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PubMed1989

[Direct detection of oxygen free radicals produced by myocardial reperfusion using electron spin resonance spectroscopy].

There is a growing evidence for the role of oxygen free radicals (OFR) in mediating myocardial tissue injury during myocardial ischemia and particularly during reperfusion. But almost all of the evidence was indirect, using electron spin resonance (ESR) spectroscopy, we have directly measured OFR generated in ischemic and reperfused isolated rabbit hearts. 17 hearts were rapidly frozen in liquid nitrogen after their arrest by cardioplegic solution and sampled after 150 min of sustained hypothermic global ischemia or after reperfusion. The ESR spectra obtained from experiment have directly demonstrated that OFR is produced in significant amounts in the isolated rabbit hearts during early stage of reperfusion but only small amount during ischemia. The mitochondrial electron transport chain appeared to be the main source of OFR. We found that superoxide dismutase scavenged OFR generated during reperfusion efficiently, but catalase did not. We believe that superoxide anion, not hydroxyl radical, is the main OFR which is responsible for myocardial reperfusion injury. We also found that Salvia, a traditional Chinese medicine, a very efficient OFR scavenger, had the similar effect as superoxide dismutase.

Animals↗
PubMed2005

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces.

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.

Avidin↗
PubMed2006

Direct detection of antibiotic resistance genes in specimens of chicken and pork meat.

Antibiotic resistance (AR) in bacteria, a major threat to human health, has emerged in the last few decades as a consequence of the selective pressure exerted by the widespread use of antibiotics in medicine, agriculture and veterinary practice and as growth promoters in animal husbandry. The frequency of 11 genes [tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), vanA, vanB, aac (6')-Ie aph (2'')-Ia, mecA, blaZ] encoding resistance to some antibiotics widely used in clinical practice was analysed in raw pork and chicken meat and in fermented sausages as well as in faecal samples from the relevant farm animals using a molecular approach based on PCR amplification of bacterial DNA directly extracted from specimens. Some of the 11 AR genes were highly prevalent, the largest number being detected in chicken meat and pig faeces. The genes found most frequently in meat were tet(K) and erm(B); vanB and mecA were the least represented. All 11 determinants were detected in faecal samples except mecA, which was found only in chicken faeces. erm(B) and erm(C) were detected in all faecal samples. The frequency of AR genes was not appreciably different in meat compared to faecal specimens of the relevant animal except for vanB, which was more prevalent in faeces. Our findings suggest that AR genes are highly prevalent in food-associated bacteria and that AR contamination is likely related to breeding rather than processing techniques. Finally, the cultivation-independent molecular method used in this work to determine the prevalence of AR genes in foods proved to be a rapid and reliable alternative to traditional tools.

Animal Husbandry↗
PubMed2002

Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum.

Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.

4-Butyrolactone↗
PubMed1993

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis.

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exams at an early age, and nine had never had colon exams (mean age, 12.1 +/- 3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommend that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.

Adenomatous Polyposis Coli↗
PubMed2005

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

2-Naphthylamine↗
PubMed2003

Direct detection of nitric oxide and its roles in maintaining gastric mucosal integrity following ethanol-induced injury in rats.

In gastric mucosal injury, nitric oxide (NO) plays both cytoprotective and cytotoxic roles, and the NO level is one determinant of these dual roles. We employed electron paramagnetic resonance (EPR)-spectrometry combined with an NO-trapping technique to directly evaluate NO production in ethanol-induced gastric injury in rats. The rat stomach, mounted on an ex vivo chamber, was perfused with ethanol (12.5 and 43%), and NO levels in mucosal tissues were measured during perfusion. Luminal nitrite/nitrate (NOx) content, mucosal blood flow, area of mucosal injury, transmucosal potential difference (PD), and luminal pH were simultaneously monitored with/without preadministration of the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). NO levels in the gastric tissue increased during ethanol perfusion, and luminal NOx levels increased after the perfusion, accompanying an increase in the area of mucosal injury and changes in physiological parameters. Preadministration of L-NAME aggravated the gastric mucosal damage and suppressed increases in mucosal blood flow in a dose-dependent manner. These results demonstrate that endogenous NO produced in ethanol-induced gastric injury contributes to maintenance of mucosal integrity via regulation of mucosal blood flow.

Animals↗
PubMed1988

Direct detection of Chlamydia trachomatis in clinical specimens by a dot-immunobinding technique using monoclonal antibody.

A dot-immunobinding technique (DIBT) has been developed to permit detection of Chlamydia trachomatis organisms or antigen in clinical specimens. This method was evaluated for the rapid diagnosis of chlamydia infections using monoclonal antibody. The membrane antigen extracted from reticulate bodies was used for the production of species-specific monoclonal antibodies by an in vitro immunization procedure. The DIBT involved spotting clinical specimen directly onto a nitrocellulose membrane followed by reaction with monoclonal antibody and a biotin-avidin-peroxidase indicator system. Specimens were tested for the presence of chlamydia by the cell culture method. Of these, 361 positives and 317 negatives were selected for detection of antigen using the DIBT method. Of 678 clinical specimens that were evaluated by DIBT, 654 (96.7%) gave identical results to the cell culture method, whereas 24 (3.5%) were positive by the DIBT but culture negative. The overall sensitivity was 100% with a specificity of 92.4%. The test could detect as little as 75 pg of chlamydial antigen.

Animals↗
PubMed1999

Observation of internucleotide NH...N hydrogen bonds in the absence of directly detectable protons.

Several structural motifs found in nucleic acids involve N-H...N hydrogen bonds in which the donor hydrogens are broadened to extinction due to chemical or conformational exchange. In such situations, it is impossible to use the well-established HNN-COSY or soft HNN-COSY experiments, which report the presence of the hydrogen bond directly on the donor proton(s). We present a pulse sequence, H(CN)N(H), for alleviating this problem in hydrogen bonds of the type NdH...Na-CH, in which the donor Nd nitrogen is correlated with the corresponding non-exchangeable C-H proton associated with the acceptor Na nitrogen. In this way, missing NdH...Na correlations in an HNN-COSY spectrum may be recovered from CH-Nd correlations in the H(CN)N(H) spectrum. By correlating a different set of nuclei relative to the HNN-COSY class of experiments, the H(CN)N(H) experiment also serves to remove ambiguities associated with degeneracies in HNN-COSY spectra. The technique is demonstrated on d(GGAGGAG)4,a quadruplex containing a novel A. (G.G.G.G). A hexad and on d(GGGCAGGT)4, containing a G.G.G.C tetrad, in which missing NH2...N7 correlations are retrieved via H8-(N2,N6) correlations in the H(CN)N(H) spectrum.

Adenine↗
PubMedDate not supplied

Direct detection of new flucytosine metabolites in human biofluids by 19F nuclear magnetic resonance.

19F nuclear magnetic resonance was used for the analysis of flucytosine (FC; 5-fluorocytosine) metabolites in biological fluids of a patient with cryptococcal meningitis who was intravenously injected with this drug at a daily dose of 7.5 g (2.5 g at 8-hr intervals). This method allows a direct, simultaneous, and quantitative determination of all the fluorinated metabolites of FC, in the range of sensitivity allowed by the spectrometer (sensitivity threshold, 0.01 mM). In urine, in addition to the already reported metabolites [unmetabolized FC and alpha-fluoro-beta-alanine (FBAL)], three new metabolites were identified: a glucuronide of FC (GLFC), 6-hydroxy-5-fluorocytosine (60HFC), and fluoride ion F-. The same metabolites (except F-) were found in plasma. In cerebrospinal fluid, only unchanged FC and GLFC were observed. The total urinary excretion during an 8-hr period between two injections of FC was 100.4% of the injected dose. Unchanged FC was the major excretory product accounting for 96.1% of the total. GLFC and 6OHFC made up, respectively, 2.7% and 1.2% of the excreted metabolites. The proportions of F- and FBAL were very low, respectively, 0.3% and 0.1% of the excreted metabolites. The global urinary excretion over a 24-hr period was 102% of the injected dose. The proportions of metabolites were very close to those obtained for the 8-hr period. In plasma, the proportions of metabolites were analogous to those determined in urine. In cerebrospinal fluid, GLFC represents 1% of the fluorinated metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Body Fluids↗
PubMed1995

Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.

As a result of X chromosome inactivation, females are mosaic for cell lineages in which either the paternal or the maternal X chromosome is active, and, if inactivation were random, each lineage should be present at approximately the same frequency. Detection of instances of non-random X inactivation can be important both clinically and for the study of X chromosome inactivation. Identification of a single-base polymorphism in an expressed region of the human XIST gene has permitted the development of a direct PCR-based assay for randomness of X inactivation. Oligonucleotide primers were designed, incorporating the variant base, and conditions established that allowed allele-specific PCR amplification. As the XIST gene is expressed only from the inactive X chromosome, differential amplification of the alleles in cDNA from heterozygotes can be used as an indicator of non-random inactivation. Using this assay, non-random X chromosome inactivation has been demonstrated in chromosomally abnormal cell lines and in lymphocytes from heterozygous, normal females. Virtually complete non-random X inactivation was also shown in a mother and her daughter, suggesting the existence of some familial factor affecting X chromosome inactivation.

Base Sequence↗
PubMedDate not supplied

Direct detection of red blood cell fragments: a new flow cytometric method to evaluate hemolysis in blood pumps.

Pump induced hemolysis is presently evaluated by measuring plasma free hemoglobin (fHb). However, this method has disadvantages because quantification of fHb depends on hematocrit (HCT) and hemoglobin (Hb) levels. The aim of this work was to devise a hemoglobin independent method, capable of quantifying cell trauma directly by measuring the number of red blood cell (RBC) fragments. Whole blood flow cytometry was used to quantify circulating RBC fragments derived from a roller pump (Sarns, Inc. Model 2 M 6,002) and a centrifugal pump (Gyro C1E3, Kyocera Corp.). The pumps were tested in a mock circuit for 2 hr (5 L/min flow against 100 mm Hg pressure head). Red blood cell fragments were quantified by a phycoerythrin (PE) labeled glycophorin A antibody specific for erythrocytes. Red blood cell fragments were smaller than the intact RBC population and overlapped in size with the platelet population (based on forward- and side-light scattering measurements). For the roller pump, the values for RBC fragments increased from 1,090 +/- 260/microl at 0 min to 14,880 +/- 5,900/microl after 120 min. In contrast, using the centrifugal pump, there was little increase in RBC fragments (from 730 +/- 270/microl at 0 min to 1,400 +/- 840/microl after 120 min). Flow cytometry can be used for the rapid, sensitive, hemoglobin independent evaluation of pump induced RBC trauma.

Erythrocytes↗
PubMed2002

An algorithm for detecting directional and non-directional positive selection, neutrality and negative selection in protein coding DNA sequences.

Positive selection or adaptive evolution is thought to be responsible, at least some of the time, for the rapid accumulation of advantageous changes in protein-coding genes. The origin of new enzymatic functions, erection of barriers to heterospecific fertilization, and evasion of host response by pathogens, among other things, are thought to be instances of adaptive evolution. Detecting positive selection in protein-coding genes is fraught with difficulties. Saturation for sequence change, codon usage bias, ephemeral selection events and differential selective pressures on amino acids all contribute to the problem. A number of solutions have been proposed with varying degrees of success, however they suffer from limitations of not being accurate enough or being prohibitively computationally intensive. We have developed a character-based method of identifying lineages that undergo positive selection. In our method we assess the possibility that for each internal branch of a phylogenetic tree an event occurred that subsequently gave rise to a greater number of replacement substitutions than might be expected. We classify these replacement substitutions into two categories - whether they subsequently became invariable or changed again in at least one descendent lineage. The former situation indicates that the new character state is under strong selection to preserve its new identity (directional selection), while the latter situation indicates that there is a persistent pressure to change identity (non-directional selection). The method is fast and accurate, easy to implement, sensitive to short-lived selection events and robust with respect to sampling density and proportion of sites under the influence of positive selection.

Algorithms↗
PubMed1993

Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay.

Screening for Chlamydia trachomatis infection in men has traditionally been limited to men who present with urethral symptoms, thereby limiting the detection of asymptomatic chlamydia infection in men. In order to effectively screen both symptomatic and asymptomatic men, we evaluated a newly developed polymerase chain reaction (PCR) assay, Amplicor C. trachomatis, from Roche Molecular Systems for the detection of C. trachomatis in urine specimens in comparison with urethral culture. A total of 530 male urine specimens were collected from 322 symptomatic and 208 asymptomatic men attending two sexually transmitted disease clinics in Baltimore, Md. The prevalence of C. trachomatis by culture was 9.8% (10.6% in symptomatic men and 8.2% in asymptomatic men). Compared with culture, the sensitivity of the PCR was 92.8%, the specificity was 94.7%, the positive predictive value was 68.4%, and the negative predictive value was 99.1%. Discrepant results between culture and PCR were further analyzed by direct fluorescent-antibody staining of elementary bodies in urine sediment and in culture transport vials and by major outer membrane protein PCR of transport media for specimens with negative culture. The revised sensitivity and specificity of PCR for urine were 95.0 and 99.8%, respectively, and the positive and negative predictive values were 98.7 and 99.1%, respectively. The sensitivity of culture compared with PCR and/or direct fluorescent-antibody staining was 68.4%. These results indicate that the PCR assay is a highly sensitive and specific assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in both symptomatic and asymptomatic men.

Bacteriological Techniques↗
PubMed2003

Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products.

Three sets of known Salmonella enterica-specific primers were used collectively, for the first time, to evaluate the use of multiplex polymerase chain reaction (m-PCR) as a diagnostic tool to detect Salmonella enterica in naturally contaminated meat and poultry products. For this purpose a total of 300 samples representing the most frequently used fresh and frozen meat (beef and lamb) and poultry (chicken) products (whole, cut, ground, and processed) were collected from eight locations within Irbid city (Jordan). After an enrichment step, DNA was extracted directly from each food sample and amplified using six Salmonella enterica specific primers. Samples were also analyzed using conventional microbiological methods for the confirmation of Salmonella enterica presence. Out of 300 samples, 93 samples were positive by m-PCR. On the other hand, 67 samples were positive by conventional microbiological methods and only 26 samples were positive by m-PCR alone. The primer pairs, used here, proved to be highly specific for Salmonella enterica. Using m-PCR, Salmonella enterica detection could be achieved easily and the results had been confirmed effortlessly within a short period of time (24-36 hours) compared to 3-8 days for the conventional microbiological methods. Our findings emphasize the value of using a combination of specific primer pairs instead of a single primer pair in the detection process to minimize any chance for any inherent experimental or natural error.

Animals↗
PubMed1992

Direct detection of activated protein C in blood from human subjects.

The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma-derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.

Antibodies, Monoclonal↗
PubMed1991

Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes.

Using 10 overlapping nested sets of primers and using peripheral blood lymphocyte (PBL) total RNA as template, we have developed a system, based on PCR, which allows the rapid production of double-stranded cDNA corresponding to the entire coding sequence of the dystrophin gene. The product can be visualized on native minigels by ethidium staining and directly sequenced after gel purification. We have used this system to analyze the structures of PBL dystrophin mRNA in 26 Duchenne, Becker, or intermediate muscular dystrophy patients who have gross rearrangements of the dystrophin gene. In each case, the effect that the genomic rearrangement has on the structure of the transcript--and, by inference, on the dystrophin protein--has been determined, and the results confirm the frameshift hypothesis. The study also identifies a series of alternatively spliced transcripts which are specific to the rearranged genotypes and which seem therefore to arise following the alteration in the context of the splice signal. The system has been used for unambiguous identification of carrier females. Furthermore, the rapid production of microgram quantities of dystrophin cDNA from a readily accessible tissue makes point-mutation screening a practical proposition.

Base Sequence↗
PubMed1996

Direct detection of peptide-dependent HLA variability by surface plasmon resonance.

Cytotoxic T lymphocytes (CTL) recognize antigens as peptides associated with molecules of the major histocompatibility complex (MHC). The accurate characterization of antigenic peptides requires knowledge of how peptides bind to MHC molecules, and hence the conformational changes they can induce. Several reports have indicated that the conformation of the MHC class I molecule plays a role in T cell recognition. We therefore studied the interaction of a series of viral epitopes with HLA-A2, -A3, -B7 and -B8 molecules to determine how peptides could induce conformational changes in HLA molecules. This was done either directly with class I heavy chains in lysates of peptide-loading deficient T2 cells, or with purified material from B-EBV transformed cell lines. The peptide-induced HLA conformations were assessed using monoclonal anti-HLA antibodies (mAbs) and detected by surface plasmon resonance (SPR). Antigenic peptides specifically bound to the HLA molecule, even when assembly occurred in a mixed solution of HLA molecules. Distinct patterns of reactivity to a given peptide-bound class I molecule were obtained with monomorphic and allele-specific anti-HLA mAbs.

Amino Acid Sequence↗