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Biomedical subjects

S A Martin

Publications and source records attributed to S A Martin.

At least 73 records · Page 4Linked to original sources
PubMed1996

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Base Sequence↗
PubMed1996

Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry to oligonucleotide analysis.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

Base Sequence↗
PubMed1996

Automated analytical system for the examination of protein primary structure.

This paper describes an automated analytical system for the examination of protein primary structure in which (i) the target protein is first purified by immunoaffinity chromatography, (ii) subsequent chromatographic and chemical reaction steps in the sequencing process are directly coupled, (iii) buffer exchange between these unit operations is achieved while the protein is absorbed on a mixed bed of strong ion exchange sorbents, (iv) proteolysis occurs in an immobilized trypsin column having a 10-1000 fold-excess of enzyme, (v) the tryptic digest is directly transferred to a perfusion dilute capture column where it is concentrated and rapidly desalted, and (vi) peptides eluted from the dilute capture column and analytical microbore and capillary perfusion reversed-phase chromatography columns are analyzed by either single-stage mass spectrometry (MS) or tandem MS/MS. Protein structure variants were easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb A0 was verified.

Amino Acid Sequence↗
PubMed1996

The study of 2,3,4-trihydroxyacetophenone and 2,4,6-trihydroxyacetophenone as matrices for DNA detection in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric study of DNA detection using 2,3,4-trihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, and their combination has been carried out systematically. The results show that a mixture of 2,4,6-trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone and ammonium citrate with molar ratios of 2:1:1 serves as a good matrix for the detection of DNA, especially for samples containing a small quantity of DNA such as polymerase chain reaction product. The resolution and shot-to-shot reproducibility using this matrix are better than, and the MALDI sensitivity comparable to, that obtained when using 3-hydroxy picotinic acid (3-HPA), PA and ammonium citrate matrix (9:1:1). The mechanism of desorption/ionization using this matrix is discussed.

Base Sequence↗
PubMed1996

Brugia pahangi: characterisation of a small heat shock protein cDNA clone.

Microfilariae of Brugia pahangi undergo a transition in their life cycle from the homeothermic mammalian host to the poikilothermic mosquito vector. Coincident with this switch, the developmental cycle of the microfilaria is reinitiated. When cultured at mammalian temperatures (37 degrees C), microfilariae express a complex of small heat shock proteins. In order to further characterise this group of proteins a cDNA library was constructed from heat-shocked microfilariae by reverse-transcriptase PCR and then screened with a heterologous probe. A clone which codes for a small heat shock protein was isolated and characterised in detail. Southern blot analysis identified a putative small heat shock protein multigene family. The expression of the small heat shock protein cDNA appears to be controlled at the level of RNA synthesis and is highly stage specific and temperature dependent.

Amino Acid Sequence↗
PubMed1996

Effect of extracellular hydrogen on organic acid utilization by the ruminal bacterium Selenomonas ruminantium.

The objective of this study was to evaluate the effect of extracellular H2 on organic acid utilization by two lactate-utilizing strains of Selenomonas ruminantium (HD4, H18). Both strains were able to grow (optical density at 600 nm > or = after 9 h) on either aspartate, fumarate, or malate in the presence of 1 atmosphere (atm) of H2. Succinate was the major end product produced in these fermentations. When cells were incubated with lactate plus 1 atm H2, growth was minimal little lactate was fermented. The electron transport inhibitor, acriflavine, was strong inhibitor of growth when either strain was incubated in the presence of organic acid plus H2. Compared with glucose- or lactate-grown cells, cellular carbohydrate levels were lower for both strains in cells grown on either organic acid plus H2. These results suggest that electron transport plays a role in organic acid utilization by S. ruminantium.

Animals↗
PubMed1996

Stage-specific gene expression in lymphatic filarial nematodes.

Lymphatic filarial nematodes remain a significant cause of morbidity throughout much of the tropics. One approach to the development of rational control methods is an improved understanding of the basic biology of these organisms in relation to the mechanisms used to complete their life cycles. In this article, Eileen Devaney, Sam Martin and Fiona Thompson review new approaches to defining stage-specific molecules in filarial nematodes, and discuss their recent work on the isolation and characterization of stage-regulated cDNAs from Brugia pahangi.

Journal Article↗
PubMed1996

Laser desorption mass spectrometry for point mutation detection.

A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment.

Cell Line↗
PubMed1996

Effects of organic acid and monensin treatment on in vitro mixed ruminal microorganism fermentation of cracked corn.

The objective of this study was to determine the effects of organic acids and monensin on the in vitro fermentation of cracked corn by mixed ruminal microorganisms. Ruminal fluid was collected from a steer fed 36.3 kg of wheat silage and 4.5 kg of concentrate supplement once daily. Mixed ruminal microorganisms were incubated in anaerobic media that contained 20% (vol/vol) ruminal fluid and .4 g of cracked corn. Incubations were carried out in batch culture for 24 h at 39 degrees C. Organic acids (L-aspartate, fumarate, and DL-malate) were added to serum bottles (n = 4) to achieve final concentrations of 0, 4, 8, or 12 mM. Monensin, dissolved in ethanol, was included in serum bottles at a final concentration of 0 or 5 ppm of culture fluid. The addition of 8 and 12 mM organic acids to cracked corn fermentations increased final pH (P < .05), tended to increase total gas production and CO2 concentration, and decreased the acetate:propionate ratio (P < .05). Organic acids tended to decrease methane concentrations and hydrogen concentration was not altered. DL-Malate addition at all levels reduced (P < .05) lactate accumulation. Additive effects of monensin and organic acids were observed in some fermentations. In conclusion, organic acid addition to in vitro mixed ruminal microorganism fermentations yielded beneficial results independent of monensin treatment by decreasing the acetate: propionate ratio and increasing final pH.

Acetates↗
PubMed1996

Ecology, metabolism, and genetics of ruminal selenomonads.

Selenomonas ruminantium is one of the more prominent and functionally diverse bacteria present in the rumen and can survive under a wide range of nutritional fluctuations. Selenomonas is not a degrader of complex polysaccharides associated with dietary plant cell wall components, but is important in the utilization of soluble carbohydrates released from initial hydrolysis of these polymers by other ruminal bacteria. Selenomonads have multiple carbon flow routes for carbohydrate catabolism and ATP generation, and subspecies differ in their ability to use lactate. Some soluble carbohydrates (glucose, sucrose) appear to be transported via the phosphoenolpyruvate phosphotransferase system, while arabinose and xylose are transported by proton symport. High cell yields and the presence of electron transport components in Selenomonas strains has been documented repeatedly and this may partially account for the energy partitioning observed between energy consumed for growth and maintenance functions. Most strains can utilize ammonia, protein, and/or amino acids as a nitrogen source. Some strains can hydrolyze urea and/or reduce nitrate and use the ammonia for the biosynthesis of amino acids. Experimental evidence suggests that ammonia assimilatory enzymes in some strains may possess unique properties with respect to other presumably similar bacteria. Little is known about the genetics of ruminal selenomonads. Plasmid DNA has been isolated from some strains, but it is unknown what physiological functions may be encoded on these extrachromosomal elements. Due to the predominance of S. ruminantium in the rumen, it is an ideal candidate for genetic manipulation. Once the genetics of this bacterium are better understood, it may be possible to amplify its role in the rumen.

Adenosine Triphosphate↗
PubMed1996

Hexose phosphorylation by the ruminal bacterium Selenomonas ruminantium.

Three strains of Selenomonas ruminantium (D, GA192, and H18) were surveyed for phosphorylation of D-glucose and 2-deoxyglucose by phosphoenolpyruvate and ATP. Cells of all three strains that had been treated with toluene had high rates of hexose phosphorylation with either phosphoryl donor; this activity was constitutive in strain D. Glucose phosphorylation that was dependent on phosphoenolpyruvate was maximal at pH 7.2, remained fairly high at pH 6.5, but decreased (> or = 65%) at pH 5.0 for all strains. Cell extracts were used to evaluate the involvement of soluble kinases in 2-deoxyglucose phosphorylation. Both glucose and 2-deoxyglucose were phosphorylated by ATP, but phosphorylation of either hexose was negligible with phosphoenolpyruvate in each bacterium. Because phosphoenolpyruvate could not serve as a phosphoryl donor, the activity dependent on phosphoenolpyruvate in cells treated with toluene might have been due to a phosphotransferase system associated with the membrane. Unlabeled 2-deoxyglucose was a strong inhibitor (> or = 59%) of [14C]glucose phosphorylation with ATP by cell extracts of all S. ruminantium strains, and unlabeled glucose was a strong inhibitor (> or = 78%) of [14C]2-deoxyglucose phosphorylation with ATP. Based on Lineweaver-Burk kinetics, 2-deoxyglucose was a competitive inhibitor of initial rates of kinase activity in strains D, GA192, and H18. These results collectively suggest that glucose phosphorylation with phosphoenolpyruvate and 2-deoxyglucose phosphorylation with ATP are common traits in these strains of S. ruminantium.

Adenosine Triphosphate↗
PubMed1996

Chronic respiratory illness as a predictor of survival in idiopathic dilated cardiomyopathy: the Washington, DC, Dilated Cardiomyopathy Study.

Although bronchial asthma and emphysema have been associated with idiopathic dilated cardiomyopathy in case-control studies, little is known about the prognostic importance of chronic respiratory disease in idiopathic dilated cardiomyopathy. To study this, we examined history of bronchial asthma, emphysema and chronic bronchitis, and respiratory medication use as possible predictors of survival in idiopathic dilated cardiomyopathy using data from a Washington, DC, population-based study (n = 129). The cumulative survival rates among patients with a history of emphysema or chronic bronchitis were 60% and 48% at 12 and 36 months, respectively, compared with 81.8% and 67.2% among patients without emphysema or chronic bronchitis. The survival rates of idiopathic dilated cardiomyopathy patients with and without a history of bronchial asthma at the time of idiopathic dilated cardiomyopathy diagnosis were similar. In multivariate analysis using the proportional hazards model, only ventricular arrhythmias and ejection fraction were found to be statistically significant predictors of survival in idiopathic dilated cardiomyopathy. The adjusted relative risk estimate for emphysema and chronic bronchitis was close to one. Thus, the results of this population-based study do not suggest that history of chronic respiratory illness is an independent predictor of survival in idiopathic dilated cardiomyopathy.

Adult↗
PubMed1996

Applying nursing theory to the practice of nurse anesthesia.

With the current movement of anesthesia education into graduate programs, changes in curriculum are inevitable. These changes will include advanced nursing theory. How the issues of nursing theory apply to the practice of nurse anesthesia are examined. Applications of Betty Neuman's systems theory are used in specific examples of the anesthesia role. The profession of nurse anesthesia may benefit significantly from the contributions of nursing theory.

Education, Nursing, Graduate↗
PubMed1995

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.

Adrenocorticotropic Hormone↗