Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens.

AIMS: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. METHODS: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. RESULTS: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. CONCLUSION: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.

The effects of temperature and pressure on the thermodynamic activity of anesthetics.

Anesthetic potency is often expressed by volume percent or partial pressure in the gas phase, concentrations in the aqueous phase, etc. However. these values do not represent the anesthetic activity at the action sites. Because the activity at the action sites is difficult to obtain. Ferguson (Ferguson, J., 1939. Proc. R. Soc. Lond. B 127 387-404) defined the thermodynamic activity, which is the ratio between the anesthetizing partial pressure and the vapor pressure of the pure anesthetic at the same temperature. This paper discusses the effects of temperature and pressure on the thermodynamic activity of anesthetics. It also discusses the limitations of the Meyer-Overton rule.

[Anesthesia for hepatectomy in a patient with glycogen storage disease].

We successfully anesthetized a 19-year-old female with type I glycogen storage disease for hepatectomy. She had hypoglycemia and severe metabolic acidosis before surgery. General anesthesia was performed with epidural anesthesia. We used acetated Ringer solution mainly as intraoperative fluids instead of lactated Ringer solution and controlled administration of glucose determining blood glucose. The patient's plasma lactate levels, pyruvate and base excess during and after operation, were unchanged compared with those before operation. Administration of sodium bicarbonate was not necessary. In the case of metabolic acidosis due to accumulation of lactate as in glycogen storage diseases, it was useful to use acetated Ringer solution for fluid therapy.

Alcohol interaction with high entropy states of macromolecules: critical temperature hypothesis for anesthesia cutoff.

Nerve excitation generates heat and decreases the entropy (review by Ritchie and Keynes (1985) Q. Rev. Biophys. 18, 451-476). The data suggest the existence of at least two thermodynamically identifiable states: resting and excited, with a thermotropic transition between the two. We envision that nerve excitation is a transition between the two states of the excitation machinery consisting of proteins and lipids, rather than the sodium channel protein alone. Presumably, both proteins and lipids change their conformation at excitation. We proposed (Kaminoh et al. (1991) Ann. N.Y. Acad. Sci. 625, 315-317) that anesthesia occurs when compounds have a higher affinity to the resting state than to the excited state of excitable membranes, and that there is a critical temperature above which the affinity to the excited state becomes greater than to the resting state. When the temperature exceeds this critical level, compounds lose their anesthetic potency. We used thermotropic phase-transition of macromolecules as a model for the excitation process. Anesthetic alcohols decreased the main transition temperature of dipalmitoylphosphatidylcholine (DPPC) membranes and also the temperature of the alpha-helix to beta-sheet transition of poly(L-lysine). The affinity of alcohols to the high- and low-temperature states of the DPPC membranes were separately estimated. The difference in the affinity of n-alcohols to the liquid (high-temperature) and solid (low-temperature) states correlated with their anesthetic potency. It is not the total number of bound anesthetic molecules that determines the anesthesia, rather, the difference in the affinity between the higher and lower entropy states determines the effects. The critical temperatures of the long-chain alcohols were found to be lower than those of the short-chain alcohols. Cutoff occurs when the critical temperature of long-chain alcohols is below the physiological temperature, such that the anesthetic potency is not manifested in the experimental temperature range.

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.

High pressure antagonism of alcohol effects on the main phase-transition temperature of phospholipid membranes: biphasic response.

The combined effects of high pressure (up to 300 bar) and a homologous series of 1-alkanols (ethanol C2 to 1-tridecanol C13) were studied on the main phase-transition temperature of dipalmitoylphosphatidylcholine (DPPC) vesicle membranes. It is known that short-chain alkanols depress and long-chain alkanols elevate the main transition temperature. The crossover from depression to elevation occurs at the carbon-chain length about C10-C12 in DPPC vesicle membranes coinciding with the cutoff chain-length where anesthetic potency suddenly disappears. Alkanols shorter than C8 linearly decreased the transition temperature and high pressure antagonized the temperature depression. Alkanols longer than C10 showed biphasic dose-response curves. High pressure enhanced the biphasic response. In addition, alkanols longer than the cutoff length depressed the transition temperature under high pressure at the low concentration range. These non-anesthetic alkanols may manifest anesthetic potency under high pressure. At higher concentrations, the temperature elevatory effect was accentuated by pressure. This biphasic effect of long-chain alkanols is not related to the 'interdigitation' associated with short-chain alkanols. The increment of the transition temperature by pressure was 0.0242 K bar-1 in the absence of alkanols. The volume change of the transition was estimated to be 27.7 cm3 mol-1. This value stayed constant to the limit of the present study of 300 bar.

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition.

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.

Use of ion-exchange membranes to measure transfer free energies of charged local anesthetics: correlation to anesthetic potency.

Ion-selective electrodes, sensitive to local anesthetic cations, were prepared with carboxylated poly(vinyl chloride) (PVC) membranes. Three plasticizers with varying degrees of polarity were used to adjust the hydrophobicity of the membrane. The affinity of the drug to the ion-exchange membrane was measured by the electromotive force of the cation-selective electrodes. The difference in the transfer free energies of the anesthetics for the membrane was estimated in reference to dibucaine. The values correlated to their clinical potencies. By comparing drugs with similar structures, the transfer free energy per methylene moiety linked to the hydrophilic domain was found to be -1.7 kJ.mol-1, and that of Cl linked to the hydrophobic domain was -3.1 kJ.mol-1. Interferences from Na+ and K+ were estimated as the selectivity coefficients against dibucaine. The values were 2.6 x 10(-5) for Na+ and 1.2 x 10(-4) for K+. The ion-exchange membrane appears to mimic the surface properties of cell membranes. These cation-selective electrodes have potential applicability in measuring charged local anesthetic concentrations (activities) in biological materials under limited conditions.

Differential affinity of charged local anesthetics to solid-gel and liquid-crystalline states of dimyristoylphosphatidic acid vesicle membranes.

Cationic local anesthetics decreased the transition temperature of the anionic phospholipid (dimyristoylphosphatidic acid, DMPA) vesicles. The counterion concentration changes the electrical double layer effect, and affects the magnitude of temperature depression caused by anesthetics. From the counterion effect on the transition-temperature depression, the partition coefficients of cationic local anesthetics to liquid-crystalline and solid-gel DMPA membranes were separately estimated. The differences in the partition coefficients between solid-gel and liquid-crystalline membranes correlated to the nerve blocking potencies. There are at least two states in the nerve membranes: resting state at higher temperature and excited state at lower temperature. We speculate that the resting state corresponds to the liquid-crystalline state, and the excited state to the solid-gel state. The difference in the partition coefficients to the resting and excited states is the cause of local anesthesia.

Depression of phase-transition temperature by anesthetics: nonzero solid membrane binding.

The anesthetic-induced depression of the main phase-transition temperature of phospholipid membranes is often analyzed according to the van't Hoff model on the freezing point depression. In this procedure, zero interaction between anesthetics and solid-gel membranes is assumed. Nevertheless, anesthetics bind to solid-gel membranes to a significant degree. It is necessary to analyze the difference in the anesthetic binding between the liquid-crystal and solid-gel membranes to probe the anesthetic action on the lipid membranes. This article describes a theory to estimate the anesthetic binding to each state at the phase-transition temperature. The equations derived here reveal the relation between the partition coefficients of anesthetics and the anesthetic effects on the transition characters: the change in the transition temperature, and the broadening of transition. The theory revealed that the width of transition temperature is determined primarily by the membrane/buffer partition coefficients of anesthetics. Our previous data on the local anesthetic action on the transition temperature of the dipalmitoylphosphatidylcholine vesicle membrane (Ueda, I., Tashiro, C. and Arakawa, K. (1977) Anesthesiology 46, 327-332) are analyzed by this method. The numerical values for the partition of local anesthetics into the liquid-crystal and solid-gel dipalmitoyl-phosphatidylcholine vesicle membranes at the phase-transition temperature are: procaine 8.0 x 10(3) and 4.7 x 10(3), lidocaine, 3.7 x 10(3) and 2.3 x 10(3), bupivacaine 4.1 x 10(4), and 2.6 x 10(4), and tetracaine 7.3 x 10(4) and 4.7 x 10(4), respectively.

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry.

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.

Effect of surface ionization of dimyristoylphosphatidic acid vesicle membranes on the main phase-transition enthalpy and temperature.

The main phase transition of phospholipid bilayers is a property expressed by the order-disorder conformational change of the lipid tails. Nevertheless, with ionizable phospholipids, changes in the surface charge have large effects on the membrane properties. The free energy of a charged phospholipid membrane depends on the degree of ionization, area per phospholipid molecule, and the temperature. Here, the effect of surface electrostatic charges on the temperature and the enthalpy of the main phase transition of dimyristoylphosphatidic acid vesicle membranes is analyzed. A simple equation is presented that describes the relationship among the surface charge density, the phase-transition temperature, the surface area ratio between solid and liquid membranes, and the excess enthalpy. The theory indicated that the pH-induced shift in the excess enthalpy is attributable to the change in the surface area ratio between the solid and liquid membranes.

Anesthetics release unfreezable and bound water in partially hydrated phospholipid lamellar systems and elevate phase transition temperature.

A dimyristoylphosphatidylcholine multilamellar system with varied water content was prepared by dessiccating sonicated vesicles in vacuo. The water content in the sample was determined by gas chromatography after dissolving the multilamellar system in water-free benzene. Differential scanning microcalorimetry revealed several endothermic peaks in the heating scan at subzero temperature, ranging from -25 to -3 degrees. The peaks that appeared in the subzero temperature range indicate the existence of water molecules bound to the lipid head groups, differing from free water that freezes at 0 degrees. The difference between the amount of water molecules that froze in calorimetry and the total amount of water detected by gas chromatography indicates the presence of unfreezable, tightly bound water molecules. The relative amount of free, intermediate, and unfreezable water was estimated by comparing the differential scanning microcalorimetry data with gas chromatography measurements. The addition of halothane and 1-hexanol significantly decreased the intermediately bound water peaks. The anesthetics dehydrated the lamellar system. The phase polymorphism of partially hydrated phospholipid multilayers is well known, and the temperature that corresponds to the main phase transition of fully hydrated lipid membranes shifts to a higher temperature. The addition of anesthetics increased the phase transition temperature when the water content was less than 18 wt%. This result is the complete reverse of the depressant action of anesthetics in fully hydrated lipid membranes. The present anesthetic effect upon the elevation of the transition temperature is apparently caused by anesthetic-induced dehydration of the lipid-water interface at the present experimental condition.

Effects of reserpine and adenosine agonist on ?(2)-adrenergic receptor of rat vas deferens examined with [(3)H]yohimbine and [(3)H]clonidine.

The changes of [(3)H]yohimbine and [(3)H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [(3)H]clonidine binding sites but had no influence on affinity and number of binding sites of ?(2)-antagonist, [(3)H]yohimbine. Amount of [(3)H]yohimbine binding sites was found to be higher than that of [(3)H]clonidine with or without the treatment. Inhibition curves of ?(2)-agonists, clonidine and norepinephrine, on [(3)H]yohimbine binding were less than unity though ?(2)-antagonist inhibited with about 1.0 of n(H). The treatment with adenosine agonist reduced the IC(50) value of agonists on the [(3)H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in ?(2)-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist. On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for ?(2)-adrenergic receptors and also increased the amount of the ?(2)-receptors.