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PubMedDate not supplied

Cardiac troponin I in feline hypertrophic cardiomyopathy.

Measurement of plasma cardiac troponin I concentration ([cTnI]) is a sensitive and specific means for detecting myocardial damage in many mammalian species. Studies have shown that [cTnI] increases rapidly after cardiomyocyte injury. The molecular structure of cTnl is highly conserved across species, and current assays developed for its detection in humans have been validated in many species. In this study, [cTnI] was quantified using a 2-site sandwich assay in plasma of healthy control cats (n = 33) and cats with moderate to severe hypertrophic cardiomyopathy (HCM) (n = 20). [cTnI] was significantly higher in cats with HCM (median, 0.66 ng/mL; range, 0.05-10.93 ng/mL) as compared with normal cats (median, <0.03 ng/mL; range, <0.03-0.16 ng/mL) (P < .0001). An increase in [cTnI] was also highly sensitive (sensitivity = 85%) and specific (specificity = 97%) for differentiating cats with moderate to severe HCM from normal cats. [cTnI] was weakly correlated with diastolic thickness of the left ventricular free wall (r2 = .354; P = .009) but not with the diastolic thickness of the interventricular septum (P = .8467) or the left atrium: aorta ratio (P = .0652). Furthermore, cats with congestive heart failure at the time of cTnI analysis had a significantly higher [cTnI] than did cats that had never had heart failure and those whose heart failure was controlled at the time of analysis (P = .0095 and P = .0201, respectively). These data indicate that cats with HCM have ongoing myocardial damage. Although the origin of this damage is unknown, it most likely explains the replacement fibrosis that is consistently identified in cats with moderate to severe HCM.

Animals↗

PubMed2006

Identification in triatomine vectors of feeding sources and Trypanosoma cruzi variants by heteroduplex assay and a multiplex miniexon polymerase chain reaction.

Feeding sources of triatomine vectors (Triatoma longipennis) collected in peridomiciles in Mexico were identified by a heteroduplex assay developed with triatomine blood meals. Trypanosoma cruzi parasites were also characterized in the same blood meal samples by multiplex-polymerase chain reaction assay of mini-exon gene inter-genic regions. The main blood meal source was from rats, but the bugs were able to feed on a wide variety of hosts, and human blood meals were identified. Trypanosoma cruzi was the only flagellate species identified in the blood meals. All populations belong to the T. cruzi I lineage, a result that is consistent with the previously assumed predominance of this lineage in Mexico. This combination of blood meal and T. cruzi lineage identification provides a powerful tool for understanding T. cruzi transmission cycles.

Animals↗

PubMed1997

Development of a radioimmunoassay for Cebus apella prolactin.

Prolactin (PRL) is a pituitary hormone that plays important roles in mammalian reproductive physiology, specially lactation. The regulation of PRL secretion shows important species differences. To study PRL regulation in a subhuman primate, the Cebus apella, we developed an heterologous radioimmunoassay using an antibody against rhesus PRL (anti-m5PRL) and a Cebus apella pituitary extract as PRL standard. The assay has a sensitivity that allows measurements of cebus PRL in small amounts of Cebus apella plasma obtained from animals in different physiological conditions. Plasma cebus PRL concentrations (+/-SEM) varied in different reproductive stages. PRL concentration in adult Cebus apella females that have regular menstrual cycles (161.6 +/- 15.0 mIU/ml) was similar to that found in adult (100.3 +/- 7.6 mIU/ml) and prepuberal males (101.2 +/- 3.9 mIU/ml). PRL concentration was higher in pregnant (677.8 +/- 11.8 mIU/ml) and in nursing (625.0 +/- 47.0 mIU/ml) Cebus apella females than in 15-d post-partum non-nursing (369.0 +/- 19.0 mIU/ml) and cycling females. PRL concentration in Cebus apella newborns (719.0 +/- 49.2 mIU/ ml) was similar to that found in pregnant and nursing females, and higher than in the other females as well as adult and prepuberal males. These differences in PRL concentration in different physiological conditions are similar to that observed in humans and other primates. A PRL response to thyrotropin releasing hormone (TRH) was demonstrated in 2 nursing Cebus apella females, similar to the response found in nursing woman and rhesus. Altogether, the data presented support the proposal that the assay developed to measure PRL in Cebus apella is an adequate tool to study the physiology of PRL in this species.

Animals↗

PubMed2003

Assays for complexed prostate-specific antigen and other advances in the diagnosis of prostate cancer.

This review discusses advances in the area of serum and tissue markers for prostate cancer. A recently developed assay for complexed prostate-specific antigen (PSA) has been found to have better specificity than that afforded by assay of total PSA. Researchers in Austria have found that lowering the PSA cutoff point for a diagnosis of prostate cancer resulted in a significant increase in identifying men with cancer at a favorable pathologic stage. Difficulties in pathologic interpretation of tissue specimens can result in both under- and over-diagnosis of prostate cancer. When in doubt, referral to a pathologist who specializes in prostate cancer is warranted. Epidermal growth factor receptor is emerging as an important therapeutic approach not only to prostate cancer but also to breast and colon cancers.

Journal Article↗

PubMed2001

Liquid chromatographic-tandem mass spectrometric urine assay for a highly metabolized cyclic ureidobenzenesulfonamide: issues concerning assay specificity and quality control preparation.

An LC-MS-MS method was validated for the quantitation of a beta(3) agonist (A) in human urine to support Phase I studies. A was designed to accelerate metabolism for weight reduction. During assay development a significant loss of A was apparent from frozen urine quality control samples. The addition of 0.75% bovine serum albumin (BSA) in urine (v/v) was required to maximize the recovery of A from urine. Urine samples were basified and extracted into methyl t-butyl ether-isopropyl alcohol (90:10, v/v). The organic layer was washed, evaporated, reconstituted, and injected onto a 5 cm, C8 HPLC column prior to MS-MS analysis. The standard curve was linear from 5 to 500 ng/ml. Intraday precision for peak area ratios from BSA urine samples at seven separate concentrations over a range of 5-500 ng/ml (n=5) was <4.0% and calculated concentrations were within 91-115% of nominal concentrations. Interday precision for BSA urine quality control (QC) samples at four separate concentrations (n=10 of each) was <5.0% and individual calculated concentrations were within 90-111% of nominal concentrations. This work emphasizes that potential metabolites and quality control standards should be prepared and assayed as early as possible in method development, especially before the sample collection section of the clinical protocol is prepared. The methods described here have wide utility to other compounds containing basic benzene sulfonamides and to beta3 agonist candidates.

Adrenergic beta-Agonists↗

PubMed2007

Measuring the rate of gross chromosomal rearrangements in Saccharomyces cerevisiae: A practical approach to study genomic rearrangements observed in cancer.

Gross chromosomal rearrangements (GCRs), including translocations, deletions, amplifications and aneuploidy are frequently observed in various types of human cancers. Despite their clear importance in carcinogenesis, the molecular mechanisms by which GCRs are generated and held in check are poorly understood. By using a GCR assay, which can measure the rate of accumulation of spontaneous GCRs in Saccharomyces cerevisiae, we have found that many proteins involved in DNA replication, DNA repair, DNA recombination, checkpoints, chromosome remodeling, and telomere maintenance, play crucial roles in GCR metabolism. We describe here the theoretical background and practical procedures of this GCR assay. We will explain the breakpoint structure and DNA damage that lead to GCR formation. We will also summarize the pathways that suppress and enhance GCR formation. Finally, we will briefly describe similar assays developed by others and discuss their potential in studying GCR metabolism.

Biological Assay↗

PubMed1998

Serum concentrations of cardiac troponin I in sudden death: a pilot study.

Sudden cardiac death due to lethal arrhythmia may be the initial presenting symptom of ischemic heart disease. In many cases, in the absence of trauma, a majority of these deaths will be visually inspected by a medical examiner and released with death being ascribed to atherosclerotic cardiovascular disease, coronary artery disease, arrhythmia, myocardial infarction, or a similar diagnosis. When an autopsy is performed, there may be significant cardiovascular disease but no gross or histologic evidence of an acute myocardial infarct unless the patient survived for several hours following the event. Biochemical assays of creatine kinase MB fraction (CKMB) performed on serum have been used to document myocardial injury in the absence of morphologic changes. Newly developed assays for cardiac troponin I (cTnI) may detect myocardial injury with a greater sensitivity than CKMB. A prospective study was performed on 28 autopsied patients at the Office of the Chief Medical Examiner of the state of Maryland. Subclavian blood was sampled for subsequent analysis of serum CKMB and cTnI. In 3 cases of cardiac-related death, there was insufficient plasma for analysis of both CKMB and cTnI, and only CKMB was quantitated. In 12 cases, hemolysis rendered interpretation questionable. Of the remaining 16 cases, the mean serum CKMB level was 857.9 ng/ml (n = 7) and the cTnI level was 93.4 ng/ml (n = 4) for cardiac-related deaths, compared with mean CKMB levels of 116.4 ng/ml (n = 9) and mean cTnI levels of 16.6 ng/ml (n = 9) for non-cardiac-related deaths. The differences in serum elevation of both CKMB and cTnI noted between the cardiac- and non-cardiac-related deaths were statistically significant. Serum cTnI concentrations >40 ng/ml were only noted in cardiac-related deaths. These data suggest that an elevated postmortem serum concentration of cTnI reflects ischemic heart disease and supports its use in determining cause of death. Quantitation of this analyte may prove useful when death may be due to an arrhythmia following a morphologically undetectable microinfarct.

Biomarkers↗

PubMed2005

Cell-based assays to detect inhibitors of fungal mRNA capping enzymes and characterization of sinefungin as a cap methyltransferase inhibitor.

The m7GpppN cap at the 5' end of eukaryotic mRNAs is important for transcript stability and translation. Three enzymatic activities that generate the mRNA cap include an RNA 5'-triphosphatase, an RNA guanylyltransferase, and an RNA (guanine-7-) -methyltransferase. The physical organization of the genes encoding these enzymes differs between mammalian cells and yeast, fungi, or viruses. The catalytic mechanism used by the RNA triphosphatases of mammalian cells also differs from that used by the yeast, fungal, or viral enzymes. These structural and functional differences suggest that inhibitors of mRNA capping might be useful antifungal or antiviral agents. The authors describe several whole-cell yeast-based assays developed to identify and characterize inhibitors of fungal mRNA capping. They also report the identification and characterization of the natural product sinefungin in the assays. Their characterization of this S-adenosylmethionine analog suggests that it inhibits mRNA cap methyltransferases and exhibits approximately 5- to 10-fold specificity for the yeast ABD1 and fungal CCM1 enzymes over the human Hcm1 enzyme expressed in yeast cells.

Adenosine↗

PubMed2002

Gene induction studies and toxicity of chemical mixtures.

As part of its mixtures program, the Agency for Toxic Substances and Disease Registry (ATSDR) supports in vitro and limited in vivo toxicity testing to further our understanding of the toxicity and health effects of chemical mixtures. There are increasing concerns that environmental chemicals adversely affect the health of humans and wildlife. These concerns have been augmented by the realization that exposure to chemicals often occurs to mixtures of these chemicals that may exhibit complex synergistic or antagonistic interactions. To address such concerns, we have conducted two studies with techniques that are being used increasingly in experimental toxicology. In the first study, six organochlorine pesticides (4,4 -DDT, 4,4 -DDD, 4,4 -DDE, aldrin, dieldrin, or endrin) were selected from the ATSDR Comprehensive Environmental Response, Compensation and Liability Act of 1980 (or Superfund) priority list and tested for their ability to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were dosed with and without selected environmental chemicals either individually or in defined combinations. Estradiol consistently elicited 10- to 23-fold dose-dependent inductions in this assay. By contrast, all six of the organochlorine pesticides showed no detectable dose-related response when tested either individually or in binary combinations. Thus, these chemicals as binary mixtures do not exhibit any additional estrogenicity at the levels tested in these assays. In the second study, arsenic [As(V)], cadmium [Cd(II)], chromium [Cr(III, VI)], and lead [Pb(II)] were tested in a commercially developed assay system, CAT-Tox (L), to identify metal-responsive promoters and to determine whether the pattern of gene expression changed with a mixture of these metals. This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal-transduction pathways. Singly, As(V), Cd(II), Cr(III, VI), and Pb(II) produced complex induction profiles in these assays. However, no evidence of synergistic activity was detected with a mixture of Cd(II), Cr(III), and Pb(II). These results have shown metal activation of gene expression through several previously unreported signal-transduction pathways and thus suggest new directions for future studies into their biochemical mechanisms of toxicity. In conclusion, the (italic)in vitro(/italic) methods used in these studies provide insights into complex interactions that occur in cellular systems and could be used to identify biomarkers of exposure to other environmental chemical mixtures.

Biomarkers↗

PubMed1988

Adaptation of biotechnology methods on the study of arthropods.

Biotechnological advances are rapidly being applied to the study of arthropods and arthropod-born diseases. Improved tools are available for species identification, study of species complexes, detection and identification of pathogens in vectors and detection and characterization of insecticide resistance. These techniques have their basis in hybridoma, DNA probe and biochemical assay technologies. Problem areas in assay development are discussed.

Animals↗

PubMed2000

Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence.

The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HMD2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation (D. A. Freedman, L. Wu, and A. J. Levine, 1999, Cell. Mol. Life Sci. 55, 96-107). Several reports have suggested that disruption of this complex in normal cells results in p53 signaling (V. Böttger, A. Böttger, A. Sparks, W.-L. Liu, S. F. Howard, and D. P. Lane, 1997, Curr. Biol. 7, 860-869; C. Wasylyk, R. Salvi, M. Argentini, C. Dureuil, I. Delumeau, J. Abecassis, L. Debussche, and B. Wasylyk, 1999, Oncogene 18, 1921-1934). A homogeneous time-resolved fluorescence (HTRF) assay has been developed to monitor p53/HDM2 binding. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, and two fluorophore-conjugated detection reagents, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody ¿Eu(K)-anti-GST. Binding of p53 to HDM2 brings the fluorophores into close proximity, allowing fluorescence resonance energy transfer to occur. Development of this assay and comparison to a traditional ELISA are described in this report. The HTRF assay was then utilized to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p73 peptide for HDM2.

Amino Acid Sequence↗

PubMed1993

Immunoassays for des-Arg9-bradykinin.

Splenocytes from a female, BALB/c mouse immunized with bradykinin conjugated to ovalbumin with toluene diisocyanate were fused with mouse myeloma cells, X63/Ag8.653, using polyethylene glycol. Seventy-nine hybridomas were identified by ELISA to be making kinin reactive antibodies. In preliminary specificity studies it was determined that all of these hybridomas were producing antibodies more reactive with des-Arg9-bradykinin than with bradykinin. ELISAs were developed with the five clones that displayed the highest affinities for des-Arg9-bradykinin. Radioimmunoassays were developed for 3 of these 5 clones as well as with 5 monoclonal antibodies previously described (Odya and Lee 1990). The most sensitive des-Arg9-bradykinin assay developed was a radioimmunoassay in which carboxypeptidase B-treated [Tyr5]-bradykinin was the labeled antigen, clone OLNBK-5 was the antibody, and dextran-coated charcoal was used to separate bound from free radioactivity. The concentration of des-Arg9-bradykinin that inhibited 50% of the radioactive peptide binding was 0.08 +/- 0.03 nM. The relative specificity of this assay (des-Arg9-bradykinin = 100%) was: 29% bradykinin and about 1% with each of the following: lysyl-bradykinin, methionyl-lysyl-bradykinin, des-Arg1-bradykinin and des-Phe8-Arg9-bradykinin.

Animals↗

PubMedDate not supplied

Pharmacokinetics of aditoprim in goats using a radioassay.

A simple, highly sensitive radioassay was developed for the activity of a newly discovered inhibitor of dihydrofolate reductase (DHFR), aditoprim. The procedure is based on the inhibition of binding of [3H]-methotrexate ([3H]MTX) with bacterial dihydrofolate reductase by the antifolate, aditoprim. The analytic sensitivity using this binding inhibition method was less than 5 ng in plasma. The procedure developed requires no extraction of the drug from the plasma. The variation of simultaneous duplicate determinations was 6.3 per cent, whereas the variability of plasma samples assayed on different days was less than 11 per cent. The assay developed was applied to study the pharmacokinetics of aditoprim in the goat. In comparison with trimethoprim (TMP), the new inhibitor of DHFR, aditoprim, had a longer half-life and a larger volume of distribution, suggesting enhanced and prolonged antibacterial activity of aditoprim over TMP.

Animals↗

PubMed1984

The development and validation of a radioimmunoassay for human pregnancy-associated plasma protein A (PAPP-A).

A specific and sensitive radioimmunoassay for human pregnancy-associated plasma protein A (PAPP-A) is described. This assay utilized tracer repurified by affinity chromatography on heparin-Sepharose. The most sensitive assay developed had a detection limit of 5 microliters PAPP-A in serum, and this protein could not be detected in sera from non-pregnant subjects. During pregnancy, mean circulating levels rose from 0.6 mg/l at 6 weeks gestation to 110 mg/l at term, and PAPP-A was detected in 44 of 101 umbilical blood samples. The use of the anticoagulants EDTA and acid citrate dextrose resulted in significantly different results in the plasma compared to the corresponding serum sample. PAPP-A was also measured in amniotic fluid.

Amniotic Fluid↗

PubMed2007

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts.

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.

Actins↗

PubMed1999

Biochemical diagnosis of aortic dissection: from bench to bedside.

Aortic dissection is an acute cardiovascular disease associated with high mortality and morbidity. Although uncommon, recent studies have shown that the incidence of this catastrophic disease is steadily increasing. Unfortunately, the disease is still not well recognized on clinical presentation due to lack of specific signs and symptoms. As early diagnosis and initial management are critical for survival, we focused on developing a biochemical diagnostic approach for this disease given its meritorious properties in use in the acute clinical situation and additional projected combined use with established imaging modalities. Studies using an assay developed against smooth muscle myosin heavy chain, a protein which is released from the aortic medial smooth muscle cells on insult to the aortic wall, showed promising results for use of this assay in the diagnosis of aortic dissection. The background of this pioneering assay in addition to its clinical use are discussed in this review.

Acute Disease↗

PubMed1993

Development of radioimmunoassays on microplate: application for CA-GI and CA-Br tumor markers.

By taking advantage of a newly available microplate counter for radioactivity and the organic solvent-resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA-GI and CA-BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8-well strips or even single wells. Single-well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions. Similar dose-response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA-Br and CA-GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA-GI and CA-Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated.

Antibodies↗

PubMed2000

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose.

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.

Chemistry, Clinical↗