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Background and Purpose: Evaluation and treatment of acute ischemic syndromes, in the heart and brain, require vessel visualization by iodinated X-ray contrast agents. However, these contrast agents can induce injury, in both the kidneys and target organs themselves. Sulfobutylether beta cyclodextrin (SBECD) added to iohexol (SBECD-iohexol) (Captisol Enabled-iohexol, Ligand Pharmaceuticals, Inc, San Diego, CA) is currently in clinical trials in cardiovascular procedures, to determine its relative renal safety in high-risk patients. Preclinical studies showed that SBECD-iohexol reduced contrast-induced acute kidney injury in rodent models by blocking apoptosis. The current study was undertaken to determine whether SBECD-iohexol is also cardioprotective, in the male rat ischemia-reperfusion model, compared to iohexol alone., Methods: After anesthesia, the left coronary artery was ligated for 30 min and the ligation released and reperfusion followed for 2 h prior to sacrifice. Groups 1-4 were injected in the tail vein 10 min prior to ischemia with: (1) vehicle; (2) iohexol; (3) SBECD; and (4) SBECD-iohexol. Infarct size, hemodynamics, and serum markers were measured., Results: An eight-fold increase in serum creatine kinase in the iohexol-alone group was observed, compared with no increase in the SBECD-iohexol group. The mean arterial pressure and rate pressure product were depressed in the iohexol-alone group, but not in the SBECD-iohexol group, or controls. No difference in infarct size or serum creatinine among the groups was observed., Conclusion: The results of this study suggest that SBECD-iohexol is superior to iohexol alone, for both the preservation of cardiomyocyte integrity and preservation of myocardial function in myocardial ischemia., (© 2021 The Authors. Journal of Neuroimaging published by Wiley Periodicals LLC on behalf of American Society of Neuroimaging.)

Here, in Part II of a duology on the characterization and potential treatment for COVID-19, we characterize the application of an innovative treatment regimen for the prevention of the transition from mild to severe COVID-19, as well as detail an intensive immunotherapy intervention hypothesis. We propose as a putative randomized controlled trial that high-dose methotrexate with leucovorin (HDMTX-LR) rescue can abolish 'PANIC', thereby 'left-shifting' severe COVID-19 patients to the group majority of those infected with SARS-CoV-2, who are designated as having mild, even asymptomatic, disease. HDMTX-LR is endowed with broadly pleiotropic properties and is a repurposed, generic, inexpensive, and widely available agent which can be administered early in the course of severe COVID-19 thus rescuing the critical and irreplaceable gas-exchange alveoli. Further, we describe a preventative treatment intervention regimen for those designated as having mild to moderate COVID-19 disease, but who exhibit features which herald the transition to the severe variant of this disease. Both of our proposed hypothesis-driven questions should be urgently subjected to rigorous assessment in the context of randomized controlled trials, in order to confirm or refute the contention that the approaches characterized herein, are in fact capable of exerting mitigating, if not abolishing, effects upon SARS-CoV-2 triggered 'PANIC Attack'. Confirmation of our immunotherapy hypothesis would have far-reaching ramifications for the current pandemic, along with yielding invaluable lessons which could be leveraged to more effectively prepare for the next challenge to global health., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: At sufficiently high doses, methotrexate (HDMTX) achieves substantial CNS penetration, whereas other tissues can be rescued from the effects of HDMTX by leucovorin rescue (LR), which does not penetrate the blood-brain barrier., Objectives: To report on the efficacy and safety of HDMTX with LR (HDMTX-LR), in the treatment of acute demyelinating inflammatory CNS syndromes refractory to conventional immunotherapy., Methods: We performed a retrospective chart review of 12 patients treated (6 multiple sclerosis [MS], 4 neuromyelitis optica [NMO], and 2 Sjogren's syndrome myelopathy [SSM]) with HDMTX-LR after failing to improve, or exhibiting worsening following conventional immunotherapy. 11 patients were followed for a total of 6months following HDMTX-LR (one was lost to follow up after 1month); and clinical findings were documented at 1month, 3months, and 6months following HDMTX-LR therapy., Results: Ten patients demonstrated both clinical and radiologic evidence of near, if not complete, abolishment of disease activity, in conjunction with impressive reconstitution of neurologic function in the 6-month period following HDMTX-LR. Mean Kurtzke Expanded Disability Status Scale (EDSS) prior to HDMTX-LR was 8.1 (±1.4). Following HDMTX-LR, mean EDSS was 6.6 (±2.4) at 1month, 5.8 (±2.3) at 3months, and 5.7 (±2.3) at 6months., Conclusions: In this retrospective assessment of treatment-recalcitrant fulminant inflammatory CNS syndromes, HDMTX-LR was observed to be a safe and highly effective treatment, producing the rapid and near complete cessation of disease activity, in conjunction with an important corresponding and 'durable remission' in the majority of our small treatment cohort., (Copyright © 2016. Published by Elsevier B.V.)

Background and Purpose: Contrast-induced acute kidney injury (CI-AKI) is a serious complication of the use of iodinated contrast agents. This problem is particularly acute in interventional neurology and interventional cardiology, probably due to the intra-arterial route of injection, high contrast volumes, and preexisting risk factors of these patients. In an attempt to develop a contrast agent that is less damaging to the kidneys, we have studied the effects of adding a small amount of the substituted cyclodextrin, sulfobutyl-ether-β-cyclodextrin (SBECD), to iohexol in rodent models of renal toxicity., Methods: Renally compromised mice and rats were injected with iohexol and iohexol-SBECD via the tail vein. The renal pathology, creatinine clearance, and survival benefits of iohexol-SBECD were studied. The safety of direct intra-arterial injection of the iohexol-SBECD formulation was studied in a dog heart model system. Mechanism of action studies in cell culture model using a human kidney cell line was performed using flow cytometry., Results: Nephrotoxicity was significantly reduced using iohexol-SBECD compared to iohexol alone, at mole ratios of iohexol:SBECD of 1:0.025. SBECD increased survival from 50% to 88% in a rat survival study. In the dog heart model, iohexol-SBECD was safe. Cell culture studies suggest that SBECD interferes with the early stages of contrast-induced apoptosis in a human renal cell line., Conclusion: We have shown that the addition of a small amount of SBECD (one molecule of SBECD per 40 iohexol molecules) significantly protects rodent kidneys from CI-AKI. Further development of this new formulation of iodinated contrast is warranted., (© 2016 The Authors. Journal of Neuroimaging published by Wiley Periodicals, Inc. on behalf of American Society of Neuroimaging.)

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal Lalpha, 435 nm for the gel L'beta, and 507 nm for the interdigitated gel LbetaI phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 x 10(4) in the interdigitated gel, 1.8 x 10(4) in the noninterdigitated gel, and 7. 6 x 10(4) in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 x 10(4) for the liquid crystal and the gel phase, and 5.1 x 10(4) in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.

Phosphatidylethanol (Peth) is formed in biological membranes when ethanol replaces water in the transphosphatidylation reaction catalyzed by phospholipase D. This charged lipid accumulates in the presence of ethanol, and it has unusual properties that can influence membrane structure and function. We have previously shown that dimyristoylphosphatidylethanol (DMPeth) and dipalmitoylphosphatidylethanol (DPPeth) form the interdigitated gel phase in the presence of Tris-HCl [O.P. Bondar, E.S. Rowe, Biophys. J., 71 (1996) 1440-1449]. In the present investigation, differential scanning calorimetry (DSC) and fluorescence have been used to investigate the effect of cholesterol on the phase behavior of DPPeth and DMPeth. Our results show that cholesterol prevents the formation of the interdigitated phase in the presence of Tris-HCl, and that ethanol counters this influence and restores the ability of these lipids to interdigitate. Pyrene-PC fluorescence probe was used in this investigation and gave results that were in agreement with the conclusions based on the DSC study., (Copyright 1998 Elsevier Science B.V.)

Recent studies have shown that the traditional paradigm relying on hydrophobic effects is not adequate to describe membrane partitioning of amphiphilic solutes. To elucidate the thermodynamics and determine the role of the hydrophobic effect in the partitioning of small amphiphilic molecules into lipid bilayers, we have used titration calorimetry to directly measure the enthalpy, partition coefficients, and heat capacity change for the partitioning of a series of n-alcohols into lipid bilayers of several lipid compositions. The incremental thermodynamic quantities have been compared with model compound data for partitioning into bulk hydrocarbon solvents. We have found that there is a large negative heat capacity change upon partitioning, indicating a major contribution from the dehydration of nonpolar solute moieties; however, these hydrophobic effects also involve changes in lipid interactions with water in the interfacial region of the bilayer. In addition, we have found that the enthalpy effects are large, indicating that the partitioning process is accompanied by significant changes in the intralipid interactions within the bilayer. Cholesterol has a large effect on partitioning thermodynamics, making both the enthalpy and entropy contributions significantly more positive, resulting in a relatively small net decrease in the negative free energy of partitioning. These results demonstrate that while hydrophobic interactions play a major role in partitioning, the process is considerably more complex than the partitioning of model compounds between water and bulk hydrocarbons, with major contributions coming from changes in the structure and thermodynamic state of the bilayer, including the interfacial region. The results are discussed in terms of both the thermodynamics of partitioning and the role of lipid properties in membrane function. Our results support a paradigm for membrane structure and function in which the thermodynamic state, which is a function of lipid composition, temperature, and dissolved solutes, is a critical membrane property.

Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy-4'-(15-azacrown-5) flavone (FRC) have been investigated as new fluorescence probes for studying the formation of the interdigitated gel phase in lipid bilayers. The formation of the interdigitated gel phase in the saturated symmetrical phosphatidylcholines (PCs) and phosphatidylethanol (Peth) in the presence of ethanol has been well studied. The present study examines the behavior of these new probes in PC-ethanol and Peth-ethanol systems, as well as in PC-cholesterol and Peth-cholesterol vesicles. The present results demonstrate that both flavonols give distinctively different spectra in interdigitated lipid compared to non-interdigitated lipids, when examined in lipids in which the interdigitation behavior is known. This makes them useful for determinations of the structural state of unknown lipids, and for following the transitions between interdigitated and non-interdigitated phases. However, in the presence of cholesterol, only FCR gave appropriate indications of interdigitation. The results with FME in the presence of cholesterol were not consistent with the known behavior of the lipids examined; instead, FME appears to be located preferentially in the cholesterol-rich non-interdigitated regions of the bilayer.

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

Phosphatidylethanol is formed when ethanol substitutes in the transphosphatidylation reaction catalyzed by phospholipase D. The structural and thermotropic properties of dipalmitoylphosphatidylethanol and dimyristoylphosphatidylethanol have been studied using differential scanning calorimetry, fluorescence spectroscopy, and 31P nuclear magnetic resonance. These lipids exist in a bilayer phase with no indication of nonbilayer phase formation, as shown by 31P nuclear magnetic resonance. It was found that the phase behavior of these phospholipids before and during the main chain melting transition is different in 50 mM Tris buffer compared to salt solutions. The phase transition behavior and the 6-propionyl-2-(dimethylamino)naphthalene (Prodan) fluorescence spectra for both lipids are consistent with the formation of the interdigitated gel phase under certain conditions. Both lipids become interdigitated in Tris-HCl, and ethanol enhances the formation of this phase. Comparative studies of the 6-propionyl-2-(dimethylamino)naphthalene spectra in dipalmitoylphosphatidylglycerol, dielaidoylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine further elucidate the value and limitations of this probe as a diagnostic tool for lipid structure.

Differential scanning calorimetry has been used to investigate the effects of vitamin D3 on the physical properties of model membranes including pure phosphatidylcholines (PC's) of chain length from 14 to 18, pure dipalmitoylphosphatidylethanolamine (DPPE), and various mixtures of these lipids. The results demonstrate that the interactions of vitamin D3 with PC's are dependent on acyl chain lengths. It was found that vitamin D3 reduces the transition temperatures of PC's and PE's, broadening the transition and reducing the enthalpy, eventually abolishing the transition. The interaction of vitamin D3 with PC varies with chain length; the transition of DMPC is abolished by only 15 mol% vitamin D3 whereas 45 mol% vitamin D3 is required to abolish the transition of DSPC. These variations in vitamin D3 lipid interactions are further explored in various mixtures. In the mixture studies it is shown that vitamin D3 affects the mixing properties of the lipids in the mixtures. The results suggest that the presence of vitamin D3 in lipids can affect the lateral phase distributions of lipids, and thereby have important effects on membrane function.

Although it is now well established that the fully interdigitated phase is induced in saturated like-chain phosphatidylcholines (PCs) by a variety of amphipathic molecules including alcohols, no systematic study of the properties of the inducing molecules has been reported. To elucidate the stereochemical features that lead to the alcohol induction of interdigitation in PCs, we have investigated the induction of interdigitation in distearoylphosphatidylcholine (DSPC) by a series of alcohols. Our previously established DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence intensity method has been expanded (P. Nambi, E. S. Rowe, and T. M. McIntosh (1988), Biochemistry 27:9175-9182) and used to determine which of the alcohols induce interdigitation and to determine the threshold concentrations for each. We have found that each of the n-alcohols up to heptanol and several branched alcohols are capable of inducing interdigitation in DSPC; octanol and nonanol do not appear to induce interdigitation by these criteria. The threshold concentrations for interdigitation for each of these alcohols up to heptanol were found to be correlated with the membrane: buffer partition coefficients. The mole fraction of bound alcohol at the threshold concentration was similar for each of the alcohols up to pentanol. These results are discussed in terms of a general mechanism of the formation of the interdigitated phase.

Differential scanning calorimetry (DSC) and DPH fluorescence anisotropy have been used to investigate the effects of trans-trans farnesol on the physical properties of model membranes and extracted cell lipids. Farnesol was shown to have a significant effect on the gel to liquid-crystal phase transition temperature, the enthalpy of the transition and the transition co-operativity for extruded vesicles of dimyristoylphosphatidylcholine (DMPC). The phase transition of DMPC vesicles was eliminated at 25 mol% farnesol. Farnesol decreased the fluorescence anisotropy of the lipids extracted from human leukemia line CEM-C1 cells.

Cytochrome c has been studied as an example of a peripheral membrane protein which interacts with the lipids as well as the proteins of the inner mitochondrial membrane. In order to elucidate the thermodynamic properties of these interactions, isothermal titration calorimetry and differential scanning calorimetry (DSC) were used to study the binding of cytochrome c to negatively charged dioleoylphosphatidylglycerol (DOPG) extruded vesicles as a function of ionic strength. The binding constant and enthalpy of association decrease with increasing ionic strength, with no binding detected above 0.5 M NaCl. The enthalpy of the binding of cytochrome c to DOPG-extruded vesicles was 15 kcal/mol, and the binding constant was 6 x 10(6) M-1 at the lowest ionic strengths. The minimum size of the lipid cluster to which the protein bound was found to be approx. 9 lipid molecules in the titration calorimetry measurements and as low as 5 lipid molecules in the DSC measurements. The stability of the bound cytochrome c was found to be reduced; the thermal denaturation temperature was lowered from 83 to 50 degrees when bound to DOPG. The results of this study support previous suggestions that cytochrome c may undergo a conformational change when it binds to charged lipids such as DOPG. The results also support the suggestion that the protein penetrates partially into the lipid bilayer.

Unilamellar liposomes are widely used as model membranes to represent and study the properties of biological membranes and as potential drug delivery systems. It is well established that ethanol and other amphiphiles induce the interdigitated L beta I phase in multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). However, all of the work on this phase has been performed using the hand shaken multilamellar preparations. In the present report, we have studied the induction of interdigitation in a series of unilamellar vesicles prepared by sonication and by extrusion. The methods used to characterize the vesicles were freeze fracture electron microscopy and quasielastic light scattering (QELS). Two fluorescence methods were used to detect interdigitation, the DPH fluorescence quenching method (Nambi, P., Rowe, E.S. and McIntosh, T.J. (1988) Biochemistry 27, 9175-9182) and the pyrene-PC fluorescence method (Komatsu, H. and Rowe, E.S. (1991) Biochemistry 30: 2463-2470). It was found that sonicated vesicles are not stable in the presence of interdigitating concentrations of ethanol; they form higher aggregates at all temperatures examined. The behavior of the extruded vesicles was different from that of the SUV; each size studied was stable in the presence of ethanol, although they exhibited an increase in size. It was shown that extruded vesicles having a 200-nm or greater diameter become interdigitated in the presence of ethanol. The threshold concentration for interdigitation in vesicles is greater than that for MLVs and it decreases with increasing vesicle size, approaching the MLV value for the largest vesicles.

The visible spectrum of Co(II)-substituted human carbonic anhydrase I (HCA I) complexed with the unique CO2-competitive inhibitor imidazole undergoes a marked alkaline intensification, with a midpoint near pH 8 [Bauer, R., Limkilde, P., & Johansen, J. T. (1977) Carlsberg Res. Commun. 42, 325-339]. This change was first attributed to the ionization of a nondisplaced water ligand of the active-site metal in a five-coordinate complex. Later proposals favored assigning it to the deprotonation of the bound imidazole itself to give a tetrahedrally coordinated imidazolate anion at high pH. We have determined by isothermal titration calorimetry the pH dependence of the enthalpy of binding of imidazole and its analogues to HCA I and Co(II)HCA I. We devised an indirect strategy whereby the enthalpy of binding of the strong sulfonamide inhibitor methazolamide was determined in the absence and presence of a constant high concentration of the competing imidazole or its analogues. The standard enthalpy of binding of deprotonated methazolamide to the "acid" form of HCA I and Co(II)HCA I was found to be pH independent over the pH range of 6.5-9.5, as expected. It was also identical for both the zinc (-13.5 +/- 1.1 kcal M-1) and the cobalt (-13.7 +/- 0.4 kcal M-1) forms. The standard enthalpy of binding of neutral imidazole (average value -6.1 +/- 0.8 kcal M-1) surprisingly did not show any marked pH dependence, varying by about 1.1 and 2.6 kcal M-1 for the zinc and cobalt enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The interactions of n-butanol with dipalmitoylphosphatidylcholine (DPPC) were studied using titration calorimetry and differential scanning calorimetry (DSC). DSC results indicated that n-butanol induces the interdigitated phase in DPPC above 10 mg/mL butanol. A new application of titration calorimetry for measuring partition coefficients of nonsaturating solutes into lipids was developed. The partition coefficients and the heat of binding of n-butanol into DPPC were measured for the L beta', P beta', L alpha, and L beta I phases of DPPC. The partition coefficients were temperature dependent and ranged from 70 to 110 for the L beta I phase, from 170 to 183 for the L alpha phase, and similar to that for the L beta I phase in the P beta' phase. The binding to the L beta' phase could not be detected, giving an upper limit for this partition coefficient of 23. The enthalpies for binding to the L beta I and L alpha phases were 1.0 and 1.5 kcal/mol, respectively. The van't Hoff enthalpy was in good agreement with the calorimetric enthalpy for the partitioning into the L alpha phase; however, it was greater than the calorimetric enthalpy for the L beta I phase, suggesting that the interaction of n-butanol with this phase is cooperative in some way.

It is now recognized that many amphiphilic molecules such as ethanol can induce the formation of the fully interdigitated gel phase (L beta I) in phosphatidylcholines (PC's). In the present study, we have developed a simple detection method for the L beta I phase using pyrene-labeled PC (PyrPC), which is a PC analogue with covalently coupled pyrene moiety at the end of one of its acyl chains. The intensity ratio of its fluorescence vibrational bands is a reflection of the polarity of the environment of the fluorophore. We have tested this fluorophore in several established interdigitated lipid systems, including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC) in the presence of high concentrations of ethanol and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC) in the absence of any additives. We have found in each of these systems that the ratio of the intensities of band III (387.5 nm) to band I (376.5 nm) is sensitive to the lipid phase change from the noninterdigitated L beta' phase to the interdigitated L beta I phase. By comparison of the III/I ratios for PyrPC in the lipid systems with the III/I ratios for methylpyrene in organic solvents, it was shown that the polarity of the PyrPC environment in the L beta I phase is similar to that of pentanol or ethanol. Using this method, we investigated the effect of cholesterol on the ethanol induction of the interdigitated gel phase in 1,2-DPPC. We found that the ethanol induction of the interdigitated gel phase is prevented by the presence of 20 mol % cholesterol.

It is well established that ethanol and other amphipathic molecules induce the formation of a fully interdigitated gel phase in saturated like-chain phosphatidylcholines (PC's). We have previously shown that the induction of interdigitation in PC's by ethanol is dependent upon the alcohol concentration, the lipid chain length, and the temperature [Nambi, P., Rowe, E. S. & McIntosh, T. J. (1988) Biochemistry 27, 9175-9182]. In the present study, we have used high-sensitivity differential scanning calorimetry to investigate the transitions of distearoylphosphatidylcholine between the noninterdigitated and the interdigitated phases. The enthalpy of the L beta' to L beta I transition is approximately half that of the L beta' to P beta' transition which occurs in the absence of ethanol. The reversibility of these transitions has also been investigated by employing both heating and cooling scans in order to establish the most stable phases as a function of temperature and ethanol concentration. It has been demonstrated that the transition to the interdigitated phase is reversible as a function of temperature. Kinetic studies on the reverse transition (L beta I to L beta') demonstrate that this transition can be very slow, requiring weeks to reach completion. The rate depends upon temperature and ethanol concentration. The slow phase changes mean that the lipid can exist for long periods of time in a phase structure which is not the most stable state. The biological significance of this type of lipid behavior is the implication that the phase structure of biological membranes may depend not only on the most stable phase structure of the lipids present but also on the synthetic pathway or other kinetic variables.

We have utilized phosphorus nuclear magnetic resonance, which provides an excellent means of characterizing the physical state of lipids, to investigate the polymorphic phase behavior of pure dielaidoylphosphatidylethanolamine (DEPE). We have observed a sharp isotropic component in the typical bilayer and inverted hexagonal P-31 NMR spectra. This component appears in the spectra of both the bilayer and inverted hexagonal lipid phases after several cycles through the bilayer-to-hexagonal phase transition. The magnitude of the isotropic component increased as a function of the number of cycles through the transition. The appearance of this component was not a function of time at constant temperature, but only a function of the number of cycles through the transition. The isotropic component is stable at all temperatures above the gel-to-liquid crystal transition, but it abruptly disappears when the lipid is cooled below the gel-to-liquid crystal phase transition. It is suggested that this isotropic phase is similar to the isotropic phase observed in dioleoylphosphatidylethanolamine (DOPE) by x-ray diffraction and identified as a cubic phase (Shyamsunder, E., S. M. Gruner, M. W. Tate, D. C. Turner, P. T. C. So, and C. P. S. Tilcock. 1988. Biochemistry. 27:2332-2336).

It is now well established that a number of amphiphilic molecules such as ethanol can induce the formation of the fully interdigitated gel phase in phosphatidylcholines. We have shown earlier that alcohols such as ethanol induce biphasic melting behavior in phosphatidylcholines [Rowe, E. S. (1983) Biochemistry 22, 3299-3305] but not in phosphatidylethanolamines [Rowe, E. S. (1985) Biochim. Biophys. Acta 813, 321-330]. Simon and McIntosh [(1984) Biochim. Biophys. Acta 773, 169-172] showed that the alcohol-induced biphasic melting behavior in phosphatidylcholines is a consequence of acyl chain interdigitation. In the present study we demonstrate the detection of the transition of DPPC and DSPC to the interdigitated phase in the presence of ethanol using the fluorescence properties of the commonly used fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). By correlating fluorescence and X-ray diffraction results, we have demonstrated the use of fluorescence to study the phase transition from the noninterdigitated to the interdigitated phase. Using this method, we have investigated the temperature and ethanol concentration dependence of the induction of the interdigitated phase in DSPC and DPPC and shown that the induction of interdigitation by ethanol is temperature dependent, with higher temperature favoring interdigitation. The temperature-ethanol phase diagrams have been determined for DPPC and DSPC.

The conformational equilibria and the kinetics of the approach to equilibrium of an IgG1 myeloma (Wes) Fab fragment (SSFab) and its mildly reduced and S-carboxyamidomethylated derivative (RAFab) were studied as a function of guanidine hydrochloride concentration. The unimolecular denaturation of SSFab, the bimolecular denaturation of RAFab, and the denaturation of Wes L chain reported previously (Rowe, E. S., and Tanford, C., (1973), Biochemistry 12, 4822) are interpreted in terms of the domain structure and evaluated in terms of the thermodynamic stability of the protein and the covalent and noncovalent interactions among its subunits. The Fd-L interactions are found to be extremely strong and are maintained at concentrations of denaturant sufficient to denature the individual domains. It is shown that all of the data are consistent with a two region structure for Fab, one composed of the vL and vH domains, and the other composed of the cL and cH domains, so that there are two sites of noncovalent Fd-L interactions. One region, identified as the C region, is found to be 10(2)-10(4) times more stable than the other; this difference in stability is attributed largely to a stronger and more extensive interaction between the domains of this region. The kinetics of the approach to equilibrium are found to be extremely slow in the center of the transitions, requiring up to a week for equilibration for RAFab, and several months for SSFab. This unusual kinetic behavior is shown to be due to the strong Fd-L interaction under conditions where the monomeric domains are unstable.

The specific effect of ethanol on several aspects of the gel-to-liquid crystal transition of dipalmitoylphosphatidylcholine was investigated using two spectrophotometric techniques, one probe method and one direct method. Ethanol shifts the phase-transition temperature to low temperature, demonstrating that ethanol interacts preferentially with the fluid phase. Thermodynamic analysis of the melting point depression leads to a calculated membrane:buffer partition coefficient of 6.25 (mole fraction units) or 0.15 mole of ethanol per kilogram of lipid:mole of ethanol per liter of solution. Careful evaluation of the transition cooperativity with temperature resolution of +/- 0.1 degrees shows that there is no reduction in transition cooperativity, and thus no reduction in size of the cooperative lipid clusters due to ethanol. The implications of these findings for the mechanism of action of ethanol in terms of current theories of anesthetic mechanisms are discussed.

We have systematically investigated the effect of short chain alcohols (methanol to n-propanol) on the phase transitions of 1,2-dihexadecylphosphatidylcholine (DHPC), a lipid that forms a stable interdigitated gel phase (L beta I) in aqueous solution. The temperature of the low-temperature L beta I to P beta' phase transition of DHPC was found to increase with alcohol concentration, showing that alcohol interacts preferentially with the interdigitated phase relative to the non-interdigitated gel. The main transition of DHPC exhibited a biphasic effect of alcohol concentration similar to that previously observed with DPPC (Rowe, E.S. (1983) Biochemistry 22,3299-3305). As alcohol concentration is increased the lower L beta I to P beta' and main P beta' to L alpha transitions of DHPC merge at the threshold concentration of the biphasic effect, so that above this concentration there is one phase transition from L beta I directly to L alpha. This is analogous to DPPC above its biphasic threshold. Similar to DPPC, the transition between L beta I and L alpha exhibits marked hysteresis.

The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.

We have been systematically investigating the physical interactions of alcohols with synthetic lipids in order to elucidate the mechanisms of alcohol intoxication. It has previously been shown that the effects of ethanol on the phase transition temperatures of saturated chain phosphatidylcholines (PC's) are biphasic [11]. The secondary high concentration effect has recently been shown to be due to a transition to a new completely interdigitated state [16]. In the present report we have investigated the influence of the chain length of the alcohol on the induction of this interdigitated state. We demonstrate that all three alcohols have a biphasic effect on the transition temperature of PC, and the comparison of the concentration dependencies of this effect for the three alcohols provides some insight into the possible mechanism of the transition. The potential biological importance of this secondary alcohol-membrane interaction is discussed.

Two independent electrooptical properties, the specific Kerr constants and the electric birefringence relaxation times, of the saturated sodium dodecyl sulfate complexes of a series of reduced polypeptides of known molecular weight are reported. Both the Kerr constants and the relaxation times are unique functions of the molecular weight of the polypeptide chain. The specific Kerr constants depend upon the square of the polypeptide molecular weight. The relaxation times of the complexes, which are proportional to the rotational diffusion constants, are dependent on the molecular weight to approximately the first power. The latter finding is inconsistent with the compact prolate ellipsoid model for sodium dodecyl sulfate-protein complexes proposed by Reynolds and Tanford ((1970) J. Biol. Chem. 245, 5161) in which the cross section is constant and the length depends on linearly on molecular weight; for this model the relaxation times would depend on approximately the 2.5 power of the molecular weight in the range of sizes investigated. Combination of the present results with other properties in the literature rules out a number of other models characterized by compactness and near inflexibility. No firm conclusions can be drawn as to the suitability of the free-draining flexible model of Shirahama, Tsujii, and Takagi ((1974) J. Biochem. (Tokyo) 75, 309).

It has previously been shown that alcohol has different effects on the gel to liquid-crystal phase transition of phosphatidylcholines (PC's) and phosphatidylethanolamines (PE's) [Rowe, E. S. (1985) Biochim. Biophys. Acta 813, 321-330]. In this investigation, the thermotropic properties of binary PE-PC mixtures were studied in the presence of ethanol in order to determine whether the differential interactions of alcohol with PC and PE would lead to lateral phase separations. Phase diagrams of the dilaurylphosphatidylethanolamine-dipalmitoylphosphatidylcholine [PE(12:0)-PC(16:0)] system were constructed in the presence and absence of ethanol. It was shown that lateral phase separations occur in the gel phase over a certain composition range in the presence of 100 mg/mL ethanol. In the absence of alcohol these two lipids are miscible in both the gel and liquid-crystal states. The data suggest that in the presence of ethanol these lateral phase separations involve the coexistence of regular bilayer gel and the fully interdigitated gel phase, which has previously been shown to occur in pure PC(16:0) under these conditions [Simon, S. A., & McIntosh, T. J. (1984) Biochim. Biophys. Acta 773, 169-172]. The biological implications of these findings are discussed.

The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.

Phase transitions of dimyristoylphosphatidylcholine (DMPC) are followed using optical density measurements, as a function of aqueous ethanol concentration. The shift of the phase transition to lower temperatures is interpreted in terms of the thermodynamics of freezing point depressions. This analysis provides a partition coefficient of ethanol into DMPC of 0.07 liter of solution/kg membrane or 2.64 moles of solvent/mole of DMPC.

The gel-to-fluid phase transitions of several phosphatidylethanolamines (PE's) and phosphatidylcholines (PC's) have been investigated in the presence of three short-chain alcohols. The effects of the alcohols on the thermodynamic reversibility of these transitions was studied and it was found that the transitions for PC's are not thermodynamically reversible at relatively high alcohol concentrations. The PE transitions are thermodynamically reversible for all alcohol concentrations, and the PE's do not exhibit the biphasic effects of alcohol on the transition temperature previously reported for the PC's (Rowe, E.S. (1983) Biochemistry 22, 3299-3305). The biphasic transition temperature effects and the thermodynamic irreversibility of PC transitions at high alcohol concentrations appear to be correlated with the induction of a fully interdigitated gel phase recently reported in the literature (Simon, S.A. and McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172). The biological significance of these findings is discussed.

We have systematically investigated the effect of short-chain n-alcohols and glycerol on the pretransition of 1,2-dipalmitoylphosphatidylcholine (DPPC) by spectrophotometry. It is found that the n-alcohols and glycerol remove the pretransition above a critical concentration for each ligand. In addition, the short-chain n-alcohols below the critical concentration decrease the pretransition temperature. The longer the aliphatic chain length of the n-alcohol (up to butanol) the greater the decrease in the pretransition temperature, and the lower the concentration necessary to remove the pretransition. However, glycerol differs from the short-chain n-alcohols in that it has no significant effect on either the pretransition or the main transition, but it is also capable of removing the pretransition above a critical concentration. It has previously been shown that alcohols have a biphasic effect on the main transition temperature of phosphatidylcholines (Rowe, E.S. (1983) Biochemistry 22, 3299-3305). At high alcohol concentrations, the main transition is not thermodynamically reversible (Rowe, E.S. (1985) Biochim. Biophys. Acta 813, 321-330). Recently, Simon and McIntosh (Biochim. Biophys. Acta (1984) 773, 169-172) have identified that at high ethanol concentration DPPC exists in the interdigitated phase. The critical ligand concentration at which the pretransition disappears coincides with the induction of main transition hysteresis and the biphasic alcohol effect in the main transition. These three effects appear to correlate with the induction of the interdigitated gel state by alcohols and glycerol.