Search PubMed⌕ Search

Biomedical subjects

E J Fernandez

Publications and source records attributed to E J Fernandez.

33 records · Page 2Linked to original sources
PubMed1994

31P NMR investigation of energy metabolism in perifused MMQ cells.

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. 31P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the 31P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.

Adenylyl Cyclases↗
PubMed1994

Detection of Streptococcus mutans by PCR amplification of spaP gene.

Synthetic oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a sequence of the spaP gene, which encodes the surface protein antigen I/II of Streptococcus mutans. A DNA fragment of c. 192 bp was amplified from lysed S. mutans cells or isolated DNA. With S. mutans cells, the lower limit of detection was 4-40 cfu. With these primers, 13 reference and 50 clinical strains of S. mutans were identified. Amplification of the 192-bp product was not demonstrated when 41 strains of other streptococcal and non-streptococcal species were tested. The spaP gene PCR has potential for the rapid diagnosis of S. mutans infections.

Adult↗
PubMed1992

1H spectroscopic imaging of rat brain at 7 tesla.

1H magnetic resonance spectroscopic imaging has been used to obtain metabolite maps of the rat brain. The spin-echo-based technique has been evaluated with respect to water and lipid suppression and sensitivity. Metabolite maps were constructed for choline, creatine + phosphocreatine, amino acids, N-acetyl aspartate, and lactate. A spatial resolution of 3 x 3 mm (in plane) with 7-mm-thick slices was achieved routinely in 60-min (16 x 16 phase encodings) acquisitions. For higher intensity resonances, metabolite maps could be constructed in as little as 10 min. Results from phantoms and from rats under normal and focal ischemia conditions are presented.

Amino Acids↗
PubMed1992

Three-dimensional 1H spectroscopic imaging of cerebral metabolites in the rat using surface coils.

Three dimensional metabolite maps of protonated metabolites were obtained using 1H magnetic resonance spectroscopic imaging at 7 T. Surface coils were used to increase sensitivity and spatial resolution significantly over a volume coil two-dimensional acquisition. Adiabatic pulses were employed to provide homogeneous B1 excitation and frequency selective refocusing over the volume of the rat brain. These techniques were employed to obtain three-dimensional spectroscopic imaging spectra from nominal voxel volumes of 9-30 microliters from rat brain. The improved spatial resolution and sensitivity are also demonstrated with studies of focal ischemia in the rat.

Animals↗
PubMed1992

Phospholipase A2 in rat gingival tissue.

Phospholipase A2 (PLA2) is a proinflammatory enzyme in the synovial fluids of all--and sera of some--patients with rheumatoid arthritis. Due to the similarities in pathogenesis between rheumatoid arthritis and periodontitis, we sought to study the enzymatic properties of PLA2 in periodontal tissue. In this study, we demonstrated PLA2 activity in rat gingival tissue, about 80% of which was present in the cytosolic fraction. We characterized the cytosolic PLA2 enzyme with respect to substrate specificity, sensitivity to detergent, Ca2+ ion dependency and optimum pH. We found that phosphatidylethanolamine, rather than phosphatidylcholine, was the preferred substrate, the Ca2+ ion was essential for the expression of PLA2 activity, the enzyme was active over a broad pH range, with the optimum at pH 9.0, and sodium-deoxycholate inhibited the enzyme activity strongly in a concentration-dependent manner. These results are consistent with those which have been obtained with synovial fluid PLA2 and suggest that gingival PLA2 may be involved in the pathogenic processes of gingivitis and periodontitis.

Analysis of Variance↗
PubMed1990

A nuclear magnetic resonance technique for determining hybridoma cell concentration in hollow fiber bioreactors.

We have developed a technique for determining cell concentration in a hollow fiber bioreactor based on 23Na nuclear magnetic resonance (NMR) spectroscopy. Cell concentrations determined with this method agreed closely with concentrations calculated from 31P NMR nucleoside triphosphate (NTP) measurements and oxygen consumption rate measurements. Oxygen transfer limitations, which can complicate cell mass determinations based on oxygen consumption rates, were shown to be negligible for the bioreactor used. Specific antibody production rates in hollow fiber culture, calculated from these cell number estimates, were similar to those found in suspension culture for this cell line.

Animals↗
PubMed1989

A comparison of three radiofrequency coils for NMR studies of conductive samples.

Three rf coil designs of equal volume (approximately 15 ml) were compared using conductive samples. Magnetic loss into the sample was the dominant noise source. At physiological conductivity the sensitivity of the horizontally aligned solenoid and loop-gap resonator was only 1.3 +/- 0.2 times that of the vertically aligned slotted tube resonator.

Equipment Design↗
PubMed1984

The appearance of rapidly flowing blood on magnetic resonance images.

The appearance of rapidly flowing blood on imaging (MRI) was evaluated using flow phantoms and dye infusion experiments. Laminar flow can be maintained at high velocities in small-diameter vessels. Under such conditions, flow-related enhancement may be observed several slices into a multislice imaging volume. Decreasing cross-sectional area of the unsaturated protons in the midstream is noted on slices further removed from the entry surface. As the velocity increases, turbulence occurs. The increased random motion of the protons causes loss of intensity on the first-echo image, although rephasing with increased intensity can be noted on the second-echo image. The flow pattern of a simple intraluminal obstruction is demonstrated by MRI and dye infusion experiments. Rephasing is noted within the eddy downstream from the obstruction. Clinical examples of the phantom findings are shown and applications are discussed.

Blood Circulation↗
PubMed1983

The effect of stasis on a microvascular anastomosis.

An experiment was done in rats to determine the effect of stopping the flow of blood across a fresh anastomosis. It was found that brief periods of stasis, up to 30 minutes, caused a minimal increase in the rate of thrombosis, but 60 minutes of stasis caused a significant risk of thrombosis in both arteries and veins. The thrombosis associated with stasis could be prevented by the systemic administration of heparin.

Animals↗
PubMedDate not supplied

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry.

We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the identification of the conformers present.

Animals↗
PubMedDate not supplied

Mapping of cerebral metabolites in rats by 1H magnetic resonance spectroscopic imaging. Distribution of metabolites in normal brain and postmortem changes.

The goal of this study was to examine metabolic differences between cortex and basal ganglia in normal rat brain and to determine postmortem changes using in vivo 1H magnetic resonance spectroscopic imaging at 300 MHz. The resonances observed were: choline, creatine + phosphocreatine, N-acetyl aspartate (NAA), lactate (Lac), and three small resonances in the amino acid region which included resonances from aspartate + NAA (Asp), glutamine + NAA (Gln), and glutamate + GABA (Glu). A previously unassigned resonance was observed at 1.13 ppm in brain of rats anesthetized with pentobarbital. Spectroscopic images in normal brain demonstrated increased NAA and Gln and decreased Glu in cortex compared to basal ganglia. The major postmortem changes were an increase of Lac, Glu and Cho and a decrease of NAA and Asp. The rise in Lac was significantly higher in cortex than in basal ganglia.

Amino Acids↗
PubMedDate not supplied

Unfolding and conformational distributions during protein precipitation.

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.

Chemical Precipitation↗
PubMedDate not supplied

Mechanical deformation of compressible chromatographic columns.

A one-dimensional model of mechanical deformation of compressible chromatography columns is presented. The model is based on linear elasticity and continuum mechanics and is compared to a more complete two-dimensional model and one-dimensional porosity profiles measured by NMR imaging methods. The model provides a quantitative description of compression and the effects of wall support during scale-up. A simple criterion for the significance of wall support as a function of both diameter and length is also developed. Although the model accounts only for mechanical deformation, flow compression can be included, and validation presented here suggests that a more complete model may be valuable for anticipating the effects of scale and aspect ratio on pressure-flow behavior of compressible columns.

Chromatography, Liquid↗