Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Quantification and purity determination of newly synthesized thioacridines by capillary liquid chromatography

Capillary liquid chromatography (CLC) was applied for quantification and impurity profile determination of ten newly synthesized acridine thioderivatives. A reversed-phase CLC system employing two different stationary phases, Nucleosil C18 and LiChrosorb RP-select B, was used. The mobile phase composition was optimized to get a satisfactory separation of impurities from the main acridine component in a reasonable analysis time. Significant differences in the chromatographic behavior between acridine derivatives containing and lacking amino groups were observed. Optimized separation conditions were used in CLC to measure the calibration curves of the acridine derivatives in a concentration range from 1.0 x 10(-6) to 1.0 x 10(-3) M at two different detector wavelengths (214 and 230 nm). Limits of detection and quantification of all the substances were determined. The detection limits went down to units of microM for most of the derivatives. CLC was also demonstrated to be a suitable method for the purity determination of test batches of the acridine thioderivatives.     

Contribution to the coincidence of malignant tumours and some allergic manifestations

Clinical observations during the past decades led us to the early empirical assumptions that adult patients affected by certain types of allergic manifestations may have a lower prevalence of malignant tumors. In order to test those observations as well as some contradictory reports (1-3), we conducted a retrospective study using 32 years of records and statistics of a medium-sized hospital (420 beds) at St. Mary's Hospital, a general, non-chronic, teaching hospital, affiliated with McGill University in Montreal. The study has been realized during two periods: (a) 1965-1989 and (b) 1990-1996.     

Assessment of spectral analysis of heart rate variability in patients with history of atrial fibrillation by means of age-dependent parameters

Heart rate variability evaluation is a useful diagnostic tool for autonomic nervous balance assessment. The role of the autonomic nervous system in aetiology of atrial fibrillation is sometimes clear as a trigger from a patient's history, but mostly it acts as a modulating factor which is not easy to detect. The present study demonstrates results of spectral analysis of short-term heart rate variability during ortho-clinostatic tests processed by means of age-dependent parameters. An original telemetric system and a unique method for heart rate variability assessment, developed by the Faculty of Physical Culture, were applied for the first time to examine patients with the history of atrial fibrillation.     

Aliphatic ionenes as gene delivery agents: elucidation of structure-function relationship through modification of charge density and polymer length

Polycation-based gene delivery agents are generally polydisperse populations whose properties are averaged among the different molecular weight species. Therefore, to understand the physicochemical properties of polycations and their relationships to cellular gene transfer, one needs to control the molecular weight of the polymer as well as its cationic charge density. To investigate the structure-function correlation of polycations with respect to the degree of polymerization (DP) and charge density, a series of model materials based on aliphatic ionenes was synthesized and fractionated into distinct molecular weight fractions with DP range from 14 to 32. The aliphatic ionene fractions and their polyelectrolyte complexes (PEC) with DNA were studied using physicochemical and biological methods. Ionene polymers were shown to possess low cytotoxicity (minimal viability of the P388D1 murine macrophage cells 80%). DP and charge density of the ionenes were shown to be the factors of effective control of PEC dissociation in water-salt solutions, with a diminished role of charge density upon lengthening the ionene chain. These polymer characteristics were also important for DNA-ionene PEC resistivity to DNase activity and the ability of ionenes to serve as gene delivery vectors in vitro and exhibited good correlation with the results of salt-induced dissociation of PEC. These data may be useful for developing correlations and mathematical models to predict synthetic gene delivery vector efficiency.     

Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.     

A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector

The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.     

Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts

Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice.