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PubMed1977

Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

Alcohols↗

PubMed1985

Immune response of cattle to Brucella abortus outer membrane proteins measured by lymphocyte blastogenesis.

Lymphocytes from cattle were tested in a blastogenesis test with outer membrane proteins isolated from smooth strain 2308 and rough strain 45/20 of Brucella abortus. The titration assay developed for measuring blastogenesis to microbial antigens (Baldwin, Antczak and Winter, this issue, pp. 319-333) was used to assess the response to both group 2 (porins) (Douglas et al., 1984) and group 3 proteins (Verstreate et al., 1982). Blastogenesis was evaluated for distinguishing cattle infected with virulent B. abortus strain 2308 from unimmunized cattle, cattle vaccinated with attenuated strain 19, or inoculated with Escherichia coli 0116:H31, known to cause serological cross-reactions with B. abortus (Nielsen et al., 1980). Strain 45/20 porin was the most effective for this purpose and data analyses utilizing the titration assay were better than those relying on a single point assay. When compared with BASA, an antigen preparation used in other studies (Kaneene et al., 1978a), responses to porin provided a more specific index of infection with B. abortus. Reactions to 45/20 porin occurred, however, in some heifers vaccinated as adults with strain 19 or inoculated with E. coli 0116:H31. Furthermore, nonpregnant heifers had negligible or only transient blastogenesis responses to the porin during the first 14 weeks after infection even though they developed strong 0 antibody responses. We do not recommend the blastogenesis test in its present form as a useful adjunct to serological tests, and could allow measurement of cell mediated immune responses relevant to protective immunity.

Animals↗

PubMed1978

Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography.

I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.

Adult↗

PubMed2001

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity.

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

Acrosin↗

PubMed1995

Quantitation of polymorphic epithelial mucin: a challenge for biochemists and immunologists.

Polymorphic epithelial mucin (PEM) is a heavily glycosylated protein present at the apical surface of glandular epithelial cells which is shed into the lumen of epithelial tissue. In carcinomas cell polarisation is lost, PEM is overexpressed and found on the entire cell surface. High amounts of PEM are shed into the circulation of cancer patients. CA 15.3 is the first commercial assay for the detection of PEM. After roughly one decade of use in clinical practice it is considered valuable for breast cancer therapy monitoring and, in the follow up, for early detection of metastatic disease. The extreme polymorphism of this molecule, with its varying number of multiple epitopes and tremendous variation in glycosylation which can mask catcher/tracer epitopes, impairs its precise measurement. A further impediment is complex formation with autoantibodies, as revealed by a recently developed assay.

Antigens, Neoplasm↗

PubMed2004

Determination of polyvinylpyrrolidone using high-performance liquid chromatography.

Polyvinylpyrrolidone (PVP) is a versatile polymer with innate surface activity. It is very difficult to accurately assay due to its wide molecular weight range and amphiphilic nature. This study evaluated a reversed-phase HPLC method to separate and quantify PVP K15. The assay used a Hicrome C18 150 mm x 3 microm HPLC column in combination with an 80/20 propanol-1-ol: deionised water, 0.01% TFA mobile phase, which resolved the polymer as a single peak, tR = 10.69+/-0.17 min (n=120) at 243 nm. The column's performance was constant throughout the study, N (theoretical plates) = 1729+/-22 and the peak symmetry remained good (As ranged from 0.74 to 0.92, n=10 over the calibration range). The developed assay proved to be accurate, sensitive and capable of recovering PVP K15 from pharmaceutical formulations. The limits of quantification and detection were calculated statistically as 2.40 and 0.72 mg ml(-1), respectively. Assay reproducibility assessed at five concentrations gave an average coefficient of variance <3.5% and the accuracy of the analytical method was 102.62+/-2.04%. The recovery of PVP K15 from directly compressed tablets and Refresh eye drops was 98.02+/-2.73 and 108.35+/-6.52%, respectively.

Chromatography, High Pressure Liquid↗

PubMed2006

Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA.

A duplex real-time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5' nuclease (TaqMantrade mark) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y-chromosome probe, male-specific. A mixture-challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real-time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real-time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.

Chromosomes, Human, Y↗

PubMed2000

Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices.

Here, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1-3-d period to form capillary tubes. Blocking antibodies to the alpha2beta1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.

Animals↗

PubMedDate not supplied

Genetic testing coverage and reimbursement: a provider's dilemma.

The rapid growth of new molecular genetic tests stimulated by the diagnostic potential of DNA/RNA analyses has resulted in the capability of molecular genetic assay technology outpacing the American Medical Association Current Procedural Terminology (CPT) codes and Medicare reimbursement. The AMA CPT Editorial Panel is poised to change the way we report genetic testing, and this change may have the potential to stimulate a governmental review of how Medicare is paying for diagnostic genetic testing. Genetic assays are costly, and those in laboratory management need to be aware of potential changes that may influence the ability of their laboratory to provide access to genetic testing services for their physician clientele. The commercialization of genetic testing has resulted in a proliferation of commercial laboratories and university medical center laboratories, CLIA-certified to perform high complexity testing, offering some level of genetic testing. The genetic tests are offered as home brew (in-house developed) assays, most of which are using analyte-specific reagents. Because these are home brew assays, there is no standardization in how the industry tests for specific mutations. As these genetic assays are billed using the generic molecular diagnostic codes, 83890 through 83912, from the Pathology and Laboratory Chemistry subsection of the CPT, payers are not able to identify the specific mutations being tested and make payment determinations based on the mutations as they relate to the diagnosis code. This article discusses the history of molecular diagnostic coding and related reimbursement, current coverage issues, and the genetic coding proposal under consideration by the AMA CPT Editorial Panel.

Centers for Medicare and Medicaid Services, U.S.↗

PubMed2005

Homogeneous assay technology based on upconverting phosphors.

Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.

Biosensing Techniques↗

PubMed2006

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis.

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.

Adenosine Triphosphate↗

PubMed2006

Use of the three-spined stickleback (Gasterosteus aculeatus) as a sensitive in vivo test for detection of environmental antiandrogens.

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17alpha-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 microg/L), linuron (at 150 microg/L), and fenitrothion (at 15 and 150 microg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.

Angiogenesis Inhibitors↗

PubMed1992

Isolation of YAC clones from the pericentromeric region of chromosome 10 and development of new genetic markers linked to the multiple endocrine neoplasia type 2A gene.

Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 alleles, PIC = 0.88), provide useful reagents for a polymerase chain reaction-based predictive genetic test that can be performed rapidly from small amounts of DNA.

Chromosomes, Fungal↗

PubMed1999

Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics.

The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamilbodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed.

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes (cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2 x 10(4) colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.

Animal Feed↗

PubMed1996

Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA.

Recent studies suggest that ouabain or a ouabain-like substance (OLS) may be present endogenously in humans. We developed a RIA for ouabain with antisera raised in goat against ouabain conjugated to keyhole limpet hemocyanin and ovalbumin. The antiserum was of high antibody titer (200,000) and was specific for ouabain, with little cross-reactivity with common steroids and structurally related compounds such as ouabagenin (4%), strophanthidin (4%), and dihydroouabain (2%). The RIA had a working range of 0.06-2.0 nmol/L, and the intra- and interassay CV was 6.5% at a concentration of 1.7 nmol/L. With this assay the effect of salt loading on urinary excretion of OLS was examined in 10 healthy volunteers (ages 18-22 years) who increased their salt intake (sodium) for 5 days and reduced it for the next 5 days. Urine was collected and OLS concentration was measured by RIA after solid-phase extraction with a Bond Elut C18 column. Excretion of OLS and sodium were maximal on day 5 and lowest on days 9 and 10. Urine excretion of OLS on day 5 (2.66 +/- 1.22 nmol/24 h) was significantly higher (P < 0.0001) than on day 10 (1.47 +/- 0.69 nmol/24 h). We conclude that (a) the assay developed has sufficient sensitivity and specificity to detect endogenous OLS present in biological fluids, and (b) salt intake increases the excretion of OLS.

Adolescent↗

PubMed1999

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

Animals↗

PubMed2004

A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.

Equilibrium binding experiments are widely used for the accurate characterization of binding and competitive binding behavior in biological systems. Modern high-throughput discovery efforts in chemical biology rely heavily upon this principle. Here, we derive exact analytical expressions for general competitive binding models which can also explain a commonly encountered phenomenon in these types of experiments, anticooperative incomplete displacement. We explore the effects of nonspecific binding behavior and parameter misestimation. All expressions are derived in terms of total concentrations determined a priori. We discuss a general framework for high-throughput screening assays based on fluorescence polarization and strategies for assay development, sensitivity regimes, data quality control, analysis, and ranking. Theoretical findings are visualized by simulations using realistic parameter sets. Our results are the basis for the discovery of small-molecule inhibitors of the protein-protein interaction between human calcineurin and NFAT transcription factors, as discussed in the subsequent paper (31).

Computer Simulation↗