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)

Investigation of the kinetics of nucleic acid release by HeLa (human cervical carcinoma cell line) and A431 (human squamous carcinoma cell line) cells is presented. The released DNA and RNA were shown to accumulate in culture medium and at the cell surface. A portion of cell surface bound RNA can be eluted with PBS/EDTA. Mild trypsin treatment is required for complete detachment of cell surface bound RNA and cell surface bound DNA. Electrophoretic analysis reveals characteristic patterns of cell-associated and free RNA and DNA molecules.
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PMID:Extracellular nucleic acids in cultures of long-term cultivated eukaryotic cells. 1525 68

Reliable data on plasmin activities in blood of patients during fibrinolytic treatment are lacking. This is due to continuing plasminogen activation by plasminogen activators after blood withdrawal. The purpose of this study was to establish a new method for stabilization of blood and to detect plasmin activity in stabilized plasma. For optimization of plasma stabilization by arginine, 50 microL pooled normal citrated plasma was incubated with 50 microL of 0 to 1500 mM arginine, pH 8.7, and 25 microL 100 IU/mL u-PA, 1250 IU/mL t-PA, 10000 U/mL reteplase, 400 U/mL plasminogen-streptokinase-activator complex, 10 microg/mL tenecteplase in 6% BSA-PBS or 25 microL 25 microg/mL plasmin in 20% glycerol. Twenty-five microliters 3 mM HD-Val-Leu-Lys-pNA were added immediately (1 step) or after 90 minutes (room temperature [RT]). The same experiment was performed with pooled normal citrated plasma supplemented with 3.2 mg/mL EDTA, preoxidized with 0 mM or 20 mM chloramine-T for 10 minutes (37 degrees C). For optimization of plasmin activity, the oxidation time of the arginine-stabilized plasma sample containing 0.5 U/mL active plasmin and the chloramine-T amount was varied. Citrated plasma is stabilized against the in vitro action of all six plasminogen activators tested if the final arginine concentration is greater than 500 mM. Neither the addition of EDTA nor the addition of chloramine-T changes this plasma-stabilizing power of arginine. The optimized functional plasmin assay consists of incubation of 10 microL arginine-stabilized plasma with 10 microL 1.5 M arginine, pH 8.7, and 10 microL 100 mM CT in PBS. After 30 minutes (37 degrees C), 75 microL 1.2 M KCl, 1.6 M Arg, 0.75 mM Val-Leu-Lys-pNA (Stop-CS Reagent), and 175 microL 6% BSA-PBS are added and the absorbance increase (DeltaA) at 405 nm is determined. With the present arginine stabilization procedure of plasma and the determination of plasmin activity in arginine-stabilized plasma as described, it is feasible to determine the activity of plasmin in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes in the samples.
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PMID:Functional determination of plasmin in arginine-stabilized plasma. 1601 16

Reliable data on plasminogen activator (PA) activities in blood of patients receiving fibrinolytic treatment are lacking. This is due to the continuing in vitro action of PA after blood withdrawal. We have elaborated a new simple stabilization technique for plasma involving the addition of arginine in final concentrations greater than 500 mM. In this study, new assays for PA in stabilized plasma are developed. The assay was performed with substrate plasma, that is, pooled normal plasma, preoxidized with chloramine-T; oxidant amount and oxidation time were optimized. The chloramine consumption by plasma was assayed with a KJ-assay (absorbance increase at 405 nm by addition of 200 microL 4 M KJ to 25 microL oxidized plasma). The substrate plasma concentration in the PA assay and the PA acting time was optimized. The inhibition of PA by the cations Na(+), K(+), Mg(2+), and Ca(2+) was evaluated. The optimized PA assay consists of incubation of 10 microL arginine-stabilized plasma with 10 microL 1.5 M arginine, pH 8.7 and 10 microL 100 mM CT in PBS. After 30 minutes (37 degrees C), 175 microL 15 mM CT oxidized EDTA plasma are added. After 40 minutes (37 degrees C), 75 microL Stop-CS Reagent is added and DeltaA at 405 nm was determined, giving PA + plasmin activity in plasma. A control value (basal plasmin activity) consists of the addition of Stop-CS Reagent before 175 microL oxidized EDTA plasma. To obtain plasmatic PA activity, the control value has to be subtracted from the PA main value. The assay is matrix-independent and linear up to 1250 IU/mL t-PA, 790 U/mL reteplase, or 199 IU/mL u-PA (37 nM). With arginine stabilization of plasma and the described determination of plasminogen activator activity in arginine-stabilized plasma, it is feasible to determine the activity of plasminogen activators in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes of the samples.
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PMID:Functional determination of plasminogen activator in arginine-stabilized plasma. 1601 17

We successfully established a novel cell line (OS-1) derived from human ovarian small cell carcinoma, hypercalcemic type secreted PTH, PTH-rP and ACTH. The OS-1 cell line was established from metastatic focus of uterus. A patient was 25-year-old Japanese woman. The first she received left ovariectomy on April 2002. The histopathological diagnosis was ovarian small cell carcinoma, pT2c, Nx, Mx. Then on June 2003, metastatic focus of uterus was ectomied. A part of the recurrent tumor of uterus was cut into small pieces with razor blades, and dissociated with 0.1% trypsin-0.02% EDTA/ PBS(-) solution at room temperature. The single cells and small cell clusters were seeded into 60mm dishes and cultured in growth medium (GM: DMEM/F12 supplemented with 20% fetal bovine serum and 0.1% non-essential amino acids solution) at 37 degrees C, 4.7% CO2 in humidified air. Medium was exchanged twice a week. The OS-1 cells grew as floating cultures in the dishes. Radioimmunoassay of the conditioned media was revealed that the cultures secreted large amount of PTH, PTHrP and ACTH simultaneously. Susceptibilities of anti-cancer drugs to the OS-1 cells were examined using oxygen electrode meter (Daikin), and the results suggested VLB and TXL were effective, and CDDP, CPT-11, VP-16, VCR, CPA, MMC and CBDCA were not effective. In our knowledge, it is the first report that the cell line secreting PTH, PTHrP and ACTH was successfully established from ovarian small cell carcinoma, hypercalcemic type. We expect that OS-1 cell line contribute to study on the mechanism of ectopic hormone secretion and susceptibility of anti cancer drugs to the small cell carcinoma.
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PMID:Establishment and characterization of a human ovarian small cell carcinoma, hypercalcemic type, cell line (OS-1) secreting PTH, PthrP and ACTH--special reference to the susceptibility of anti-cancer drugs. 1603 5

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID(50) at 37 degrees C showed a stable HA of maximum 2.6 log HA/100 microL for 2 weeks. Peak TCID(50) titers of 10(7.7) viruses/mL were achieved 20h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca(2+) addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3-2.6 log HA units/100 microL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 microL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 x 10(6) cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.
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PMID:Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media. 1678 Oct 22

The anchoring of thiolated single-stranded DNA (HS-ssDNA) monolayers onto platinum substrates was investigated by sum-frequency generation spectroscopy. Different buffer solutions were used for the preparation of the adlayers. Vibrational fingerprints in the 2700-3100 cm(-1) spectral range showed the intercalation of Tris/EDTA (TE) buffer molecules within the HS-ssDNA self-assembled monolayer. Buffer contribution to SFG can be quenched either by using SFG inactive molecules like KH(2)PO(4)/K(2)HPO(4)/NaCl (PBS) or by repeated rinsing of the DNA layer with pure water. Comparing the SFG spectra of HS-ssDNA and mercaptohexanol (MCH), which had been self-assembled onto the same substrate, enabled us to infer ordering of the anchor arms and strong disordering of the DNA strands of HS-ssDNA monolayers self-assembled on platinum.
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PMID:Sum-frequency generation spectroscopy of DNA monolayers. 1711 92

Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called the Chi2x - Test. The P < .05 significance barrier calculated with the Chi2x - Test is equivalent to that calculated with the Fisher's Exact Test. The FIATA might be considered an interesting screening test for inactive fibrinogen forms or soluble fibrin, as eg in disseminated intravascular coagulation. Fibrinogen precipitation by vancomycin within the blood vessel might explain why vancomycin has to be infused slowly (< 10 mg/min) to prevent nephrotoxicity. The FIATA is of such a simplicity that the determination of fibrinogen antigen in plasma can be performed anywhere--even outside a hospital--within seconds. Thus, the presented FIATA might contribute to extra hospital testing of patients for assessing their risk for myocardial or cerebral ischemia/infarction.
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PMID:The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean. 1716 98

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.
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PMID:Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL. 1728 Jun 83

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.
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PMID:Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract. 1741

Puerarin, a natural isoflavonoid found in Chinese Pueraria lobata (Wild.) Ohwi, has received increasing attention because of its possible role in the prevention of osteoporosis. However, the relationship between puerarin and bone formation remains unknown. In the present study, rat osteoblasts isolated from newborn Wistar rats were used to investigate the effect of puerarin on osteoblasts, and its possible molecular mechanism. Data showed that puerarin caused a significant increase in cell viability, alkaline phosphatase (ALP) activity and mineral nodules formation in osteoblasts, suggesting that puerarin had a stimulatory effect on osteoblastic bone formation. This functional improvement by puerarin was accompanied by activation and nuclear translocation of Akt. Furthermore, puerarin-stimulated osteoblastic growth, Akt activation and redistribution were significantly blocked by the specific PI3K inhibitor, LY294002. These results strongly suggested that puerarin stimulated osteoblastic proliferation and Akt activation in a PI3K-dependent manner. In summary, puerarin derived from Chinese Pueraria lobata (Wild.) Ohwi can promote bone formation in cultured rat osteoblasts, which might be mediated by activation of the PI3K/Akt pathway. DMEM:Dulbecco's modification of Eagel's medium PBS:phosphate buffered saline DMSO:dimethyl sulfoxide EDTA:ethylene diamine tetraacetic acid SDS:sodium dodecyl sulfate SDS-PAGE:sodium dodecylsulfate polyacrylamide gel electrophoresis FITC:fluorescein isothiocyanate HRP:horseradish peroxidase PI3K:phosphatidylinositol 3-kinase.
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PMID:Stimulatory effect of puerarin on bone formation through activation of PI3K/Akt pathway in rat calvaria osteoblasts. 1744 35


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