeLaval cell counter (DCC) is a portable device designed for on-farm somatic cell count (SCC) analysis in bovine milk. This study evaluated the performance of the DCC when analyzing ovine milk. A total of 29 composite ovine milk samples, ranging between 20 x 10(3) and 2,200 x 10(3) cells/mL, were divided into 15 aliquots/milk sample corresponding to 5 SCC methods using 3 types of preservation (unpreserved, azidiol, and bronopol). The SCC methods were the Fossomatic (FSCC), the DCC in undiluted samples, and the DCC in samples diluted 1:1 in 3 different types of diluents (PBS + Triton X-100, PBS + ethidium bromide + Triton X-100, and PBS + propidium iodide + Triton X-100). All analyses were carried out in duplicate. In addition, each sample was analyzed in quadruplicate by the direct microscopic method (DMSCC) using Pyronin Y-methyl green as a stain. Comparison of methods was based on overall accuracy studies (means comparison, repeatability, and regression studies vs. DMSCC and FSCC as reference methods). The DCC methods used to analyze milk samples diluted in staining solution (with ethidium bromide or propidium iodide) showed large coefficients of regression (b = 0.91 to 1.01) and correlation (r > 0.99) when compared with the DMSCC and FSCC methods. In these samples the DCC gave repeatability values (s(r) = 33 to 48 x 10(3) cells/mL) similar to the DMSCC (s(r) = 36 x 10(3) cells/mL), and their log SCC means (5.52 to 5.54) did not differ from the reference value (5.54). However, undiluted samples analyzed by the DCC method showed large standard deviations of repeatability and SCC values lower than those by the DMSCC or FSCC methods, probably because of the high solids content in ovine milk. The type of preservation did not affect the outcomes. Consequently, the DCC was determined to be accurate when analyzing diluted ovine milk based on comparison with the SCC reference methods.
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PMID:Evaluation of the overall accuracy of the DeLaval cell counter for somatic cell counts in ovine milk. 1710 93

An increase in oxidizing response above a certain threshold produces, in the absence of a concomitant rise in antioxidant/reducing response, oxidative stress that is associated with complications in diabetes. A simple technique involving reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in order to determine quantitatively the antioxidant status of plasma. MTT (50microL; 5.0mg/mL in PBS) was incubated with plasma (100microL) in PBS for 30, 60 or 120min at 37 degrees C, the reaction terminated by addition of 1.0mL of 0.04M hydrochloric acid in isopropanol and the absorbance measured at 570nm. The modulation by plasma of the generation of reactive oxygen species (ROS) in 12,13-phorbol dibutyrate (PDB)-stimulated granulocytes was evaluated using a chemiluminescence luminol-dependent assay. Plasma from healthy subjects (n=15) showed significantly higher antioxidant status (p<0.05) over all time periods studied compared with plasma from diabetic patients (n=27). MTT was directly reduced by plasma although platelets were not involved. Moreover, the reduction of MTT by bovine serum albumin at levels equivalent to the concentration of human serum albumin in plasma was much lower. The antioxidant status of plasma, as evaluated by MTT dye reduction, may reflect an antioxidant response since ROS generation in PDB-stimulated granulocytes was rapidly down-regulated by the presence of plasma (3.3-fold in diabetic patients and 5.8-fold in healthy subjects) confirming the lower antioxidant activity of plasma from diabetic patients. The results demonstrate that extracellular reduction of MTT by plasma may occur via enzymatic and non-enzymatic processes.
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PMID:Determination of the antioxidant status of plasma from type 2 diabetic patients. 1727 Mar 9

The tumor suppressor p14(ARF) and protooncogene epidermal growth factor receptor (EGFR) play an important role in the development of laryngeal squamous cell carcinoma (LSCC). We explored the inhibition of proliferation and induction of differentiation in human larynx cancer cells (Hep-2) in vitro when p14(ARF) couples with antisense complementary DNA of EGFR to transfect into Hep-2 cells via the AdEasy-1 vector system. In vitro studies, using standard isobologram analyses, identified whether Ad-antisense EGFR is synergistic with Ad-14(ARF). To evaluate the cytotoxicity of these agents the gold standard clonogenic survival assay was used. Western blotting analyses, 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometer (FCM) analysis was used to detect protein expression, proliferation, and cell cycle distribution of Hep-2 cells, respectively. Meanwhile, empty vector and PBS were set as a control. The activity of proliferation of Hep-2 cells was inhibited markedly by infection of Ad-p14(ARF) combined with Ad-antisense EGFR compared with Ad-p14(ARF) or Ad-antisense EGFR alone (P = 0.001, P = 0.002, respectively), with Ad-sense EGFR (P = 0.0005), with vector control (Ad-Ctrl) (P = 0.0001), and with PBS (P = 0.0001). FCM revealed that the proportion in the G(0)/G(1) phases increased by up to 86.9% when Ad-p14(ARF) was associated with Ad-antisense EGFR to transfect Hep-2 cells. A weakened expression of EGFR protein and P14 (ARF) protein overexpression was observed. Our study in vitro indicated that association of Ad-p14(ARF) with Ad-antisense EGFR remarkably inhibited activity of proliferation and inducted differentiation of Hep-2 cells. Therefore, not only EGFR, but also p14(ARF), plays a major role in the genesis and in modulating cell growth and differentiation of LSCC, and their synergistic effect was obvious. An effective potential target of gene therapy to prevent LSCC proliferation was provided.
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PMID:Combined p14ARF and antisense EGFR potentiate the efficacy of adenovirus-mediated gene therapy in laryngeal squamous cell carcinoma (LSCC). 1732 65


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