Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
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Two kit preparations of the organometallic precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in aqueous media are presented. Method A uses gaseous carbon monoxide and amine borane (BH(3).NH(3)) as the reducing agent. In method B CO(g) is replaced by K(2)[H(3)BCO(2)] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 degrees C. HPLC and TLC analyses revealed 7 +/- 3% of unreacted (188)ReO(4)(-) and <5% of colloidal (188)ReO(2). Solutions of up to 14 GBq/mL Re-188 have been successfully carbonylated with these two methods. The syntheses of two tailor-made bifunctional ligand systems for the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) are presented. The tridentate chelates consist of a bis[imidazol-2-yl]methylamine or an iminodiacetic acid moiety, respectively. Both types of ligand systems have been prepared with alkyl spacers of different length and a pendent primary amino or carboxylic acid functionality, enabling the amidic linkage to biomolecules. The tridentate coordination of the ligands to the rhenium-tricarbonyl core could be elucidated on the macroscopic level by X-ray structure analyses and 1D and 2D NMR experiments of two representative model complexes. On the nca level, the ligands allow labeling yields >95% with [(188)Re(H(2)O)(3)(CO)(3)](+) under mild reaction conditions (PBS buffer, 60 degrees C, 60 min) at ligand concentrations between 5 x 10(-4) M and 5 x 10(-5) M. Thus, specific activities of 22-220 GBq pe micromol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 degrees C revealed stabilities between 80 +/- 4% and 45 +/- 10% at 24 h, respectively, and 63 +/- 3% and 34 +/- 3% at 48 h postincubation in human serum depending on the chelating system. Decomposition product was mainly (188)ReO(4)(-). The routine kit-preparation of the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in combination with tailor-made ligand systems enables the organometallic labeling of biomolecules with unprecedented high specific activities.
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PMID:Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [(188)Re(H(2)O)(3)(CO)(3)](+) and synthesis of tailor-made bifunctional ligand systems. 1212 Nov 30

The prokaryotic expression vector PEGX-3X was used to express hepatitis E virus (HEV) open reading frame 2(ORF2 402-660). The recombinant protein was soluble in PBS buffer and was purified by the glutathione Sepharose 4B affinity column. When the purified recombinant protein was applied to detect HEV antibodies in sera of clinical patients, the results were quite consistent with the HEV diagnostic kit from Diagnostic Biotechnology Ltd(DBL), Singapore. The recombinant protein was more reactive with HEV antibodies than DBL reagent.
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PMID:[Cloning, sequencing and expression of hepatitis E virus structural gene in E. coli and application of the recombinant products for diagnosis]. 1251

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.
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PMID:Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine. 1755 44

In order to characterize the bacterial microbiota present within oral cancerous lesions, tumorous and non-tumorous mucosal tissue specimens (approx. 1 cm(3)) were harvested from ten oral squamous cell carcinoma (OSCC) patients at the time of surgery. Any microbial contamination on the surface of the specimens was eliminated by immersion in Betadine and washing with PBS. Bacteria were visualized within sections of the OSCC by performing fluorescent in situ hybridization with the universal oligonucleotide probe, EUB338. DNA was extracted from each aseptically macerated tissue specimen using a commercial kit. This was then used as template for PCR with three sets of primers, targeting the 16S rRNA genes of Spirochaetes, Bacteroidetes and the domain Bacteria. PCR products were differentiated by TA cloning and bacterial species were identified by partial sequencing of the 16S rRNA gene fragments. A total of 70 distinct taxa was detected: 52 different phylotypes isolated from the tumorous tissues, and 37 taxa from within the non-tumorous specimens. Differences between the composition of the microbiotas within the tumorous and non-tumorous mucosae were apparent, possibly indicating selective growth of bacteria within carcinoma tissue. Most taxa isolated from within the tumour tissue represented saccharolytic and aciduric species. Whether the presence of these bacteria within the mucosa has any bearing on the carcinogenic process is a concept worthy of further investigation.
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PMID:A molecular analysis of the bacteria present within oral squamous cell carcinoma. 1803 35

The aim of study was to investigate the synergetic effect of B7-1 and CD40L co-stimulating pathway in the immunotherapy for lymphoma and to explore the effective manner of tumor vaccine for treating lymphoma. The lymphoma cell line A20 cells were inoculated into BALB/c mice as to establish A20-bearing mice model, the B7-1 and CD40L expression vector were alone or in combination directly injected into lymphoma of mice model, the PBS, vector pcDM8 and pcDNA3.1 were selected as controls so as to observe tumor growth. The pathological section and HE staining of tumor tissue were performed to observe the histological characteristics and the cell infiltration of lymphoma, the CCK-8 detection kit was used to analysis the splenic CTL cytotoxicity. The results showed that the intratumor injection of B7-1 and CD40L resulted in reduction of tumor size. Morphological observation of tumor revealed inflammatory cell infiltration in the tumors, massive necrosis and localization of tumor. CCK-8 kit detection indicated significant enhancement of splenic CTL cytotoxicity, the effect of B7-1 combined with CD40L was stronger than that of B7-1 or CD40L alone. It is concluded that B7-1 and CD40L show immunotherapeutic effect on lymphoma, and the effect becomes stronger when they are combined in treating lymphoma. Meanwhile, the intratumor injection may be considered as a safe and effective way for tumor vaccine.
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PMID:[Synergetic effect of B7-1 and CD40L in the immunotherapy for lymphoma]. 1842 56

A simple sensor method was developed for aflatoxin M(1) analysis to be applied directly with milk by using antibody modified screen-printed carbon working electrode with carbon counter and silver-silver chloride pseudo-reference electrode. A competitive ELISA assay format was constructed on the surface of the working electrode using 3,3,5',5'-tetramethylbenzidine dihyrochloride (TMB)/H(2)O(2) electrochemical detection scheme with horseradish peroxidase (HRP) as the enzyme label. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to milk samples. Extensive interference to the electroanalytical signal was observed upon the analysis of milk. Through a series of chemical fractionations of the milk, and testing the electrochemical properties of the fractions, the interference was attributed to whey proteins with focus towards alpha-lactalbumin. A simple pre-treatment technique of incorporating 18 mM calcium chloride, in the form of Dulbucco's PBS, in a 1:1 ratio to the milk sample or standards and also to the washing buffer stabilised the whey proteins in solution and eliminate the interfering signal. The resulting immunosensor was interference free and achieved a limit of detection of 39 ng l(-1) with a linear dynamic detection range up to 1000 ng l(-1). The developed immunosensor method was compared to a commercial ELISA kit and an in-house HPLC method. The immunsensor was comparable, in term of sensitivity, but vastly superior in term of portability and cost therefore a key instrument for the detection of aflatoxin M(1) at the source of the contamination.
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PMID:Development of an electrochemical immunosensor for aflatoxin M1 in milk with focus on matrix interference. 1916 7

We validated a whole-body cortisol extraction technique for channel catfish, Ictalurus punctatus, fry. Three volume enhancement methods were tested: CAL method (zero calibrator A diluent added to lipid extract), PBS method (phosphate buffered saline added to lipid extract), and VO method (food grade vegetable oil added to lipid extract). The volume enhancement extracts were evaluated using a commercial radioimmunoassay kit. Sensitivity, accuracy, precision, reproducibility, and parallelism could not be determined for the PBS method as cortisol levels were not detected in any of the extracted samples. Intra-assay coefficient of variation (CV) for the CAL and VO methods were 7.3 and 8.3%, respectively, while inter-assay CV were 9.6 and 10.6%, respectively. Based on the sensitivity, accuracy, precision, reproducibility, and parallelism results, we conclude that the CAL method is the most appropriate method for volume enhancement of catfish fry lipid extract. Using the CAL method to detect cortisol in catfish fry, fish were stressed daily for 2 weeks. Fry weights were similar throughout the study while whole-body cortisol levels were higher (P < 0.01) in stressed fish after 1 day of stress. These data show the CAL method can effectively measure whole-body cortisol in catfish fry.
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PMID:Validation of a whole-body cortisol extraction procedure for channel catfish (Ictalurus punctatus) fry. 1968 Jul 67

Although the role of microglial activation in neural injury remains controversial, there is increasing evidence for a detrimental effect in the immature brain, which may occur in response to release of neurotoxic substances including pro-inflammatory cytokines. However, the signaling mechanisms involved in microglial-induced neuronal cell death are unclear. Microglia isolated from the brains of wild-type (WT) or MyD88 knockout (KO) mice were exposed to PBS or the TLR4-ligand LPS (100 ng/mL) for 2, 6, 14, or 24 h, and the microglia-conditioned medium (MCM) collected. Detection of multiple inflammatory molecules in MCM was performed using a mouse 22-plex cytokine microbead array kit. Primary neuronal cultures were supplemented with the 14 or 24 h MCM, and the degree of neuronal apoptosis examined after exposure for 24 h. Results showed a rapid and sustained elevation in multiple inflammatory mediators in the MCM of WT microglia exposed to LPS, which was largely inhibited in MyD88 KO microglia. There was a significant increase in apoptotic death measured at 24 h in cultured neurons exposed to CM from either 14 or 24 h LPS-stimulated WT microglia (p<.05 vs. WT control). By contrast, there was no increase in apoptotic death in cultured neurons exposed to CM from 14 or 24 h LPS-stimulated MyD88 KO microglia (p=.15 vs. MyD88 KO control). These data suggest that MyD88-dependent activation of microglia by LPS causes release of factors directly toxic to neurons.
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PMID:Microglial MyD88 signaling regulates acute neuronal toxicity of LPS-stimulated microglia in vitro. 1990 19

In order to research the survivin gene's action on an animal tumor, we used an adenovirus-mediated siRNA system to inhibit the expression of survivin in an animal model of hepatocarcinoma using nude mice. We constructed a hepatocarcinoma model with nude mice using the hepatocarcinoma cell line HepG2 and divided the mice into four groups depending on the injection dose of AdsiRNA-survivin. We injected the constructed survivin-siRNA adenovirus into tumor-bearing nude mice, observed tumor growth, and determined the tumor growth curve. We then detected tumor cell apoptosis using a TUNEL kit that can assay sliced DNA in tumor cells. The growth of tumors injected with a high or low dose of AdsiRNA-survivin was obviously inhibited, and this level of inhibition was positively correlated with the injected dose of adenovirus. Results of the TUNEL test showed that many of the apoptotic cells were brown in color with concentrated nuclei and an irregular cell shape for both the high and low injection doses. The number of apoptotic cells decreased by group in the order of the high dose group, the low dose group, the AdsiRNA-U6 group, and the PBS group. In conclusion, our results demonstrated that an adenovirus-mediated siRNA system can be used for animal experiments in vivo. AdsiRNA-survivin efficiently inhibited tumor growth and induced tumor cell apoptosis, and it did so in a dose-dependent manner.
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PMID:Adenovirus-mediated siRNA inhibited survivin gene expression induces tumor cell apoptosis in nude mice. 2010 33

An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RT-PCR. Compared with other methods, it showed great advantage of low cost and high efficiency and can be applied to RNA extraction of activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods. This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge.
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PMID:[Rapid method to extract high-quality RNA from activated sludge]. 2032 49


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