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PubMed2005

Electrochemical/electrospray mass spectrometric studies of I- and SCN- at gold and platinum electrodes: direct detection of (SCN)3-.

Results on the electrochemistry of I- and SCN- at gold and platinum electrodes using an electrochemical cell coupled to an electrospray mass spectrometer are reported. We demonstrate that our apparatus is capable of these very challenging electrochemical/electrospray experiments and that B(C6H5)4- is a suitable internal standard for negative-ion studies in acetonitrile. With I- at a platinum electrode, we observe well-behaved oxidation to I3-. Experiments on I- at gold electrodes are more complex, showing AuI2- as well as I3-. The AuI2- mass spectrometric ion intensity varies in a complex way throughout the applied electrochemical voltage range studied; we propose that this variation involves the adsorption of I- on the gold electrode surface. In experiments on SCN- from (C4H9)4NSCN at gold electrodes, we observe Au(SCN)2-. Finally, at platinum electrodes, we directly observe (SCN)3-, a species analogous to I3- and (CN)3- that has been previously postulated but unverified. This important finding was confirmed by the isotope pattern and demonstrates the stability of the anion.

Journal Article↗
PubMed2000

Direct detection of pulsations of the Cepheid star zeta Gem and an independent calibration of the period-luminosity relation.

Cepheids are a class of variable (pulsating) stars whose absolute luminosities are related in a simple manner to their pulsational periods. By measuring the period and using the 'period-luminosity' relationship, astronomers can use the observed visual brightness to determine the distance to the star. Because these stars are very luminous, they can be observed in other galaxies, and therefore can be used to help determine the expansion rate of the Universe (the Hubble constant). Calibration of the period-luminosity relation is a necessary first step, but the small number of sufficiently nearby Cepheids has forced the use of a number of indirect means, with associated systematic uncertainties. Here we present a distance to the Cepheid zeta Geminorum, determined using a direct measurement (by an optical interferometer) of its changes in diameter as it pulsates. Within our uncertainty of 15 per cent, our distance is in agreement with previous indirect determinations. Planned improvements to the instrument will allow us to calibrate directly the period-luminosity relation to better than a few per cent.

Journal Article↗
PubMed1992

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Acrylamides↗
PubMed1994

Peptide-induced conformational changes in class I molecules. Direct detection by flow cytometry.

Activation of a T cell in response to peptide bound to class I MHC occurs by the sum of interactions across the area of contact between the TCR, the peptide, and class I MHC. It has been observed recently that substitution of the peptide residue at a position that is not accessible from the exterior of the class I molecule modulates T cell responses, raising the possibility that there may be indirect structural effects in the peptide-class I complex as a consequence of peptide binding. This report describes the use of mAbs to probe the conformation of the alpha 1 and alpha 2 domains of the mouse class I molecule Kb when bound to ovalbumin peptide and a panel of 19 peptide analogues that differ at position 2 (P2). By crystallographic data, side chains of this position are buried in the Ag binding cleft and have no direct access to the TCR. Substitution of position 2 results in a measurable change in conformation of the class I molecule, a change that correlates with the ability to stimulate T cells. This leads to a model that T cell activation by the peptide-class I complex may occur in three ways: 1) direct interaction of the TCR with the class I heavy chain, 2) direct interaction of the TCR with solvent-accessible peptide side chains, and 3) indirect interaction of peptide with TCR mediated via conformational perturbations in the class I complex.

Amino Acid Sequence↗
PubMed1992

Gonadal mosaicism in a family with adrenoleukodystrophy: molecular diagnosis of carrier status among daughters of a gonadal mosaic when direct detection of the mutation is not possible.

Adrenoleukodystrophy is a severe, X-linked neurological disease that has been shown to be linked to DNA markers from Xq28. We tested several families with these markers and, in one family, found two apparent recombination events between DXS52 and the disease. Expansion of the study to include other tests and several others markers from Xq28 led us to conclude that recombination probably had not occurred and that, instead, the mutation in this family had a mitotic origin and that the grandmother was a gonadal mosaic. For genes that have been cloned, it is often possible to demonstrate the presence or absence of a specific mutation in such families and to determine carrier status on that basis. This is not possible when the gene has not been cloned. We therefore describe a method that can be employed by a molecular diagnostic laboratory to discriminate between people who inherit the same RFLP haplotype, with or without the mutation, from a parent with gonadal mosaicism in diseases where direct gene analysis is not yet possible.

Adrenoleukodystrophy↗
PubMed2001

Direct detection of a microlens in the Milky Way.

The nature of dark matter remains mysterious, with luminous material accounting for at most approximately 25 per cent of the baryons in the Universe. We accordingly undertook a survey looking for the microlensing of stars in the Large Magellanic Cloud (LMC) to determine the fraction of Galactic dark matter contained in massive compact halo objects (MACHOs). The presence of the dark matter would be revealed by gravitational lensing of the light from an LMC star as the foreground dark matter moves across the line of sight. The duration of the lensing event is the key observable parameter, but gives non-unique solutions when attempting to estimate the mass, distance and transverse velocity of the lens. The survey results to date indicate that between 8 and 50 per cent of the baryonic mass of the Galactic halo is in the form of MACHOs (ref. 3), but removing the degeneracy by identifying a lensing object would tighten the constraints on the mass in MACHOs. Here we report a direct image of a microlens, revealing it to be a nearby low-mass star in the disk of the Milky Way. This is consistent with the expected frequency of nearby stars acting as lenses, and demonstrates a direct determination of a lens mass from a microlensing event. Complete solutions such as this for halo microlensing events will probe directly the nature of the MACHOs.

Journal Article↗
PubMed1989

Direct detection of a major mutation responsible for phenylketonuria in the population of the Federal Republic of Germany.

Forty-six individuals having phenylketonuria (PKU) alleles at the phenylalanine hydroxylase (PAH) locus were tested for the haplotype 2 PKU mutation by allele-specific hybridization following in vitro DNA amplification. Patients and carriers previously shown to have a mutant haplotype 2 PAH allele demonstrated conservation of this mutation. In vitro DNA amplification greatly facilitated this analysis and provides the possibility of population screening for 37% of the mutant German PAH alleles.

Alleles↗
PubMed1996

Direct detection of the antioxidant activity of a new flavonic derivative using the chemiluminescence method.

The antioxidant potential of a new water soluble flavonic derivative, namely theophylline rutoside (TR-1722) has been tested using the chemiluminescence method. The method is based on the oxidative degradation of luminol by the hydrogen peroxide in Tris-HCl-buffer, when reactive species of oxygen are being obtained: O2-., HO., 1O2, and allows for the capability of substances to inhibit the free radical processes in this test system to be quantified and hence for their antioxidant properties in respect to a standard substance (in our case quercetin) to be compared. The results obtained reveal that TR-1722 has antioxidant action comparable to that of quercetin, the highest efficacity being registered at the concentration of 2 mumol/l, the conditions being: H2O2 16,2 mmol/l; luminol 2 mumol/l, in Tris-HCl buffer 20 mmol/l, pH 8.3. The antioxidant potential of TR-1722 is also maintained when the conditions of the system are modified, that is, the concentration of the hydrogen peroxide, the intensity of the action being dependent on the hydrogen peroxide concentration, but no direct proportionality is registered.

Antioxidants↗
PubMed1992

Direct detection of circulating hepatitis C virus RNA using probes from the 5' untranslated region.

Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards.

Base Sequence↗
PubMed2003

Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy.

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.

Computer Simulation↗
PubMed1996

Hepatitis C and B virus infections in hepatocellular carcinoma. Analysis of direct detection of viral genome in paraffin embedded tissues.

BACKGROUND: Although there have been many seroepidemiologic studies on hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) occurrence, the actual role of HCV in hepatocarcinogenesis is unknown. METHODS: We have previously reported on a highly sensitive method of detecting and identifying sequences of RNA genome in formalin fixed, paraffin embedded (FFPE) tissue by polymerase chain reaction (PCR) assay. Using this method, we carried out a retrospective study to determine the prevalence of HCV and hepatitis B virus (HBV) genomes in FFPE specimens from 102 Japanese patients with HCC. RESULTS: HCV-RNA was detected by nested PCR reverse transcription (RT) in 64 of the 102 patients (62.7%), and 78.1% (50/64) of those HCV-RNA-positive patients had HCV genotype II. HCV-RNA was present in 54 of 70 (77.1%) anti-HCV-positive patients, and also in 5 of 20 (25%) anti-HCV-negative patients. HBV-DNA was detected by nested PCR in FFPE liver specimens from 21 of 102 (20.6%) patients. HBV-DNA positivity was consistent with seropositivity for serum HBV markers in 17 of these 21 patients (80.9%). HBV-DNA was present in FFPE samples from 2 patients who were seronegative for HBV markers, and in 1 patient who was seropositive for anti-HBs. Double infection of these two viruses was found in 6 patients (5.9%). Three patients (2.9%) were negative for both hepatic viral genomes and serum viral markers. CONCLUSIONS: The precise prevalence of HCV and/or HBV infection among HCC patients can be determined by studying routinely-processed FFPE HCC samples preserved for up to 11 years using the technique of nested PCR. HCV-RNA was detected in the majority of our HCC cases; type II was the most common genotype of HCV encountered. The incidence of HCV-associated HCC was three times greater than that of HBV. Thus, the hepatitis virus infection most frequently associated, and probably ectologically implicated, with HCC in Japanese people is HCV infection.

Adult↗
PubMed2000

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants.

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.

Chromatography, High Pressure Liquid↗
PubMed1999

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry.

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.

Amino Acid Sequence↗
PubMed1997

The power of iterated generalized least squares (GLS) method to detect direct relationships in the analysis of correlated quantitative traits.

We examined the power of the stepwise iterated generalized least squares (GLS) method by modeling the relationship between quantitative traits and other variables using the simulated data for Problem 2A. The comparison between the generating model provided by the workshop and the results of the stepwise iterated GLS model showed that this method could be used as a first step in identifying the underlying model for genetic data. The estimated covariance matrices also provide useful information about the genetic properties of the traits.

Computer Simulation↗
PubMed1998

Restriction-PCR fingerprinting of the immunoglobulin VH repertoire: direct detection of an immune response and global analysis of B cell clonality.

Here we describe a method for fingerprinting the mouse immunoglobulin heavy chain variable region (VH) repertoire. Using a novel combination of existing techniques, large numbers of expressed VH genes can be simultaneously displayed as a fingerprint of VH gene fragments on a sequencing gel. This is achieved using isotype-specific reverse transcription-PCR amplification, restriction digestion and end-labeled primer run-offs. This technique (Res-PCR) allows analysis of the immune response, in this case to phenyloxazolone, in several different tissues and enables molecular cloning and sequencing of the VH genes involved in vivo. In addition, with Res-PCR, a "global" picture of expressed immunoglobulin genes is represented. This elucidates B cell clonality in different tissues and isotypes and detects a preference for shorter complementarity-determining region 3 in IgM-expressing cells. Res-PCR is a rapid and revealing method which will allow analysis of the complexity and sequence composition of the B cell immunoglobulin repertoire and so perhaps better define the B cell memory compartment and immune responses in vivo.

Amino Acid Sequence↗
PubMed1999

Separation and direct detection of raw and gelatinized starch hydrolyzing activities of glucoamylase on isoelectric focusing gels.

A procedure to detect raw and gelatinized starch activities of glucoamylase on isoelectric focusing (IEF) gels by using 2, 3, 5-triphenyltetrazolium chloride is described. The reagent reacts with the reducing group of glucose released by glucoamylase from the substrate starch. Using the reaction, production of glucoamylase by Aspergillus niger was detected on 10% IEF gels within a pH range of 2.5-9.5. Since the method can detect raw and gelatinized starch activities of glucomylase associated with 1 microg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.

Aspergillus niger↗
PubMed1999

Whipple's arthritis: direct detection of Tropheryma whippelii in synovial fluid and tissue.

We describe 2 patients presenting with polyarthritis in whom the synovial fluid (1 patient) or synovial tissue (1 patient) was positive for Tropheryma whippelii, the Whipple's disease-associated bacillus, when examined by polymerase chain reaction (PCR) and DNA sequencing. Histopathologic findings were consistent with articular Whipple's disease in the synovial fluid of 1 patient and the synovial tissue of the other. In both patients, bowel mucosal specimens were negative for Whipple's disease features by histologic and PCR methods. One patient was positive for T whippelii in the peripheral blood. Control synovial fluid specimens from 40 patients with other arthritides, including Lyme arthritis, were negative. Sequencing of a 284-basepair region of the 16S ribosomal RNA gene confirmed that the sequence is closely related to the known T whippelii sequence. Both patients responded to treatment with antibiotics.

Actinobacteria↗