安石榴甙分子印迹聚合物微球的制备及识别性能

以安石榴甙为模板分子,丙烯酰胺为功能单体,乙二醇二甲基丙烯酸酯为交联剂,偶氮二异丁腈(AIBN)为引发剂,乙腈为溶剂,使用沉淀聚合方法,制备分子印迹聚合物,得到纳米级微球.等温吸附实验研究表明,印迹聚合物与空白聚合物相比,对目标分子具有更好的吸附性能,在研究浓度范围内,印迹聚合物对目标分子的最大吸附量为32.6μmol/g;Scatchard分析表明,印迹聚合物具有两种不同性能的结合位点,空白聚合物有一种结合位点,印迹聚合物的最大表观结合量为243μmoL/g,空白聚合物的最大表观结合量为25.3μmoL/g;底物选择实验表明分子印迹聚合物对安石榴甙具有更高的选择性能.     

Conditions for biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation

The conditions affording biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation was analyzed theoretically. It was shown that when a competing ligand was subjected to transformation to a ligand that showed higher affinity than the parent compound, biphasic competition curves, which might wrongly be interpreted as indicating the presence of two receptor sites, could be observed in binding assays containing a homogenous receptor population. Biphasic competition curves were seen if the conversion of the competitor occurred according to zero and second order kinetics, as well as by enzymatic catalytic processes. However, when the conversion occurred according to a first order kinetics, the competition curves were uniphasic and resolved only into one-site fits, with the apparent affinity of the competitor reflecting the degree of conversion of the competitor to its metabolite. When the metabolic conversion resulted in a metabolite that showed lower affinity for the receptor than the parent compound, the competition curves became supersteep for conversions according to zero and second order kinetics, as well as for conversion by enzymatic catalytic processes.     

Comparison of radioligand assay and immunostaining for epidermal growth factor receptor in human breast cancer

Epidermal growth factor (EGF) receptor status is a useful prognostic indicator in women with breast cancer. Lack of standardization and correlation of methodology for the detection of EGF receptor has hampered its further evaluation. EGF receptor status was ascertained by immunohistochemistry and radioligand assay in 120 breast cancers. Of 52 tumours negative for EGF receptor on radioligand assay, 47 were negative on immunohistochemistry and, of 68 tumours positive for the receptor on assay, 52 were positive on immunohistochemistry. If the more widely evaluated radioligand assay is assumed to be the 'gold standard', immunohistochemistry has a sensitivity of 81 per cent and a specificity of 91 per cent.     

Competitive binding assay for determination of rat insulin-like growth factor binding protein-3

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63+/-0.28 mg/l (mean+/-SEM) in young rats and increased to 3.41+/-0.26 mg/l (p < 0.05) in old rats (n = 5-6). Fasting for 3 days reduced rIGFBP-3 from 2.41+/-0.27 to 1.33+/-0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16+/-0.04 mg/l; p < 0.05) and diabetic rats (1.04+/-0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49+/-0.04 mg/l; ns), when compared to controls (2.79+/-0.22 mg/l). Changes in levels of IGFBP-3 parallelled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.     

Dry polyacrylamide batch method for studying drug protein binding

The validity of a new technique for studying drug protein binding is tested. By this gel diffusion or batch method using dry polyacrylamide, sulfadiazine and sulfafurazole are bound by human plasma proteins to the same extent as reported by other authors with different techniques. The binding of warfarin to fresh human plasma was quantitatively less than expected. This might be due to the possibility that other plasma constituents of non-fasted test persons displace warfarin from protein binding sites, since it is bound more in buffer solutions containing physiological concentrations of human albumin. Phenylbutazone displaced warfarin from its binding sites in diluted human serum albumin concentrations. Quinidine was found to be adsorbed by the otherwise inert polyacrylamide gel and the method is therefore not applicable to quinidine binding studies.     

A two-phase model for controlled drug release from biphasic polymer hydrogels

A comprehensive two phase model is developed to describe the sustained release of a solute or drug from a biphasic hydrogel substrate. Such a material consists of a continuous hydrophilic phase (polymer backbone in water) and a dispersion of spherical microdomains made of the hydrophobic side chains of the polymer organised in a micelle like fashion. The solute or drug is assumed to be encapsulated within the dispersed microdomains, and to diffuse from the interior to the surface of the microdomain where it exchanges following a Langmuir isotherm. Mass transfer to the bulk phase occurs by desorption of the drug from the surface through a driving force that is proportional to the difference of surface and bulk concentration. Accordingly the drug is released to the surroundings by diffusion through the bulk. Depending on the values of the Langmuir constant and assuming well stirred behaviour in the interior of the microdomain, the present model results in either of the two asymptotic models developed in previous studies. The results of a parametric study show that the desired steady state flux of a specific drug to the surroundings may be obtained given appropriate values of structural properties of the material. This conclusion is further supported when using this model to simulate earlier experimental results. The polymer structural properties can be manipulated easily during the fabrication of dispersed-phase networks, as indicated by preliminary experiments.     

New procedure for analysing drug binding data, exemplified by warfarin binding to human serum albumin

Determination of binding constants for multiple binding of a ligand usually results in highly variable figures. We have found that the variations depend mainly upon cooperativity of ligand binding, and that cooperativity is generally absent on binding to human serum albumin. When this is taken into account it becomes possible to obtain binding constants with only slight variation. A computerized curve fitting procedure for analysing binding data has been established consisting of the following steps. (1) Fitting of Scatchard's equation to observed binding equilibrium data to obtain a best-fit set of Scatchard binding constants. (2) Repetition of the fitting procedure, not to obtain a best fit but to generate 30 acceptable sets of Scatchard binding constants. (3) Fitting of Adair's equation to the observed points to obtain a best fit. If the sum of weighted and squared deviations is significantly smaller than the fitting of Scatchard's equation, cooperativity should be considered. If not, cooperativity cannot be demonstrated and the binding constants obtained by fitting Scatchard's equation can be accepted, with the variations found. (4) Final transformation of all Scatchard constants to Adair's. To illustrate the method warfarin data obtained by equilibrium dialysis was used.     

Scanning electron microscopy of the myosin-coated surface of polystyrene beads in a force-movement assay system for ATP-dependent actin-myosin sliding

An outbreak of Cryptosporidiosis occurred over three months in a British Columbia community, peaking in December 1990. Results of the case-control study and illness surveys support the hypothesis that transmission occurred in a public children's pool at the local recreation centre. Analysis using lab-confirmed cases revealed a matched odds ratio of 4.5 [95% CI 0.97, 20.83], and using clinical cases an unmatched odds ratio of 12.8 [95% CI 3.68, 46.77], associated with swimming in the children's pool within two weeks prior to onset of illness. Other risk factors were not significant. Attack rates in various groups of children's pool users ranged from 8% to 78%. The children's pool was closed for steam cleaning and disinfection. Unusually frequent defecations including liquid stools had occurred before and during the outbreak. Improvements were instituted for removal of feces and superchlorination of pool water.     

An in vitro transcription assay for probing drug-DNA interactions at individual drug sites

In this study, we show that oligodendrocyte differentiation is powerfully inhibited by activation of the Notch pathway. Oligodendrocytes and their precursors in the developing rat optic nerve express Notch1 receptors and, at the same time, retinal ganglion cells express Jagged1, a ligand of the Notch1 receptor, along their axons. Jagged1 expression is developmentally regulated, decreasing with a time course that parallels myelination in the optic nerve. These results suggest that the timing of oligodendrocyte differentiation and myelination is controlled by the Notch pathway and raise the question of whether localization of myelination is controlled by this pathway.     

Spectrophotometric assay using streptavidin-alkaline phosphatase conjugate for studies on the proteolysis of polymer bead-bound peptides

Over the past 20 years ovarian cancer has provided a vivid illustration of the successes, failures and challenges for the medical oncologist. During that time the results of treatment have substantially improved; in the West of Scotland for example, for women aged under 55, 3-year survival rates have increased from 36% to 50%. One reason for this was probably the introduction of effective agents such as cisplatin in the mid-1970s and then carboplatin in the mid-1980s. The recent introduction of taxoids promises further improvement in the future. It is important to remember, however, that the best results will be obtained by an optimal organization for the delivery of treatment; national audit studies have shown that factors such as management in integrated clinics can have a major impact on outcome. Nevertheless, the majority of patients still die from the disease; when relapse occurs, clinical drug resistance eventually proves fatal despite further treatment. What are the fundamental mechanisms by which this resistance develops, and what means are available to attempt its circumvention? Factors involved could be described as pharmacological or cellular. Pharmacological resistance might best be addressed by increasing the doses of the drugs used, particularly, cis- or carboplatin. Three years ago we published the results of a randomized trial of 2 doses of cisplatin in 191 patients. At that stage a highly significant median survival advantage for the higher dose (100 mg/m2) of cisplatin was seen. However, a recent updated analysis with a median follow-up of 4 1/2 years shows a reduction in the survival benefit, with 4-year overall survival rates for high- and low-dose cisplatin of 32.4% and 26.6%, respectively. This suggests that a population of drug resistant ovarian cancer cells will eventually emerge despite the use of initial higher doses of cisplatin. A more dose-intensive approach is being pursued with carboplatin, and it seems clear that dose-increments over standard therapy of at least 4-fold will be necessary, to justify further randomized trials. Meanwhile, the alternative approach to delivering high drug concentrations, i.e. intraperitoneal (i.p.) chemotherapy, clearly merits further study, particularly in the light of a recently reported study in patients with minimal disease, which showed a significant survival benefit for i.p. cisplatin treatment. Cellular factors will probably prove to be crucial; studies using various cell lines suggest that multiple mechanisms are likely to be involved and these will need to be examined in relevant clinical material. After DNA damage induced by a range of cytotoxic agents has taken place in ovarian cancer cells, the key to sensitivity/resistance may well be the ability of these cells to engage the process of apoptosis. Several genes are involved in control of this process; these include the p53 gene, mutations of which have been linked to cisplatin resistance in our laboratory studies, as well as in clinical trials with carboplatin. We have also demonstrated an association in ovarian cancer cell lines between cisplatin resistance and microsatellite instability (indicative of defective mismatch repair) and the clinical relevance of this link is also being pursued. A thorough understanding of underlying mechanisms may lead to the rational development of therapeutic means for circumventing cisplatin-resistance in ovarian cancer; the emergence of new classes of drug such as taxoids as topoisomerase I inhibitors offers further promise of improvement in outcome in the next few years.     

Magnetic resonance spectroscopy or imaging for a diagnosis of poliomyelitis or neurovirulence assay of poliovirusese

An integrated database and search system has been developed for protein family identification and information retrieval, as an approach to undertake the highly complex, genomic-scale problem of molecular sequence database search and organization. AVAILABILITY: http://diana.uthct.edu CONTACT: wu@uthct.edu     

Isolation of the [3H]gabapentin-binding protein/alpha 2 delta Ca2+ channel subunit from porcine brain: development of a radioligand binding assay for alpha 2 delta subunits using [3H]leucine

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.     

An in vitro messenger RNA binding assay as a tool for identifying hybridization-competent antisense oligonucleotides

Targeting, use of appliances, and standards of outcome for General Dental Service orthodontic cases collected between 1990 and 1991 were compared with a sample of cases from an earlier study, collected between 1987 and 1988, using the PAR index and IOTN. Comparisons are made generally and in relation to the changes in prior approval regulations for cases started since October 1987. More lower-need cases were treated, but there were no more "unnecessary' treatments and there has been a limited improvement in outcomes, as assessed by the indices, associated with increased use of fixed appliances since 1987. Prior approval appeared to give no tangible benefits in terms of levels of unnecessary treatment or quality of outcome.     

Micellar electrokinetic chromatography stability indicating assay and content uniformity determination for a cholesterol-lowering drug product

This study describes a specific, linear, precise, accurate and sensitive method for the determination of a developmental cholesterol-lowering drug formulated in capsules. The method can also determine two known hydrolytic degradants of the drug. Samples are dissolved in acetonitrile-phosphate buffer pH 4.5, diluted with water and assayed by micellar electrokinetic chromatography (MEKC) in a buffer containing 0.1 M borate-0.025 M SDS at 30 degrees C with an applied voltage of 25 kV. Detection is by UV absorbance at 200 nm. The method was cross validated by comparison with a gradient elution HPLC method. The MEKC method gave at least equivalent precision, accuracy and sensitivity to HPLC but was superior in the resolution of the known impurities and gave a considerably shorter analysis time. The method has been accepted as part of a regulatory submission to the US Food and Drug Administration (FDA).     

Trends in odor intensity for human and electronic noses: relative roles of odorant vapor pressure vs. molecularly specific odorant binding

Response data were collected for a carbon black-polymer composite electronic nose array during exposure to homologous series of alkanes and alcohols. The mean response intensity of the electronic nose detectors and the response intensity of the most strongly driven set of electronic nose detectors were essentially constant for members of a chemically homologous odorant series when the concentration of each odorant in the gas phase was maintained at a constant fraction of the odorant's vapor pressure. A similar trend is observed in human odor detection threshold values for these same homologous series of odorants. Because the thermodynamic activity of an odorant at equilibrium in a sorbent phase is equal to the partial pressure of the odorant in the gas phase divided by the vapor pressure of the odorant and because the activity coefficients are similar within these homologous series of odorants for sorption of the vapors into specific polymer films, the data imply that the trends in detector response can be understood based on the thermodynamic tendency to establish a relatively constant concentration of sorbed odorant into each of the polymeric films of the electronic nose at a constant fraction of the odorant's vapor pressure. Similarly, the data are consistent with the hypothesis that the odor detection thresholds observed in human psychophysical experiments for the odorants studied herein are driven predominantly by the similarity in odorant concentrations sorbed into the olfactory epithelium at a constant fraction of the odorant's vapor pressure.     

Development and preliminary validation of a microtiter plate-based receptor binding assay for paralytic shellfish poisoning toxins

More than 20 countries have either established or proposed regulatory limits for one or more of the paralytic shellfish poisoning (PSP) toxins as they occur in seafood products. PSP toxin levels are generally estimated using the standard AOAC mouse bioassay, yet because of various limitations of this method [e.g. high variability (+/-20%), low sensitivity, limited sample throughput and use of live animals], there remains a need for alternative testing protocols. A sensitive and selective, high capacity assay was developed for the PSP toxins which exploits the highly specific interaction of these toxins with their biological receptor (i.e. voltage-dependent sodium channel) and is thus based on functional activity. This receptor binding assay provides a radioactive endpoint, and is performed in a microtiter filter plate format with results determined by standard liquid scintillation counting within 24 hr. The Ki for the assay is 3.66 +/- 0.86 nM saxitoxin, with a limit of detection of c. 5 ng saxitoxin/ml in a sample extract. Good quantitative agreement of the assay with both mouse bioassay and high-performance liquid chromatographic analysis of crude extracts of contaminated shellfish, as well as PSP toxin-producing algae, was observed. Our findings indicate that the receptor binding assay has a strong predictive value for toxicity determined by mouse bioassay, and that this approach warrants consideration as a rapid, reliable and cost-effective alternative to live animal testing for detection and estimation of PSP-related toxicity in seafood and toxic algae.