A DNA polymerase stop assay for G-quadruplex-interactive compounds

We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2, 6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K+by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.     

A novel assay for hepatitis B e markers

The evaluation of a novel assay that allows simultaneous testing for hepatitis B e antigen and its antibody in a single well is described. The results of routine application and sequential studies on patients with acute hepatitis B and chronic hepatitis B treated with interferon are presented. The specificity and sensitivity of the assay and its ability to be used to follow the response of a patient during the whole seroconversion episode has been evaluated. The assay proved to give useful information about the reactivity of the sample, especially in those patients who were changing their "e" status.     

A colorimetric assay method for the evaluation of neurotrophic activity in vitro

A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.     

Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme

In this study, the efficacy of an anti-ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were transfected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H-ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo. When the transformants were transfected by a ribozyme designed to cleave only activated H-ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.     

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.     

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors

BACKGROUND: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets. RESULTS: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors. CONCLUSIONS: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.     

An assay method for nitric oxide-related compounds in whole blood

The tibialis anterior muscle and soleus muscle of apolipoprotein-E-deficient mice were examined by light and electron microscopy. By light microscopy, sarcoplasmic inclusions were seen in tibialis anterior muscle and 40% of type 2 myofibers were affected in all animals over 8 months of age. These inclusions reacted for nonspecific esterase, cytochrome oxidase, and myoadenylate deaminase and were also periodic acid Schiff positive and stained basophilic with hematoxylin. Moreover, they reacted immunocytochemically with an antibody specific to fragment 17 to 24 of the published sequence of Alzheimer's cerebrovascular amyloid peptide. Immunoreactivity was lost when the antibody was adsorbed with the appropriate synthetic peptide. Ultrastructurally, the inclusions consisted of tubular arrays and were similar to those observed in human muscle in several pathological conditions. In type 1 myofibers of both tibialis anterior and soleus muscle, however, mitochondrial abnormalities including an increase in their number and size were detected, but tubular aggregates were not seen. These large mitochondria possessed an electron-dense inner chamber with an increased number of tightly packed cristae. The results obtained suggest that in these mice there is a disturbed lipid metabolism in skeletal muscle fibers that manifests itself with an accumulation of phospholipid in the form of sarcoplasmic reticulum tubules in the type 2 fibers and enlarged mitochondria with tightly packed cristae in the type 1 fibers. In addition, beta-amyloid protein was closely associated with the accumulated tubules and vesicles of sarcoplasmic reticulum and may represent dysregulation of amyloid precursor protein metabolism.     

A human whole blood assay for clinical evaluation of biochemical efficacy of cyclooxygenase inhibitors

In this study, PGE2 levels in lipopolysaccharide (LPS)-challenged human whole blood and TxB2 levels following blood coagulation were measured as biochemical index for cyclooxygenase (Cox)-2 and Cox-1 activity respectively. Incubation of human mononuclear cells isolated from whole blood with LPS (100 mu g/mL) induced a time-dependent increase in the expression of Cox-2 protein (>100 fold at 24 hr). This is associated with increases in PGE2 production and free arachidonate release in the plasma. Cox-1 protein was detected in the human mononuclear cells at time zero but was not induced by either LPS or PBS. Most non-steroidal anti-inflammatory drugs (NSAIDs) are more potent at inhibiting Cox-1 than Cox-2. Five experimental compounds CGP-28238, Dup-697, NS-398, SC-58125 and L-745,337, have a greater selectivity for Cox-2. Indomethacin at a single oral dose (25 mg) inhibited approximately 90% the whole blood Cox-2 and Cox-1 activities ex vivo in healthy subjects. These results support the use of this assay to assess the biochemical efficacy of selective Cox-2 inhibitors in clinical trials.     

A novel assay for examining the molecular reactions at the eukaryotic replication fork: activities of replication protein A required during elongation

Studies to elucidate the reactions that occur at the eukaryotic replication fork have been limited by the model systems available. We have established a method for isolating and characterizing Simian Virus 40 (SV40) replication complexes. SV40 rolling circle complexes are isolated using paramagnetic beads and then incubated under replication conditions to obtain continued elongation. In rolling circle replication, the normal mechanism for termination of SV40 replication does not occur and the elongation phase of replication is prolonged. Thus, using this assay system, elongation phase reactions can be examined in the absence of initiation or termination. We show that the protein requirements for elongation of SV40 rolling circles are equivalent to complete SV40 replication reactions. The DNA produced by SV40 rolling circles is double-stranded, unmethylated and with a much longer length than the template DNA. These properties are similar to those of physiological replication forks. We show that proteins associated with the isolated rolling circles, including SV40 T antigen, DNA polymerase alpha, replication protein A (RPA) and RF-C, are necessary for continued DNA synthesis. PCNA is also required but is not associated with the isolated complexes. We present evidence suggesting that synthesis of the leading and lagging strands are co-ordinated in SV40 rolling circle replication. We have used this system to show that both RPA-protein and RPA-DNA interactions are important for RPA's function in elongation.     

Multicenter evaluation of a second-generation assay for cardiac troponin T

We report on the evaluation of the second-generation assay for cardiac troponin T (cTnT) on the Enzymun system. This new assay is completely specific for the cardiac isoform of TnT, utilizing two cardiospecific monoclonal antibodies. The assay time is reduced to 45 min. The interassay precision shows a median CV of 5.5%; 20% interassay CV was found between 0.05 and 0.1 microg/L. The cardiosensitivity of the second-generation cTnT assay in patients with ischemic myocardial injury appears equivalent when compared with the first-generation assay. We found no falsely positive results in patients with skeletal muscle damage including multitraumas, surgery patients, and marathon runners who showed highly increased values with the unspecific first-generation assay. In Duchenne disease cTnT was still increased, but to a much lower extent. cTnT remains increased in renal failure, but to a lesser degree than with the first-generation assay. The cause of this increase remains unclear. Although a cross-reactivity of skeletal muscle TnT in the second-generation assay could be excluded by our findings, minor myocardial damage or expression of the cardiac isoform of TnT in regenerating muscles cannot be ruled out in those cases with apparently falsely increased cTnT values. The second-generation cTnT assay is a step forward in the combination of cardiosensitivity and cardiospecificity in biochemical markers for diagnosis of heart disease.     

Clinical evaluation of the BDProbeTec strand displacement amplification assay for rapid diagnosis of tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96. 2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

A novel beta-adrenoceptor ligand for positron emission tomography: evaluation in experimental animals

The present study characterized the rat colonic secretory response to 5-hydroxytryptamine (5-HT) and determined alterations in this response following stress. 5-HT stimulated rat colonic short-circuit current in a concentration-dependent fashion (pD2 = 5.19). This response was subject to desensitization and was mimicked by the indolealkylamines with a rank order potency of 5-HT approximately alpha-methyl-5-HT > 5-carboxytryptamine approximately 5-methoxytryptamine. 2-Methyl-5-HT was a partial agonist. The colonic response to 5-HT was unaltered by methysergide (10 microM), ritanserin (0.1 microM), ondansetron (1 microM) or clozapine (10 microM), but was antagonized by the 5-HT4 receptor antagonists SB204070 (pD'2 = 9.32), GR113808 (pKb = 8.56), DAU6285 (pKb = 6.07) and SDZ205557 (pKb = 6.80). The response of colonic epithelial and oesophageal tunica muscularis mucosae to 5-HT is therefore mediated by a similar 5-HT4 receptor. Following wrap restraint stress, the colonic response to 5-HT became bimodal. Half of the preparations were hyper-responsive, while the rest were hypo-responsive to 5-HT. This 5-HT4 receptor may therefore be involved in stress related changes in fluid transport.     

Therapeutic evaluation of compounds in the SCID-RA papillomavirus model

A previous study by Kreider (Kreider et al., 1979) indicated that rabbit skin, which had been transplanted to immunodeficient nude mice, could be successfully infected with cottontail rabbit papillomavirus (CRPV). We have extended this observation in developing a rodent model for evaluation of compounds for activity against the papillomaviruses. In this model (called the SCID-Ra model), rabbit ear skin is transplanted to the dorsum of SCID mice and allowed to heal for 3 weeks. Infection with CRPV by scarification leads to the growth of warty lesions within 2 3 weeks in >95% of the animals. Topical and/or systemic therapy can be initiated at various times post infection (PI). Weekly lesion scores are recorded and compounds are evaluated for their ability to suppress wart growth when compared to untreated control mice. Ribavirin, which has had a suppressive effect both in the clinic for the treatment of respiratory papillomatosis and on the growth of warts in the rabbit back model, was evaluated and showed significant anti-proliferative activity with oral dosing. Both antiviral and antiproliferative compounds including podophyllin and 5-fluorouracil, which have been used clinically for the treatment of human papillomavirus (HPV) infections, were evaluated in this model. The anti-mitotic compound, Navelbine (vinorelbine tartrate), which is used for the treatment of non-small cell lung carcinoma was evaluated in this system and showed significant inhibition of wart growth with somewhat less topical cytotoxicity when compared to podophyllotoxin.     

Immobilization of small molecules on solid matrices: a novel approach to enzyme-linked immunosorbent assay screening for saxitoxin and evaluation of anti-saxitoxin antibodies

A novel enzyme-linked immunosorbent assay (ELISA) technology was developed for detecting saxitoxin or evaluation of anti-saxitoxin antibodies, which is based on non-covalent immobilization "free' saxitoxin to Maxisorp microtitre plates. The effect of pH on immobilization was studied in media with wide-range buffering capacities (piperazine-glycylglycine and barbiturate buffers). Increasing pH resulted in better responses, although this was mainly due to non-specific interactions. At pH 10.0, however, saxitoxin immobilization was quite effective and specific. The same pattern was found under four different conditions; absence vs presence of bovine serum albumin precoating and absence vs presence of 150 mM NaCl. The best results (high specific response) were achieved with bovine serum albumin precoating in the presence of 150 mM NaCl. The method of choice involved precoating Maxisorp with 5 micrograms/ml albumin followed by addition of 5 microM saxitoxin in 0.01 M piperazine-glycylglycine buffer, pH 10.0. The efficacy of this technology was demonstrated on a polyclonal rabbit anti-saxitoxin antibody and compared with a conventional ELISA of saxitoxin using saxitoxin-bovine serum albumin conjugate as the coating antigen. In the experiments investigating cross-reactivities of various saxitoxin derivatives based on a competitive assay, significantly greater sensitivity was achieved with the novel approach, e.g. 35 pM saxitoxin could be detected (3 x 10(4) times lower concentrations than using the conjugate). The assay works well with mussel tissue homogenates, and because it does not require the use of the covalent saxitoxin-carrier conjugates it offers a simpler alternative to the traditional ELISA for saxitoxin.     

Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis

Employing subtype-specific antisera, we measured the relative immunoreactivity of five muscarinic acetylcholine receptor (mAChR) subtype proteins (m1-m5) in the human iris. The most intensive FITC immunofluorescence was detected by the anti-m3 antibody, followed by anti-m1 and -m5 antisera, in the iris sphincter muscle cells. Only very weak fluorescence was obtained by anti-m2 and -m4 antibodies. In dilator muscle cells, weak but not consistent immunoreactivity was found by anti-m1 and -m5 antibodies. The results suggest that the m3 muscarinic receptor is the predominant subtype in sphincter muscle cells of the human iris.     

High-throughput assay for G2 checkpoint inhibitors and identification of the structurally novel compound isogranulatimide

Treatment of cancer cells lacking p53 function with G2 checkpoint inhibitors sensitizes them to the toxic effects of DNA damage and has been proposed as a strategy for cancer therapy. However, few inhibitors are known, and they have been found serendipitously. We report the development of a G2 checkpoint inhibition assay that is suitable for high-throughput screening and its application to a screen of 1300 natural extracts. We present the isolation of a new G2 checkpoint inhibitor, the structurally novel compound isogranulatimide. In combination with gamma-irradiation, isogranulatimide selectively kills MCF-7 cells lacking p53 function.     

模糊数学评判金属产品质量新方法及其在铅锭产品质量评判中应用

本文介绍了金属产品质量评判的常规方法 ,提出了常规评判的数学模型 ,针对常规评判中所存在的临界值、次要和一般指标评判问题 ,提出了模糊数学法评判金属产品质量的新方法 ,并将该二级评判模型用于铅锭产品质量的评判 ,结果很好地反映了客观实际。     

A novel assay for evaluating glycogenolysis in rat adipocytes and the inability of insulin to antagonize glycogenolysis in this cell type

We report here on a novel procedure for measuring glycogenolysis in rat adipocytes. In this procedure, cells are incubated for 30 min at 37 degrees C with insulin or vanadate, and with [U-14C]glucose to label the glycogen pool with radioactive glucose. The cells are washed and preincubated for an additional 1 h, before being assayed. The extent of glycogenolysis is determined by the decrease in radioactivity in precipitated glycogen, which was quite substantial under experimental conditions facilitating glycogenolysis. From the assay, we determined the following. (a) Glycogenolysis is activated in rat adipocytes in response to lipolytic hormones (i.e. catecholamines and adrenocorticotropic hormone). (b) Other agents and conditions elevating intracellular adenosine 3',5'-monophosphate levels (i.e. cholera toxin, dibutyryladenosine 3',5'-monophosphate, and isobutylmethylxanthine) also activate glycogenolysis. (c) Glycogenolysis (as opposed to lipolysis) is activated at concentrations of adrenocorticotropic hormone or isoproterenol 7-11-fold lower and at adenosine 3',5'-monophosphate concentrations 7-fold lower. (d) Calyculin A, a specific inhibitor of protein phosphatase 1, activates glycogenolysis as well. Calyculin A also activates lipolysis at an equimolar potency. (e) Insulin does not antagonize glycogenolysis in rat adipocytes. In conclusion, the assay allowed us to compare glycogenolysis to lipolysis within the same cell, and to find that the sensitivity to hormones and adenosine 3',5'-monophosphate was about 1 order of magnitude higher for glycogenolysis than for lipolysis. A more striking finding was the inability of insulin to antagonize glycogenolysis in the rat adipose cell, an effect which occurs readily in liver and muscle cells via protein phosphatase 1-activating machinery. This rules out a role for adipose protein phosphatase 1 activation in the mechanism by which insulin antagonizes lipolysis and supports the contention that the insulin effect in lowering adenosine 3',5'-monophosphate levels is the central mechanism by which insulin antagonizes lipolysis.     

Application of a stable cell culture assay for the functional assessment of novel MR contrast agents

We report the results in 33 patients who had nerve grafting of the axillary or the suprascapular nerve or of both. There were 32 men and one woman; their mean age was 21 years and the average interval between injury and operation was three months. At a mean follow-up of 27 months, the deltoid had recovered to M3 or better in 23 of 30 patients (77%) and the infraspinatus in 18 of 25 patients (72%). Shoulder elevation had reached 120 degrees or more in 27 patients (82%), with external rotation of 30 degrees or more in 27 (82%). Twenty-six patients (79%) could reach to the top of their head with their hand. Recovery of muscle strength, range of movement and shoulder function were satisfactory when surgery was performed within four months of the injury. Early exploration and nerve grafting can lead to a good functional recovery, but thorough exploration and careful repair of both nerves are essential.