Biomedical subjects
Publications and source records attributed to M C Fitzgerald.
The utility of mass spectrometry for the analysis of biological molecules has been enhanced by the development of two techniques that generate gas-phase ions via nondestructive vaporization and ionization. These techniques can be used not only to determine the primary structure of biological molecules with unprecedented accuracy, but also to map noncovalent biomolecular interactions.
In the past several years, significant progress has been made in the application of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to the analysis of large biopolymers, including nucleic acids. By isolating analyte molecules in an appropriate matrix and irradiating the sample with a high-intensity, pulsed laser beam, MALDI can generate intact, gas-phase ions of these analytes. Primarily used with time-of-flight mass spectrometers, this relatively new, soft ionization technique has allowed for the routine analysis of oligonucleotides up to 60 or so nucleotides in length. Recent results have also shown that base specific, matrix-dependent fragmentation is an important factor in the MALDI analysis of oligonucleotides. Further extension of the technique to longer oligonucleotides will rely on both the continued search for new matrix materials and an increased understanding of the desorption and ionization process in MALDI.
We have produced a line of transgenic mice expressing human hepatic triglyceride lipase (hH-TGL) to examine the in vivo effects of hepatic lipase expression on high density lipoprotein catabolism. Activation of metallothionine I promoter-hH-TGL cDNA transgene produced high levels of lipase mRNA in liver, heart, and kidney and elevated enzyme activity as assayed in post-heparin plasma. In a series of hyperlipidemic diet studies in which zinc was included in the diet to induce the transgene, hH-TGL expression was associated with a 34% lowering of plasma HDL-cholesterol levels (p < 0.01) when compared with animals on the same hyperlipidemic diet without zinc. This lowering of HDL cholesterol was paralleled by a decrease in total cholesterol and a decrease in HDL particle size. SDS-polyacrylamide gel electrophoresis analysis of the smaller HDL particles revealed that apolipoprotein AI was still the major apoprotein associated with the HDL. Quantitative analysis of abdominal aortic cholesterol content from the same animals suggests that the observed changes in plasma HDL by hH-TGL over-expression correlated with a decrease in the accumulation of aortic cholesterol (42%, p < 0.01). These data support the hypothesis that hH-TGL mediates a non-receptor pathway for the clearance of cholesterol from the plasma compartment.
beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.
In order to examine the importance of pH in the matrix-assisted laser desorption/ionization (MALDI) analysis of proteins and oligonucleotides, 37 highly substituted pyrimidine, pyridine, and benzene derivatives containing basic amino groups were screened as potential matrices. Several of these compounds were useful for the preparation and analysis of samples under neutral and basic pH conditions. The matrix, 2-amino-4-methyl-5-nitropyridine, gave good results with small proteins (up to approximately 12,000 Da). The best results with oligonucleotides were obtained with 2-amino-5-nitropyridine, which was a useful matrix for the analysis of mixed-base oligonucleotides smaller than 20 nucleotides in length and homopolymers of thymidine. In general, oligonucleotide analysis using basic matrices was found to be subject to similar constraints of size and base composition that hold when acidic matrices are used. A major advantage of these newly discovered matrix materials over the more commonly used acidic matrices is that they extend the utility of MALDI to the analysis of acid-sensitive species.
A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).
Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene.
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To determine the effects of a school-based, nurse-run prenatal counseling program, records of prenatal care visits of students and their infants' birth weights (i.e., cases) were obtained from the state live birth certificate tape for 1985-1987. From the same tape, a mother residing in the same community but not enrolled in the program was matched with each case on eight criteria to serve as a control. A total of 288 matched case-control pairs were obtained. Using McNemar's test, a significantly higher percentage of the cases received adequate prenatal care than did the controls.
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This report describes the characterization and complete sequence of the human ornithine decarboxylase (ODC) gene. Genomic Southern blot analysis shows only a single gene hybridizing at high stringency, in contrast to the murine multigene family. A Pst I restriction fragment length polymorphism was identified and an allele of the human ODC gene containing the polymorphic Pst I site was cloned and sequenced. The ODC gene is divided into 12 exons and spans 8 kb. Comparison of the human, rat, and mouse ODC genes shows striking conservation of genomic organization, as well as 82% identity in the first 148 bp of the 5'-flanking region. This region contains a TATA box, cAMP-responsive element, CCAAT box, and AP-2 binding site and is consistent with induction of ODC gene expression by both the cAMP and protein kinase C-mediated signaling pathways. The first intron of the human gene is 2,849 bp in length, and contains two putative Sp1 binding sites, as well as an Ap1 binding site, suggesting a role for the first intron in transcriptional regulation. The 5' noncoding region of the predicted mRNA contains regions of virtual identity with that of mouse and rat ODC mRNA, suggesting sequences involved in translational regulation. In addition, it was found that the exon segments corresponding to the amino and carboxyl termini of Saccharomyces cerevisiae and Trypanosoma b. brucei are unrelated to their mammalian counterparts, whereas the middle segments of the protein are conserved. These differences may influence the difference in protein half-life seen between T. b. brucei and mammalian ODC.
Distinct hemoglobins are synthesized in goats at different stages of development, similar to humans. Embryonic hemoglobins (zeta 2 epsilon 2 and alpha 2 epsilon 2) are synthesized initially and are followed sequentially by fetal (alpha 2 beta F2), preadult (alpha 2 beta C2), and adult (alpha 2 beta A2) hemoglobins. To help understand the basis of these switches, the genes of the beta-globin locus have been cloned and their linkage arrangement has been determined by the isolation of lambda phage carrying overlapping inserts of genomic goat DNA. The locus extends over 120 kilobase pairs and consists of 12 genes arranged in the following order: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Comparison of the nucleotide sequence of the 12 genes shows that the locus is organized into three homologous four-gene sets that presumably evolved by the triplication of an ancestral set of four genes (epsilon-epsilon-psi beta-beta). Interestingly, the three genes (beta C, beta A, and beta F) located at the ends of the four-gene sets are expressed at different stages of development. Therefore, the goat beta F-, beta C-, and beta A-globin genes appear to have evolved by a mechanism that includes the triplication of 40-50 kilobase pairs of DNA and the recruitment of newly formed genes for expression in fetal, preadult, and adult life.
Increasing use is being made of microprocessors in medicine to perform routine clinical measurements. Physiological parameters whose measurement involves data processing on the part of hospital technicians can in many cases be better obtained using such instrumentation. The application of a microprocessor to the measurement of ST-segment elevations in the electrocardiogram is described to illustrate the use of programmable instrumentation in medicine and demonstrate some of the ensuing benefits.
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Proteins have evolved to carry out very specific functions within the cell by interacting with a diverse set of biomolecules. Understanding how a protein's higher order structure relates to its function is important for defining the molecular basis of these interactions. In recent years, mass spectrometry has become an important tool for dissecting protein structure and function. Using electrospray ionization (ESI)- and matrix-assisted laser desorption/ionization (MALDI)-based approaches, it has been possible to monitor protein folding, characterize noncovalent protein complexes, and assess the contribution of individual amino acid residues to a protein's function. Here, it is our goal to summarize these approaches and highlight recent, biologically relevant applications where mass spectrometry has provided unique insight into the mysteries of protein structure and function.
The reaction of sulphite with formaldehyde to form hydroxymethylsulphonate (HMS), which is very stable under the controlled conditions of this assay, was used as the first step in an analytical procedure to determine foodborne sulphite. The effect of mobile-phase pH on the stability of HMS during high-performance liquid chromatography was studied. It was found that on-column HMS dissociation to formaldehyde and bisulphite increased with the pH of the mobile phase; therefore the relatively low pH 4.7, at which the dissociation of HMS was approximately 2%, was selected for the analysis. In addition, the release of sulphite from its reversibly bound forms in wine and other foods was examined as a function of the pH of the extraction medium by following the appearance of HMS formed from the reaction of the freed sulphite with formaldehyde. The rate of dissociation of the reversibly bound sulphite was relatively slow at pH 3 but very rapid at pH 7. This difference in kinetics was exploited to develop a procedure to determine free and reversibly bound sulphite in food. The method was challenged by post-reagent spiking studies, i.e. adding the sulphite spike after the food has been blended with the sulphite-protective formaldehyde solution but before proceeding with the remainder of the assay. An average recovery of 100% with a standard deviation of 5.2% (n = 45) was realized at levels of 5, 10 and 20 ppm by weight as sulphur dioxide. Recovery of the sulphite added as the bisulphite addition product of acetaldehyde, a model compound for reversibly bound sulphite, was 95%.