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Biomedical subjects

I C Smith

Publications and source records attributed to I C Smith.

At least 73 records · Page 4Linked to original sources
PubMed1991

Biological and NMR markers for cancer.

The search for a universal tumor marker continues. Present markers range from tumor products (polyamines, glycoproteins, peptides, hormones or carbohydrate-linked markers) to reaction products produced by the host tissues during tumor invasion. Techniques used to identify them include the classical methods of histology and cytochemistry as well as the more recent radioimmunoassay and metabolic probes. The in vivo techniques of increasing use for patient monitoring are MRS (magnetic resonance spectroscopy) and MRI (magnetic resonance imaging). The efficiency of some markers and statistical methods used in analyzing data are discussed, as are the ethical problems surrounding the use of new testing methods. Recent developments in MRI and MRS, marker elucidation, and evidence for a new autocrine differentiation-inhibiting factor (ADIF) are reviewed. Future needs and approaches focus on greater utilization of indicators of the preneoplastic state and of risk to cancer, as well as more careful attention to statistical analysis.

Biomarkers, Tumor↗
PubMed1991

Slow motions in lipid bilayers. Direct detection by two-dimensional solid-state deuterium nuclear magnetic resonance.

Two-dimensional solid-state 2H NMR spectroscopy of specifically deuteriated lipids is used to detect and to characterize the rate and mode of slow motions in two lipid bilayer systems. Lateral diffusion of lipid molecules over the curved surface of dipalmitoylphosphatidylcholine liposomes can be detected by two-dimensional exchange 2H NMR and it is shown that molecular orientational exchange is complete on the timescale of 100 ms. In contrast, it is shown that for the glycolipid 1,2-di-O-tetradecyl-3-O-Beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL), there is no evidence of a corresponding orientational exchange in the liquid-crystalline phase suggesting that this lipid forms relatively flat bilayers. In the gel phase of hydrated multibilayers of beta-DTGL, a slow (10(3) s(-1)) whole molecule axial motion is demonstrated at 40 degrees C. Comparison of the experimental and simulated 2D-NMR ridge patterns suggests that large angle jumps about the long molecular axis, rather than small step Brownian diffusion, can best account for the 2D-exchange spectra of beta-DTGL in the gel phase. The significance of this technique for the study of dynamics in other biological systems is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Dynamics of glycolipids in the liquid-crystalline state. 2H NMR study.

It has been shown previously that two types of motion are adequate to describe the partially relaxed 2H NMR line shapes (inversion recovery experiment) for the backbone portion of the glycolipid 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in the highly ordered gel phase (Auger, M.A., D. Carrier, I.C.P. Smith, and H. C. Jarrell. 1990. J. Am. Chem. Soc. 112:1373-1381). This study extends the latter investigation to the more fluid liquid-crystalline phase, where more complex motions are anticipated. Analyses of the powder line shapes and oriented sample relaxation data for both the glycerol backbone and head group regions of this lipid have been performed. The dynamics of glycerol at the C3 position in the gel state have been described by large angle jumps about the C2-C3 bond with a correlation time in the fast-limit motional regime (omega o tau c much less than 1) and site populations 0.46, 0.34, and 0.20. The present data show that in the liquid-crystalline phase the internal jump rate is maintained, and two additional motions are necessary to describe the dependence of the relaxation rate on the orientation of the director with respect to the magnetic field direction. These are rotation about the molecular long axis with a correlation time in the slow-limit motional regime very near to the T1 minimum (omega o tau c approximately 0.65), and molecular fluctuations about the order director (modeled by a Maier-Saupe restoration potential). This treatment was also extended to the glucose head group where additional segmental motion about the glycosidic bond has been reported previously. While the two motions dominating relaxation at the glycerol C3 segment reproduce the general relaxation features of the glucose head group, the results suggest that additional motion about the glycosidic linkage must be present. This study is a stringent test of the motional model chosen earlier because relaxation data were obtained at two 2H NMR frequencies using two relaxation experiments (T1Z and T1Q) and two types of sample preparation (oriented and dispersed multibilayers). The results strongly uphold the choice of model and indicate the utility of both oriented samples and the T1Q experiment.

Biophysical Phenomena↗
PubMed1991

Molecular details of anesthetic-lipid interaction.

By means of isotopic labeling with deuterium (2H) and observation by nuclear magnetic resonance (NMR), it has been possible to measure the conformational ordering and dynamics of lipids and anesthetics in membranes. The local anesthetic tetracaine causes disordering in the acyl chains of the lipids, very little effect near the glyceryl residue, and minor ordering effects in the charged head group. The anesthetic itself is well ordered within the lipid matrix. The magnitudes of these effects depend upon the nature of the lipid, and upon whether or not the anesthetic is charged. A model can be constructed for the location of the anesthetic within the lipid bilayers. Confirmation of the model comes from Fourier transform infrared spectroscopy (FTIR), which also shows directly the pressure-driven exclusion of local anesthetic from lipid, and the augmentation of this effect by the presence of cholesterol. Future studies by solid-state NMR of protein, and neutron diffraction of specifically deuterated components of anesthetic-nerve systems, should reveal even more precise details of the molecular events involved in anesthesia.

Anesthesia↗
PubMed1991

Identification of a multiprotein complex interacting with the c-fos serum response element.

The serum response element (SRE) is essential for serum and growth factor stimulation of the c-fos gene. We have examined the nuclear proteins, obtained from tissues with elevated expression of the c-fos gene (proliferating rat liver and hepatocarcinoma), that bind to the SRE sequence. A synthetic oligonucleotide containing the SRE sequence from the mouse c-fos gene promoter (-299 to -322) was radioactively labeled, used as a probe for the mobility shift assay and Southwestern (DNA-protein) blotting, and also used for sequence-specific affinity chromatography. We have identified a group of nuclear proteins of molecular sizes 36, 45, 62, 67, 72, and 112 kDa capable of interacting with the SRE sequence. The 36-, 67-, and 112-kDa proteins have DNA-binding properties, but the presence of the others in the SRE-protein complex could be the result of protein-protein interaction. All of these protein factors were present in nuclei obtained from intact and proliferating rat liver as well as from 5123tc Morris hepatoma. The DNA-binding activity (on Southwestern blots) of the 67- and 112-kDa proteins was not affected by alkaline phosphatase treatment, but the ability of the dephosphorylated nuclear proteins to form the complex with the SRE sequence under gel shift assay conditions was severely impaired. The same alkaline phosphatase treatment completely abolished the DNA-binding properties of the c-fos cyclic AMP-responsive element-specific proteins. Therefore, transcriptional activation of the c-fos gene at the SRE must require the presence of a multiprotein complex the formation of which is governed by phosphorylation. The binding of the 67- and 62-kDa proteins to the c-fos SRE has been previously reported; however, the 36-. 45-, 72-, and 112-kDa proteins are novel factors involved in the multifaceted regulation of c-fos gene expression in vivo.

Animals↗
PubMed1990

Glycerolipids: common features of molecular motion in bilayers.

In the present study, analysis of 2H NMR line-shape and spin-lattice relaxation behavior has been used to investigate the dynamics of several glycolipid and phospholipid bilayers. The gel-phase spectra of these lipids labeled at the C3 position of the glycerol backbone are broad (approximately 90 kHz) and characteristic of fast-limit axially asymmetric motion. Moreover, anisotropic spin-lattice relaxation is observed in all of these systems. The line-shape and relaxation features of the lipids in the gel phase were best simulated by using a fast-limit three-site jump model, with relative site populations of 0.46, 0.34, and 0.20. This motion is associated with an internal jump about the C2-C3 bond of the glycerol backbone. A second motion, rotation about the long axis of the molecule, is needed to account for the observed temperature dependence of the quadrupolar echo amplitude and the spectral line shape above and below the gel to liquid-crystalline phase transition temperature. On the other hand, the gel-phase spectra of phospholipids labeled at the C2 position of the glycerol backbone are also characterized by a fast internal motion, which is simulated by a two-site librational jump. The results indicate that the glycerol backbone dynamics of the glycolipid and phospholipid systems investigated in this study can be described in terms of common fast internal motions and a slower whole molecule axial motion. These results are compared with previous dynamic studies of similar systems.

Computer Simulation↗
PubMed1990

High-pressure infrared study of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine.

High-pressure Fourier-transform infrared (FT-IR) spectroscopy was used to study the barotropic behavior of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine. The model membrane systems studied were multilamellar aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) in the absence and the presence of tetracaine at pH 5.5 and 9.5. The infrared spectra were measured at 28 degrees C in a diamond anvil cell as a function of pressure up to 25 kbar. The results show that the barotropic behavior of the negatively charged phosphatidylserine bilayers is very similar to that observed for zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, with corresponding acyl chains. The results also indicate that the local anesthetic partitions into phosphatidylserine bilayers in an environment close to the membrane-water interface and interacts electrostatically with the lipid head group. Application of high hydrostatic pressure on the lipid-anesthetic systems results in the pressure-induced expulsion of the anesthetic from a membrane to an aqueous environment. The pressures required for expulsion of anesthetic from bilayers are much higher for the unsaturated lipid (DOPS) than for the saturated lipid (DMPS) (approximately 6 kbar vs approximately 2 kbar, respectively). Whereas incorporation of the anesthetic into DOPS bilayers does not affect significantly the structural and dynamic properties of the disordered acyl chains in the liquid-crystalline phase, it orders the DMPS acyl chains in the gel phase.

Lipid Bilayers↗
PubMed1990

Optimization of magnetization transfer measurements: statistical analysis by stochastic simulation. Application to creatine kinase kinetics.

A systematic study was performed to optimize the accuracy of kinetic parameters derived from magnetization transfer measurements. Three techniques were investigated: time-dependent saturation transfer (TDST), saturation recovery (SRS), and inversion recovery (IRS). In the last two methods, one of the resonances undergoing exchange is saturated throughout the experiment. The three techniques were compared with respect to the accuracy of the kinetic parameters derived from experiments performed in a given, fixed, amount of time. Stochastic simulation of magnetization transfer experiments was performed to optimize experimental design. General formulas for the relative accuracies of the unidirectional rate constant (k) were derived for each of the three experimental methods. It was calculated that for k values between 0.1 and 1.0 s-1, T1 values between 1 and 10 s, and relaxation delays appropriate for the creatine kinase reaction, the SRS method yields more accurate values of k than does the IRS method. The TDST method is more accurate than the SRS method for reactions where T1 is long and k is large, within the range of k and T1 values examined. Experimental verification of the method was carried out on a solution in which the forward (PCr----ATP) rate constant (kf) of the creatine kinase reaction was measured.

Adenosine Diphosphate↗
PubMed1990

Biosynthesis and characterization of a series of deuterated cis,cis-octadeca-6,9-dienoic acids.

[4,4-2H2]-, [5,5-2H2)-, [6-2H]-, [7-2H]-, [8,8-2H2)-, [11,11-2H2]-, [14,14-2H2]- and [18,18,18-2H3]-cis,cis-octadeca-6,9-dienoic (isolinoleic) acid were synthesized by supplementing cultures of the protozoan Tetrahymena with the corresponding deuterated cis-octadeca-9-enoic (oleic) acids. The cultures were harvested, the deuterated isolinoleic acids isolated and analyzed for purity by GC and TLC, and the structure and the level and position of deuteration of each fatty acid determined by 13C-NMR spectroscopy. The 13C resonances of all 18 carbons were also assigned based upon alpha-carbon deuterium isotope shifts and by comparison of the spectra to those of other polyunsaturated fatty acids. The results illustrate the utility of a biological approach for the synthesis of deuterated polyunsaturated fatty acids in yields suitable for 2H-NMR studies of membranes and possibly human metabolism.

Carbon Isotopes↗
PubMed1990

The effects of inhibitors of arachidonate metabolism on spontaneous and evoked endplate potentials at normal and crotoxin-treated frog neuromuscular junctions.

Aspirin (1 mM), a selective cyclooxygenase inhibitor, decreased endplate potential (e.p.p.) amplitude but did not affect either the biphasic changes in m.e.p.p. frequency or triphasic changes in e.p.p. amplitude induced by crotoxin. Nordihydroguaiaretic acid (30 microM), a dual inhibitor of cyclooxygenase and lipoxygenase pathways, increased e.p.p. amplitude but did not affect the characteristic changes in m.e.p.p. frequency and e.p.p. amplitude in response to crotoxin. The incidence of giant m.e.p.p.s was reduced by aspirin and increased by NDGA. Arachidonate metabolism is not a major factor in the induction of neurotoxicity by crotoxin at the frog neuromuscular junction.

Animals↗
PubMed1990

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions.

Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca2+ plus tubocurarine or 0.5 mM Ca2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Direct and indirect contractile responses of the human vas deferens and actions of noradrenaline and of calcium antagonists.

Contractile responses of the isolated human vas deferens, obtained from vasectomy operations, were measured. Large single electrical shocks gave a twitch response with short latency (0.36 s) which was insensitive to prazosin (5 microM) or TTX (0.2 microM) and was thus identified as due to direct muscle stimulation. A train of 100 low intensity shocks gave a response with a longer latency (1.9 s) which was substantially sensitive to both prazosin and TTX; we assume this response is dominated by an indirect nerve-induced contraction. Relaxations, presumably caused by activation of circular muscle, were recorded from regions of some preparations both by direct and indirect stimulation. Noradrenaline (10-20 microM) induced a tonic contracture, spontaneous contractions and a large potentiation of the response to direct stimulation--but not to indirect stimulation implying a strong presynaptic inhibition. Noradrenaline also speeded the relaxation from contractions. Verapamil (1-100 microM) and nifedipine had no effect on the direct responses but verapamil (10 microM) inhibited the indirect response. Calcium removal prevented most, and 5 mM-EDTA all, of the direct response. However, even with EDTA, noradrenaline was able to support spontaneous and stimulus-induced contractions. Thus contraction of the vas, though sustained by external calcium, does not appear to directly depend on it.

Calcium↗
PubMed1990

Magnetic resonance spectroscopy in cancer research.

Magnetic resonance spectroscopy (MRS), a useful complement to magnetic resonance imaging (MRI), yields a wealth of information about the biochemical content of tissue and the dynamic behavior of its component molecules. When used for colonic tumors, it can distinguish between those with and those without metastatic potential, through the relaxation time (T2) of the resonance at 1.3 ppm in the proton (1H) MRS spectrum. The presence of peaks near 3.2 ppm correlates well with tumor stage and allows identification of premalignant tissue. Two-dimensional MRS provides an independent method for staging colonic tumors by means of a crosspeak between 1.3 and 4.2 ppm. With a combination of MRS and MRI (localized spectroscopy) it should soon be possible to locate and stage a colonic tumor without physical intervention. In the plasma of patients who have cancer there often exists a lipoprotein band with MRS characteristics similar to those of colonic tumors. It also occurs occasionally in healthy persons and in those who have diseases other than cancer. The lipoprotein appears to be lipoprotein (a), which also has been associated with cardiac disease. We have used a simple MRS measurement on unfractionated plasma, which has been suggested as a reliable indicator of the presence of cancer, on approximately 1800 persons who were healthy, ill with cancer, and ill with other diseases. We found it unsatisfactory as a screening method for an asymptomatic population, because essentially it measures the plasma triglyceride level, especially that in very low-density lipoprotein, which can be elevated for a variety of reasons unrelated to cancer.

Animals↗