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Biomedical subjects

C H Becker

Publications and source records attributed to C H Becker.

At least 19 recordsLinked to original sources
PubMed1999

Laser desorption of DNA oligomers larger than one kilobase from cooled 4-nitrophenol.

A unique matrix system consisting mostly of 4-nitrophenol has shown to be very effective for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of large DNA oligomers when a cooled sample stage was used to prevent the sublimation of this matrix under vacuum. Using this 4-nitrophenol matrix with UV laser desorption, detection of picomole quantities of DNA oligomers containing up to approximately 800 nucleotides was routinely achieved. The effectiveness of this matrix was further demonstrated by the observation of a double-stranded DNA oligomer larger than 1000 base pairs, seen as a denatured single-stranded species, with a molecular ion mass exceeding 300 000 Da. The potential applications of 4-nitrophenol as a matrix for DNA sizing are discussed.

DNA↗
PubMed1999

Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry.

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for detecting SNPs are laborious and time-consuming, often prohibiting large-scale analysis. A rapid, accurate, and cost-effective method is needed to meet the demands of a high-throughput DNA assay. We demonstrate here that analysis of these genetic markers can now be performed routinely in a rapid, automated, and high-throughput fashion using time-of-flight mass spectrometry and a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single-base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass difference between the SNP primer and the extension peak reveals which nucleotide is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymorphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an A/T heterozygote with extension products differing by only 9 Da. Multiplex SNP determination is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.

DNA Primers↗
PubMed1998

Reliable genotyping of short tandem repeat loci without an allelic ladder using time-of-flight mass spectrometry.

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.

Alleles↗
PubMed1997

Cryogenic frozen solution matrixes for analysis of DNA by time-of-flight mass spectrometry.

We present an alternative matrix system for mass analysis of high molecular weight biomolecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Single-stranded DNA oligomers are embedded in thin films of frozen solutions containing photoabsorbing substituted phenols. Ultraviolet laser desorption from these cryogenically cooled matrixes generates internally colder ions than from the equivalent room-temperature crystalline matrixes containing no solvent, as evidenced by a substantial reduction in the extent of fragmentation in the corresponding mass spectra of oligonucleotides. Furthermore, the homogeneity of the frozen solution samples allows analyte signals to be obtained uniformly throughout the sample. Sensitivity and mass resolution approaching that obtained using the most effective room-temperature crystalline matrix for DNA (3-hydroxypicolinic acid) are consistently and reproducibly obtained in both positive and negative ion mass spectra. As fragmentation is a principle factor limiting the maximum attainable mass resolution in MALDI-TOF mass analysis of DNA, this improvement in generating intact DNA ions is advantageous.

DNA↗
PubMed1996

Effect of impurities on the matrix-assisted laser desorption mass spectra of single-stranded oligodeoxynucleotides.

The effect of impurities on the analysis of single-stranded DNA oligomers by the technique of matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry has been studied using the matrix 3-hydroxypicolinic acid and 355-nm pulsed light. By mixing the DNA oligomers with different concentrations of impurities and recording mass spectra, limits are set on the tolerable level of a given impurity in a sample. The tolerance limits for sodium chloride, potassium chloride, sodium acetate, sodium fluoride, sodium dodecyl sulfate (SDS), and manganese(II) chloride were found to be approximately 10(-2) M. It was found that magnesium salts degraded the mass spectrum at much lower levels of 10(-4) M. The organic compounds tris(hydroxymethyl)aminomethane (Tris), urea, dithiothreitol (DTT), glycerol, and ethylenediaminetetraacetic acid (EDTA), when present as its ammonium salt, were tolerable at concentrations into the range of 0.25-0.5 M, while the organic polyamine compound spermine substantially degraded the mass spectrum at concentrations above 10(-2) M. When comparing these results for DNA analysis with previously reported limits for protein analysis, large differences are seen for some of the impurities tested.

Base Sequence↗
PubMed1996

Homocysteine induced arteriosclerosis-like alterations of the aorta in normotensive and hypertensive rats following application of high doses of methionine.

Following oral administration of methionine in high doses to normotensive (NR) and spontaneously hypertensive (SHR) rats, its degradation product, homocysteine (HC), which is markedly elevated in serum, exerts an angiotoxic action directed to the aorta. This is accompanied by considerable loss of endothelium and degeneration, partly with dissolution of the media cells with formation of characteristic processes of the degenerating mitochondria, and by elevated HC and cystathion (CT) values in the aortic wall. At the arterial vessels of other organs similar alterations did not occur. There are quantitative differences between NR and SHR. In SHR, serum shows higher HC and CT concentrations than in NR, and the methionine-related aortic alterations are considerably more pronounced and develop earlier, with the additional formation of connective tissue. Here, a certain dependence on the methionine dose is noted, in contrast to NR, for which the magnitude of the reaction appears to be more related to the length of time of methionine application. Additional administration of atherogenic substances (cholestane-3 beta, 5 alpha, 6 beta-triol, cholesterol, angiotensin II, cholic acid with methylthiouracil) in SHR causes an exacerbation of the methionine-related aortic alterations. Only cholestane-triol has the same effect on the aortic wall in NR and SHR, with more accentuation in SHR. Cholestane-triol has, in NR as well as in SHR, a high coincidence with methionine-induced morphological reactions including the formation of mitochondrial processes. Simultaneous application of these two substances did not cause a potentiation of the effect. High doses of cholesterol bring about aortic alterations in SHR but not in NR. Thus, in addition to the disorder of fat and carbohydrate metabolism, disturbed protein metabolism is of decisive importance as a risk factor for coronary and other vascular diseases.

Angiotensin II↗
PubMed1995

Analysis of enzymatic DNA sequencing reactions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The products from base-specific, dideoxy-nucleotide chain-termination DNA sequencing reactions catalyzed by the modified T7 DNA polymerase have been analyzed by using the technique of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Preliminary experiments were performed to determine detection limits for a synthetic mixture of mixed-base single-stranded DNA which contained a 14-mer, a 21-mer, and a 41-mer; acceptable spectra, showing peaks for each component, were obtainable for samples that contained as little as 5 fmol per component. Initial sequencing reactions were therefore carried out on 2-pmol amounts of a short synthetic template that was 45 nucleotides in length, employing 2 pmol of 12-mer as the primer strand. This provided readable sequence information out to the 19th base past the primer. Using a 21-mer primer, nearly the entire sequence of the template could be read.

Base Sequence↗
PubMed1994

Time-of-flight mass spectrometry of underivatized single-stranded DNA oligomers by matrix-assisted laser desorption.

Matrix-assisted laser desorption with concomitant ionization (MALDI) in conjunction with time-of-flight mass spectrometry (TOF-MS) has been used to analyze underivatized random-base single-stranded DNA (ssDNA) oligomers ranging from 10 to 89 nucleotides in length by embedding them in a solid matrix of 3-hydroxypicolinic acid. At 355-nm desorption wavelength, mass spectra of positive and negative ions measured by reflecting and linear time-of-flight mass spectrometers are compared. Results from the linear system show the ionization yield is approximately equal for each polarity. Metastable ion decay is significant for the larger ssDNA oligomer ions, which results in a decrease in signal intensity and the broadening of mass peaks. In order to obtain an acceptable signal-to-noise ratio on a reflecting TOF system, a higher laser irradiance is needed, which consequently causes further degradation of mass resolution. With the apparent advantages of better sensitivity and mass resolution, it is concluded that a linear TOF system is better suited for the mass spectrometric analysis of ssDNA oligomers larger than about a 25-mer. The current system permits one-base resolution up to about a 40-mer. Mass accuracy for a 20-mer or smaller is within +/- 0.05%. Comparison of mass spectra from 5-ns and 35-ps pulse widths at the same energy density shows no significant differences. Mechanisms for oligonucleotide ion production in these experiments are discussed.

DNA, Single-Stranded↗
PubMed1993

Matrix-assisted laser desorption time-of-flight mass spectrometry of oligonucleotides using 3-hydroxypicolinic acid as an ultraviolet-sensitive matrix.

Matrix-assisted laser desorption time-of-flight mass spectrometry has been used to produce quasi-molecular ion signals from underivatized mixed-base single-stranded DNA oligomers ranging from 10 to 67 nucleotides in length. These results were obtained with a new matrix material, 3-hydroxypicolinic acid (3-hydroxypyridine-2-carboxylic acid) which showed significant improvement over many previously reported matrices studied in terms of mass range available, signal-to-noise ratio, and the ability to analyze mixed-base oligomers. The desorption and simultaneous ionization was by pulsed laser light at 10 to 50 mJ/cm2, studied at 266, 308, and 355 nm. Spectra taken at 266 nm provided the smallest amounts of doubly charged and dimer ions--characteristics desirable for DNA sequencing by this technology. Negative-ion spectra were uniformly superior to positive-ion spectra. This new matrix also is quite effective for molecular weight determinations of peptides and proteins in both positive- and negative-ion modes.

Base Sequence↗
PubMed1992

Matrix-assisted laser desorption mass spectrometry of oligodeoxythymidylic acids.

Ferulic acid, sinapinic acid and 2,5-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T)12 and a mixture of oligonucleotides pd(T)12 through pd(T)18 using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.

Mass Spectrometry↗
PubMed1990

[Submicroscopic indication of pathological cell growth in hypertrophic cardiomyopathy (HCM) in humans].

The increasing application of transvascular endomyocardial biopsy for diagnostic verification of various heart diseases and elucidation of their etiology relies mainly on light microscopical methods. It is shown by hypertrophic cardiomyopathy as an example that also submicroscopical findings may be of value in diagnostic work, prognosis, and investigating research for etiological relationships. Specific morphological markers for idiopathic HCM were not demonstrable; nevertheless, some characteristic alterations such as fibrillary disorder, excessive folding of nuclear-, intercalated disc-, and sarcolemmal membranes, contraction bands, accumulation of mitochondria, glycogen, and lipid droplets, among others as well as sometimes the thickening of vascular walls with its unknown relations to the so-called "small vessel disease" may be helpful when endomyocardial biopsies are examined with the electron microscope too. Though many cytomorphologic findings are similar to those in dilatative cardiomyopathy, this disease probably is not a final state of HCM. In order to prove the actual cause of both heart failures, more intensive investigating research especially at the molecular biological level is required.

Biopsy↗
PubMed1987

Action of cholestane-3 beta,5 alpha,6 beta-triol on rats with particular reference to the aorta.

Cholestane-3 beta,5 alpha,6 beta-triol, administered orally to rats in different doses and for varying lengths of times, effected toxic cell damage on aortic smooth muscle cells and endothelium. Cholesterol, applied in the same doses, did not lead to appreciable alterations of the aorta. After parenteral application of lipids with simultaneous administration of cholestane-triol there were no demonstrable fat deposits in the damaged aortic wall with angiotensin II (AII) induced hypertension. Thus, there was no recognizable influence of hypertension on increased fat passage in the arterial wall, or any action of lipids to enhance the permeability of vessels. However, the hypertension had an exacerbating effect in so far as in animals with AII-induced blood pressure rise alterations of the media were more pronounced after cholestane-triol, although we were unable to rule out a primary effect of AII. A potentiation of the cholestane-triol action by simultaneous application of cholesterol demonstrated for the rabbit did not occur in rats. Blood content was lowered mostly by cholestane-triol, also by cholesterol. HDL-cholesterol was little affected; almost no influence was observed in triglycerides. The strong cytotoxic action of cholestane-triol underlines its health-damaging role. Due to its action on the aorta of the rat, despite the animal's resistance to arteriosclerosis, involvement of this cholesterol derivative in the pathogenesis of arteriosclerotic alterations can not be excluded.

Administration, Oral↗
PubMed1984

Pectin-gelatin complex coacervates II: Effect of microencapsulated sulfamerazine on size, morphology, recovery, and extraction of water-dispersible microglobules.

Spherical medicated microglobules were prepared by complex coacervation of Type A gelatin with pectin, having nominal diameters of 5, 10, and 25 micron and containing 37.3, 44.9, and 45.2% (w/w) sulfamerazine, respectively. They were recovered as water-insoluble powders and were spontaneously revertible to highly disperse systems when reconstituted in water or physiological electrolyte solution. The conditions affecting microglobule formation were studied. For complete formation, the crystals must be dispersed at greater than or equal to pH 5. The effect of the sulfamerazine mass added on microglobule morphology, yield, and contents were investigated. As much as 37.3, 44.5, and 69.1% (w/w) sulfamerazine in 5-, 10- and 25-micron microglobules could be formed without loss of spherical shape. The microglobule yield versus drug-to-colloid ratio curves were nonlinear below the critical drug-to-colloid ratio for loss of sphericity. Addition of sulfamerazine suppressed coacervation by 10-15% but it had no significant effect on microglobule size. The extraction of medicated microglobules in various media demonstrated the existence of a porous matrix that required hydration to facilitate extraction of the microglobular drug. Fifteen percent of the encapsulated sulfamerazine was extracted from 25-micron microglobules as opposed to 9% from 10-micron microglobules after equilibration for 24 h in replacement electrolyte solution.

Capsules↗
PubMed1984

Different behaviour of normotonous and spontaneously hypertensive rats with vitamin D intoxication.

Spontaneously hypertensive rats (SHR) were found to tolerate the consequences of vitamin D intoxication much better than Wistar Kyoto rats (WKY). This is obvious from the 10-fold higher death rate of WKY rats and from the considerable differences of the tissue reactions occurring in the aorta, the cardiac muscle and the arterial vessels between WKY rats and SHR. Thus, myocardial alterations are much rarer in SHR; this holds also for the aorta calcification which is less pronounced in SHR and which is age-dependent in so far as it is almost absent from one year onward. Giant granulomas as a result of vitamin D intoxication were found in juvenile SHR at a time where they would otherwise no yet have occurred at a frequency observed in old untreated SHR. Common to both WKY rats and SHR is the formation of vascular alterations (plasmatic vasculosis) which reveal--though less pronounced with SHR--the angiotoxic action of vitamin D. For these considerable differences in the behaviour of WKY and SHR in vitamin D intoxication the different Ca-binding and transport properties of the arterial plasma membranes should be responsible, which is likely to be due to a defect in the Ca-transport mechanism of the smooth vascular muscle. The importance of these findings for the use of vitamin D intoxication in arteriosclerosis research as well as for studies on the pathogenesis of permeability disorders and alterations of the arterial vascular system is pointed out.

Aging↗
PubMed1983

[Autoradiographic analysis of proliferative processes in the arterial terminal vascular system in experimental hypertension].

The participation of various vascular wall cells in the proliferation process and their distribution in the arterial terminal vascular bed were investigated by 3H-thymidine autoradiography in rats during an acute angiotensin hypertension. The results show that both the proliferative pattern and the distribution of labeled cells have distinct differences according to the functional importance and the histologic structure of the vessels. A common feature of all vascular parts is the enhancement of the proliferation of smooth muscle cells. In particular, the proliferation is increased in the arterioles, suggesting a structural adaptation to the raised vascular tonus. The strongly altered endothelium of peripheral vessels show a retarded reparative cell proliferation. This implies a considerable risk factor for the development of an arteriosclerosis in the circulatory periphery.

Acute Disease↗