Ontogenetic changes of water states and structural organization in growing kidney beans: parameter-imaging based on the diffusion measurements.

Physical states of cell-associated water in growing kidney beans was studied by NMR microscopy. 1H-NMR images indicated the changes of water amount in the individual tissues. The seed gradually reduced water and water in the tissues of the pod varied according to growth stages. Images that reflect diffusive motion provide different contrast from that of the proton images; signal intensity was maintained in the tissues containing small amounts of water and showing weak signal on the proton images. These images are related to the activity of metabolism and physiological functions. The parameter-images provided the structural information about cellular matrix, the barrier spacing of water compartments and the reduced permeability of cell-membranes surrounding the compartment, were created based on the restricted diffusion of cell-associated water. The properties of the water compartment changed with the stages of growth in individual tissues of the fruits. Two types of water compartments were detected; one had approximately 65 microm spacing and constant permeability (20-25%) throughout the stages of growth, and another had the smaller and variable spacing (approximately 35 microm), and a lower permeability. The former belongs to the seed and the mesocarp, the exocarp and the epidermis of the pod. While the latter characterizes the properties of the primary tissue in the pod, the endocarp, which increased with development of the pod then showed reduction with the decrease of water in the pod with the fruit growth.

Information forgotten or overlooked: fundamental flaws in the conventional view of the living cell.

Old ideas often persist long after sound evidence dictates otherwise. I attempt to report one such case in the life sciences, by pointing out what are perceived to be fundamental flaws or questions in conventional wisdom. It is my experience that much evidence not in support of the well accepted membrane pump view of the living cell has been overlooked, forgotten or even ignored. In presenting this idea, the evolution of our knowledge from the establishment of cellular and protoplasmic theory to the emergence of solution theory is presented. The universal hypothesis based on physical chemical principles is presented, followed by the advent of the membrane-situated energy-requiring pump. The experimental demonstration of an inadequate energy supply for the first pump is discussed, followed by a review of new evidence that calls to question the use of dilute solution theory in describing adequately cellular function. Finally, roles for cellular water are suggested to explain the cellular exclusion of sodium and to serve as a barometer for the healthy state. Within the context of a metaphor, I attempt to qualitatively embrace the physical findings. It is concluded that the mobility of water molecules may be considered to change with the progression of normal tissue to a state of disease. These changes in the mobility of water molecules are "fingerprinted" by changes in the molecular motion of the solids.

Effect of electric field on physical states of cell-associated water in germinating morning glory seeds observed by 1H-NMR.

Morning glory seeds in dry conditions (0.099 g H2O/dry wt.) were exposed to electric fields and germinated. The physical state of water in the germinating seeds of both control and exposed groups were examined using 1H-NMR spectroscopy and NMR microscopy. Three water fractions were observed which were characterized by different relaxation times (T1) and chemical shifts. The average region containing long T1 fractions was approximately 50 micrometer in diameter and consisted of half-permeable barriers. The maximum intracellular water transport rate was 2.3x10-5 cm2/s. The treatment with electric field (500 kV/m for 60 min) increased the fraction with the shortest T1 and decreased that with the longest T1. Because the total water content in the treated seeds (3.4 g H2O/dry wt.) was similar to that in the untreated seeds (3.9 g H2O/dry wt.), the treated seeds held more water in a condition in restricted motion than the untreated seeds. It is thought that the membrane systems were affected by the electric polarization which led to an unusual accumulation of water and the hydration of stored macromolecules during the imbibition process. This set of events led to excessive swelling of stored macromolecules, resulting in the disruption of membrane systems and irregular organization of tissue structures.

Response of pain to static magnetic fields in postpolio patients: a double-blind pilot study.

OBJECTIVE: To determine if the chronic pain frequently presented by postpolio patients can be relieved by application of magnetic fields applied directly over an identified pain trigger point. DESIGN: Double-blind randomized clinical trial. SETTING: The postpolio clinic of a large rehabilitation hospital. PATIENTS: Fifty patients with diagnosed postpolio syndrome who reported muscular or arthritic-like pain. INTERVENTION: Application of active or placebo 300 to 500 Gauss magnetic devices to the affected area for 45 minutes. MAIN OUTCOME MEASURE: Score on the McGill Pain Questionnaire. RESULTS: Patients who received the active device experienced an average pain score decrease of 4.4 +/- 3.1 (p < .0001) on a 10-point scale. Those with the placebo devices experienced a decrease of 1.1 +/- 1.6 points (p < .005). The proportion of patients in the active-device group who reported a pain score decrease greater than the average placebo effect was 76%, compared with 19% in the placebo-device group (p < .0001). CONCLUSIONS: The application of a device delivering static magnetic fields of 300 to 500 Gauss over a pain trigger point results in significant and prompt relief of pain in postpolio subjects.

Benzoyl peroxide-induced damage to DNA and its components: direct evidence for the generation of base adducts, sugar radicals, and strand breaks.

Benzoyl peroxide is a known tumor promoter and progression agent in mouse skin, though it is not an initiator or complete carcinogen. Previous studies have suggested that this activity may be due to the generation of strand breaks in cells exposed to this compound. This may be as a result of free radical generation, though there is controversy as to which radicals are responsible for this damage; previous workers have variously implicated benzoyloxyl (PhCO2,) phenyl (Ph.), and hydroxyl radicals (HO.) as the initiating agent. In the present study a detailed examination of the radicals generated on reaction of benzoyl peroxide with Cu(I) has been carried out by electron paramagnetic resonance (EPR) spectroscopy and spin trapping; the results obtained are consistent with the formation of PhCO2. and Ph. but not HO. The subsequent reactions of these benzoyl peroxide-derived radicals with nucleobases, sugars, nucleosides, nucleotides, RNA, and DNA have been examined and the intermediate species have been identified in many cases. Comparison of these data with those obtained with Ph. alone has allowed the reactions of PhCO2. and Ph. to be distinguished. Evidence has been obtained which is consistent with both the addition of these radicals to the C5-C6 double bond of the pyrimidines to give adduct species, and hydrogen abstraction from the sugar rings. The former process is the major reaction for nucleosides and nucleotides. Studies with RNA and DNA also provide strong evidence for the formation of base adducts, though the exact identity of the species detected in these cases could not be determined due to the complexity of the spectra. Hydrogen abstraction at the sugar-phosphate backbone is also believed to occur with these substrates as strand breakage is observed; the extent of the latter is dependent on the radical flux and the attacking species, with PhCO2. appearing to be a much more effective inducer of fragmentation than Ph. The nature of the species detected with all the substrates examined, with the exception of the isolated sugars where essentially random attack by both radicals is observed, suggests that of the two possible radicals generated by benzoyl peroxide, PhCO2. and Ph., it is the former which is responsible for the majority of the observed degradation. The results obtained in this study are consistent with the genetic damage produced by this compound being due to the formation of both strand breaks and high yields of altered bases via the formation of base adducts.

Scanning tunneling microscopy study of poly-L-proline.

Differences in its peptide bonds allow the imino acid poly-L-proline to exist in two significantly different geometric structures. Form I with cis peptide bonds is supposed to be a right-handed helix and form II with trans peptide bonds a left-handed helix. Cis/trans isomerization about the proline imide is believed to cause the denaturation of a number of proteins and may be a key step in protein folding. Using scanning tunneling microscopy (STM), we present high-resolution images of air-dried poly-L-proline. It is found that the electric conductivity of one monolayer of poly-L-proline is sufficient to allow for STM imaging without significant tip-sample interaction. Only at locations where stacking of poly-L-proline chains occurs, a direct contact of the probing tip to the molecules becomes apparent and prevents us, at present, from resolving the atomic structure of the topmost layer. Our STM images of poly-L-proline show that form II is relatively rigid and forms aggregates in most cases. Form I, which is occasionally observed, is very flexible and exhibits sharp bends as well as 180 degrees backfolding. These observations confirm theoretical predictions on the existence of two peptide bond conformations of poly-L-proline.

A hypothesis defining an objective end point for the relief of chronic pain.

A three-part hypothesis for an objective end point for pain is presented: 1) chronic pain results in a characteristic, but reproducible, pattern for the distribution of T lymphocytes in the various phases of their cell cycle; 2) Significant reduction or complete loss of chronic pain will cause a reproducible change in the distribution of T lymphocytes in their cell cycle; 3) The change in T lymphocytes cell cycle distribution will be a function of the degree of recovery from the pain experience. A preliminary test of the hypothesis is presented. The cell cycle distribution of T cell lymphocytes was determined in a group of 10 subjects (experiencing chronic pain) before and after participating in a 10-day educotherapy program given by a master teacher. Associated with a significant reduction of pain was a highly significant shift of the T cell lymphocytes into the S phase of the cell cycle. This observation is consistent with parts one and two of the hypothesis.

Acute ethanol decreases NMR relaxation times of water hydrogen protons in fish brain.

The traditional belief about ethanol's mechanism of action is based on ethanol's lipophilicity and capability to penetrate and disorder lipid bilayers. This traditional belief is now being supplanted by growing evidence that ethanol has relatively selective actions on certain synaptic receptors, such as those for NMDA, serotonin, and GABA. It was recently argued that these receptor specificities are secondary to a preferential ability of ethanol to displace membrane bound water in the domains of certain receptors. The data obtained in this study are consistent with the original hypothesis: any disorganization of cellular water by ethanol will be detectable by proton nuclear magnetic resonance (NMR) spectroscopy. In particular, the relaxation times of water hydrogen protons reflect how constrained water molecules are by the macromolecules within cells. The relaxation time of "bulk" water is lengthened relative to water molecules that are under the influence of electromagnetic fields of macromolecular surfaces within cells. Here, we tested this hypothesis in living fish, which dosed themselves by swimming in water that had added ethanol. Estimates of brain alcohol at 5 min after initial exposure revealed that the brain concentration was only about 1/3 that of the water in which they were swimming. The average value of the NMR relaxation time T1, but not T2, was decreased at 5 min (when brain concentrations were on the order 100 mM) and reached statistical significance at 10 and 30 min after initial exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of potassium, lipids, and proteins from nonionic detergent treated chicken red blood cells.

The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K+ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K+, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values).

Diffusion of water in tissues and MRI.

The purpose of this paper is to present an overview of the diffusive behavior of water in biological tissues. The analysis shows that obstructive barriers would have to occupy very large volume fractions in order to account for the reduction in the diffusion coefficient (D) observed in biological systems. Generally, most models lead to the supposition that a substantial fraction (20-40%) of the cell water is hydration water, or that the diffusion coefficient of the cytoplasmic water is reduced substantially from the free water value. Thus, the conclusion that a substantial fraction of cell water has diffusive properties that are altered by the macromolecules of the cytoplasm seems inescapable. The impact of these findings on MRI remain to be determined.

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100.

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.

Diffusion of water in biological tissues.

A method is presented for obtaining simple approximate solutions for the problem of self-diffusion in an ordered array of obstacles. Our results are compared with some previous exact and approximate solutions, and we find that our method agrees well with the exact results over a large range of the volume fraction of the obstructions. It is shown that there is an important distinction between measurements of the diffusion coefficient by the capillary flow method and the spin-echo method. The modifications for the spin-echo case are given and applied to recent measurements on the anisotropy of the self-diffusion of water in striated muscle and to measurements on cysts of the brine shrimp. The analysis shows that very large volume fractions of obstructive barriers are required in order to account for the reduction in the diffusion coefficient in biological systems. Thus this model analysis leads to the supposition that a substantial fraction (20-40%) of the cell water is hydration water, or that the diffusion coefficient of the cytoplasmic water is reduced substantially from the free water value. In either case, the conclusion that a substantial fraction of cell water has diffusive properties that are altered by the macromolecules of the cytoplasm seems inescapable. In the case of NMR methodology, the measuring times are such that the values for diffusion are often influenced by the presence of macromolecular structures (obstructions) within the cells. This suggests that obstructions make a significant contribution to the value of the NMR diffusion coefficient and that NMR may have practical value for the evaluation of obstruction effects.

The state of potassium in skeletal muscle and in non-muscle cells.

The relationship between ions, water, and the electrical properties are fundamental to our understanding of cellular function. This paper is primarily directed at reviewing the theoretical explanations for the changes in cellular potential and ionic composition which are associated with early postnatal development of skeletal muscle. The findings are: (a) a two-fold reduction in tissue hydration and a significant reduction of the diffusive motion of cellular water; (b) ten-fold decrease in cellular sodium; (c) six-fold decrease in tissue chloride; (d) the concentrations of intracellular potassium, and of extracellular sodium, potassium and chloride were constant; and (e) the cellular potential changed by 55 mV. A review of the literature concerning the physical state of potassium and water is made. The theoretical explanations of these findings are evaluated in terms of the classical membrane theory and the association induction hypothesis.

Characterization of skeletal muscles by MR imaging and relaxation times.

Magnetic resonance (MR) images of three major flight muscles of chicks were obtained with surface coils using a 0.3 Tesla whole body imaging system (FONAR Beta 3000). The two fast muscles, pectoralis major (PM) and posterior latissimus dorsi (PLD), and a slow muscle, anterior latissimus dorsi (ALD), were identified in the axial, coronal, and sagittal images. The signal intensity (SI) of each muscle was electronically measured and its ratio to the background noise (S/N) was determined. Although visually the three muscles showed intermediate SI, the slow and fast muscles could be differentiated on the basis of their S/N values. These values were invariably higher in the slow muscles than in the fast muscles. To understand these differences, the muscles were excised and their mono- and multiexponential MR relaxation times (T1 and T2) were determined at 30 MHz. Multiexponential analysis enhanced the differences between the muscle types. With the sole exception of short T2, all relaxation components of the slow muscles were significantly longer than those of the fast muscles. These results suggest that elevation in the S/N, T1 and T2 values of muscles may not necessarily indicate a pathologic event, but may reflect the preponderance of slow fibers.

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance.

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of water in cysts hydrated with D(2)O have been measured for hydrations between 1.5 and 0.1 g of D(2)O per gram of dry solids. When the relaxation rates (I/T(1), I/T(2)) of (2)H and (17)O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D(2)O (or H(2) (17)O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The (2)H and (17)O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H(2)O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T(1) as a function of hydration in the vicinity of 0.6 g H(2)O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

Release of hemoglobin and potassium from human red blood cells treated with Triton X-100 under the critical micellar concentration.

The action of detergents is thought to be connected primarily with micelle formation. However, detergent monomers can also effect biological systems. It was found in this study that human red blood cells can be disintegrated with Triton X-100 non-ionic detergent at a concentration of 0.007%, lower than the critical micellar concentration (CMC). The time dependent release of hemoglobin and potassium was detected at 37 degrees C and both were sigmoid in character. Although potassium was released faster than hemoglobin, a cooperative relationship between potassium and hemoglobin within the intact red blood cell is suggested by this observation.

Liposomes of enviroxime and phosphatidylcholine: definition of the drug-phospholipid interactions.

Interaction of the antiviral compound, enviroxime (E), with natural and synthetic phosphatidylcholines in organic and aqueous media was studied. Although insoluble in chloroform, E dissolved in chloroform solutions containing phosphatidylcholines. Solvation was directly related to the length of the fatty acid chains of the phospholipid. Proton spin resonance studies suggested an interaction of the fatty acid chains with the aromatic rings of E. Suspension of E-phosphatidylcholine mixtures of molar ratios up to 0.7:1.0 in aqueous media resulted in the formation of multilamellar liposomes. Liposomes containing E were more stable permeability barriers than those prepared with phospholipid alone, a property previously observed with cholesterol. Competition experiments suggested that E bound to the same sites in lipid bilayers as does cholesterol. These data indicate that E is incorporated into lipid bilayers of liposomes and that it alters the physical properties of the liposomes in a manner similar to that of cholesterol.

Developing skeletal muscle: the importance of the physical properties of water.

We are proposing that cellular water becomes organized through its interaction with the various macromolecular (particularly protein) matrices of the cell. And a physical consequence of this interaction results in an orderly exclusion of solutes from the aqueous cytoplasm. This orderly solute exclusion, dependent on physical properties of each solute, along with selective electrostatic adsorption of ions determine the total cellular solute concentration in mature cells. We are proposing further that during early postnatal development the water-macromolecular interaction induces less order and high concentrations of sodium can occur. Finally, our current view of cell water may have been in Albert Szent-Gyorgyi's mind when he stated in 1972 that: "Sixty years of research has taught me to look upon water as part and parcel of the living machinery, if not the hub of life. Water is the most extraordinary substance! Partically all its properties are anomalous, which enabled life to use it as building material for its machinery. Life is water dancing to the tune of solids".