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PubMed1994

Preservation of surface lipids and determination of ultrastructure of Mycobacterium kansasii by freeze-substitution.

The cell wall architecture of a slowly growing mycobacterium, Mycobacterium kansasii, was examined by freeze-substitution following growth in vitro. Freeze-substituted bacteria were marked by the presence of an electron-translucent space (or electron-transparent zone [ETZ] described by previous workers [T. Yamamoto, M. Nishiura, N. Harada, and T. Imaeda, Int. J. Lepr. 26:111-114, 1958]) surrounding the majority of cells. At least two morphotypes of mycobacteria were revealed by freeze-substitution. In the first, a relatively thin (11 +/- 2.3 to 3.5 +/- 3.1 nm), uniform ETZ surrounded intact cells which contained cytoplasm filled with well-stained ribosomes and a DNA nucleoid distributed throughout the cell. The second morphotype consisted of a small proportion of organisms that were distorted in shape and were surrounded by a much thicker (59 +/- 2.6 to 198 +/- 2.5 nm) ETZ in areas of the cell which appeared to have retracted from the space it had originally occupied, leaving depressions in the ETZ. The lipid nature of the ETZ was demonstrated because cells were devoid of an ETZ when organisms were freeze-substituted in the absence of osmium tetroxide in the substitution medium or treated with neutral lipid solvents (acetone or ethanol) before freeze-substitution. Moreover, thin-layer chromatography of acetone or ethanol extracts obtained from solvent-treated cells identified a lipid component which corresponded to the M. kansasii-specific phenolic glycolipid. In contrast, negligible amounts of glycolipids were detected in extracts obtained from control HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer-treated cells, and these cells retained an ETZ. These results demonstrate that species-specific phenolic glycolipids are essential components in the architecture of the M. kansasii ETZ. Furthermore, we show that freeze-substitution is a reliable technique for the retention and precise preservation of lipid-containing polymers in the mycobacterial cell wall.

Freeze Substitution↗
PubMed1992

pH-dependence of the structure and degradation of 8-alkyl-substituted pterins.

Structures of 8-(2-hydroxyethyl)pterins (8-11) investigated using 1H- and 13C-NMR and UV/vis spectroscopies showed a complex dependence on pH, solvent and 6,7-substitution pattern. In acid, only one cation was observed for all the pterins. In neutral aqueous solution, only one neutral form, the normal quinonoid tautomer, was observed for 7-unsubstituted pterins 9 & 11, but two neutral tautomeric forms, quinonoid and 7-exo-methylene, were observed for 7-CH3 substituted pterins 8 & 10 with 70% and 92%, respectively, of quinonoid. The neutral pterins in MeOH, however, showed a different distribution of structural forms: quinonoid and a five-membered intramolecular ether forms were observed for 7-unsubstituted pterins 9 & 11 as 60% and 25%, respectively, of quinonoid, while quinonoid and 7-exomethylene forms were observed for 7-CH3 substituted pterins 8 & 10 as 10% and 50%, respectively, of quinonoid. In base, for 7-unsubstituted pterins 9 & 11 only the intramolecular ether forms were observed, while for the 7-CH3 substituted pterins 8 & 10 two anion forms, the 7-exo-methylene and intramolecular ether, were observed in the ratio 2:1. Investigation of the distinctive proton-resonance pattern of the ethanomoiety of the intramolecular ether anion of 9 using 600 MHz NMR and spectrum simulation, indicated all four protons have different chemical environments. One conformation of the cyclic-ether ring satisfying the experimental data has been deduced, and the conformational energetics of the ring studied further using AM1 semiempirical quantum chemical calculations. Structural distributions of 8-methylpterins 12-15 were also studied in base only. These showed the 7-unsubstituted pterins 13 & 15 existed solely as the hydrated anion forms, while the 7-CH3 substituted pterins 12 & 14 existed predominantly as the 7-exo-methylene anions. Spectroscopic investigations of the degradation processes of 8-(2-hydroxyethyl)pterins and 8-methylpterins in base indicated a complex pattern of oxidation, ring opening and elimination reactions as a function of time. Using authentic samples, the 7-oxo compounds 16 & 17 and ethanolamine were identified, and evidence for ring-opened forms was obtained by comparison with the relevant 2,5-diamino-6-alkyl-aminopyrimidin-4(3H)-ones 1 & 2. Characteristically different degradation pathways for 7-CH3 and 7-unsubstituted compounds were established.

Ethers, Cyclic↗
PubMed2005

Photochemistry of substituted dibenzothiophene oxides: the effect of trapping groups.

[reaction: see text] Photolyses of dibenzothiophene sulfoxides (DBTOs) with intramolecular trapping functionalities attached in the 4-position show higher quantum yields of deoxygenation. Deoxygenation quantum yields are also less solvent dependent for the substituted DBTOs. Product analysis shows a detectable amount of intramolecular O-trapped products and suggests that solvent effects observed in previous studies of DBTO derive at least mainly from the reactivity between the oxidizing species that is released, presumably O((3)P), and the solvent, rather than from other macroscopic solvent parameters.

Journal Article↗
PubMed2006

Mutations in alpha-helical solvent-exposed sites of eglin c have long-range effects: evidence from molecular dynamics simulations.

Eglin c is a small protease inhibitor whose structural and thermodynamic properties have been well studied. Previous thermodynamic measurements on mutants at solvent-accessible positions in the protein's helix have shown the unexpected result that the data could be best fit by the inclusion of residue- and position-specific parameters to the model. To explore the origins of this surprising result, long molecular dynamics simulations in explicit solvent have been performed. These simulations indicate specific long-range interactions between the solvent-exposed residues in the eglin c alpha-helix and binding loop, an unexpected observation for such a small protein. The residues involved in the interaction are on opposite sides of the protein, about 25 A apart. Simulations of alanine substitutions at the solvent-exposed helix positions, arginine 22, glutamic acid 23, threonine 26, and leucine 27, show both small and large perturbations of eglin c dynamics. Two mutations exhibit large impacts on the long-range helix-loop interactions. Previous stability measurements (Yi et al., Biochemistry 2003;42:7594-7603) had indicated that an alanine substitution at position 27 was less stabilizing than at other solvent-exposed positions in the helix. The L27A mutation effects observed in these simulations suggest that the position-dependent loss of stability measured in wet bench experiments is derived from changes in dynamics that involve long-range interactions; thus, these simulations support the hypothesis that solvent-exposed positions in helices are not always equivalent.

Computational Biology↗
PubMed1981

Clinical comparison of ethyl acetate and diethyl ether in the formalin-ether sedimentation technique.

A substitute for the volatile solvent diethyl ether has been actively sought for the Formalin-ether sedimentation technique. Ethyl acetate has recently been shown to be a comparable substitute. In an effort to verify these findings and evaluate ethyl acetate under clinical conditions, comparison studies with 62 fresh human stool specimens were performed. Parallel concentrates with diethyl ether and ethyl acetate were prepared for each specimen, and the quantity and appearance of recovered parasite species were determined. Ethyl acetate was found comparable to diethyl ether in the quantitative recovery of parasite eggs, cysts, and larvae; no distortion or alteration of parasite morphology was observed with either solvent. More care was required, however, to completely remove the interface plugs of ethyl acetate and prevent their remixing the concentrate sediment. In addition, wet mounts prepared from ethyl acetate concentrates were occasionally obscured by liquid bubbles probably composed of remaining insoluble ethyl acetate. Clinical laboratories considering substituting ethyl acetate for diethyl ether in the Formalin-ether sedimentation technique should be aware of these problems and take the appropriate precautions to avoid them.

Acetates↗
PubMedDate not supplied

Importance of solvent drag and diffusion in bile acid-dependent bile formation: ion substitution studies in isolated perfused rat liver.

Ion substitution studies were carried out in the isolated perfused rat liver to define the importance of solvent drag and diffusion in bile acid-dependent bile formation. Two different methods, namely single injection (20 mu moles) and continuous infusions at 0.4, 0.8, 1.2, and 1.6 mu moles per min taurocholate (TC), were used to determine the bile acid-dependent bile flow (BADF). Both methods gave essentially the same results. Replacement of Na+ (146mM) by 120 or 146 mM Li+ and Cl-(127mM) by 120 mM NO3- increased BADF significantly. On the other hand, replacement of Na+ by 120 mM choline and Cl- y 120 mM isethionate decreased the BADF. The osmolarity of TC solution was not different when Na+ was replaced by 120 mM Li+ or choline and TC did not affect the osmotic activity of NaCl, and choline-Cl differently. Thus, the observed effect of Na+ replacement on BADF is not due to any change in the osmotic activity of the secreted TC. Substitution of HCO3- by equimolar tricine also decreased BADF. Under this condition, BADF increased when NaCl was replaced by equimolar NaNO3. Thus, HCO3- does not seem to be essential for TC choleresis. Since Li+ and NO3- are more permeable, and choline and isethionate are less permeable than Na+ and Cl-, respectively, these results suggest that the BADF is dependent on the permeability of the substituting cations and anions and thus support the hypothesis that solvent drag and diffusion play an important role in BADF.

Animals↗
PubMed2006

Effect of hydroxylic solvent on the fluorescence behavior of some bioactive 9-oxo-imidazo[1,2-a]purine derivatives.

The spectral and photophysical behavior of four fluorescent 9-oxo-imidazo[1,2-a]purine derivatives containing pyridyl, pyridylphenyl, phenyl, and biphenylyl substituents at the C(6) position of the tricyclic skeleton is described. The studies were performed in several aprotic and protic organic solvents using absorption spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy. The results are also presented of TDDFT calculations on singlet-singlet excitation energies and oscillator strengths for two models of 9-oxo-imidazo[1,2-a]purine, with phenyl or pyridyl substituents, both in the gas phase and in methanol solution. While the derivatives with aryl substituents did not show any significant dependence of their static and dynamic fluorescence properties on the nature of the solvent, the compounds containing a pyridine residue exhibited a remarkable reduction of their fluorescence quantum yields and lifetimes in the alcoholic solutions. The solute-solvent hydrogen-bonding interaction in the first excited singlet state is responsible for the fast radiationless decay rates determined for pyridyl- and pyridylphenyl-substituted compounds in protic solvents. The results of experimental and theoretical studies show that the hydrogen of the alcohols' hydroxyl group and the nitrogen atom of the pyridine moiety are involved in the interaction. The fluorescence-quenching experiments performed for the pyridyl-substituted 9-oxo-imidazo[1,2-a]purine derivative using trifluoroethanol, methanol, and butanol as quenchers revealed that the quenching efficiencies, expressed by the Stern-Volmer quenching constants, correlate with the H-bond donating abilities of the alcohols. The quenching is a dynamic process, and the H-bonded complex formed is nonfluorescent. The experimentally determined and the calculated values of the dipole moment change associated with the electronic excitation indicate that the excited S(1) states of all of the molecules studied in this work have an intramolecular charge-transfer character and that electronic charge is transferred to the C(6) substituent upon excitation. Thus, the ability of the pyridyl substituent nitrogen atom to act as an H-bond acceptor in the excited S(1) state is enhanced. The 6-pyridyl-9-oxo-imidazo[1,2-a]purine presents a novel fluorophore, which, besides its medical applications, may be useful as a sensor of hydroxyl groups in microorganized systems.

Alcohols↗
PubMed2005

Rapid and efficient synthesis of poly-substituted quinolines assisted by p-toluene sulphonic acid under solvent-free conditions: comparative study of microwave irradiation versus conventional heating.

A rapid and efficient method for the preparation of various poly-substituted quinolines has been developed through the Friedländer condensation of 2-aminoarylketone or 2-aminoarylaldehyde with carbonyl compounds in the presence of p-toluene sulphonic acid, which was achieved by both microwave irradiation and conventional heating under solvent-free conditions.

Aldehydes↗
PubMed2005

Role of a surface tryptophan in defining the structure, stability, and DNA binding of the hyperthermophile protein Sac7d.

Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein-DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (DeltaG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 degrees C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.

Archaeal Proteins↗
PubMed2005

Paradoxical solvent effects on the absorption and emission spectra of amino-substituted perylene monoimides.

In N-(2,5-di-tert-butylphenyl)-9-pyrrolidinoperylene-3,4-dicarboximide (5PI) the absorption and emission spectra display large solvatochromic shifts, but, remarkably, the Stokes shift is practically independent of solvent polarity. This unique behavior is caused by the extraordinarily large ground-state dipole moment of 5PI, which further increases upon increasing the solvent polarity, whereas the excited-state dipole moment is less solvent dependent. In the corresponding piperidine compound, 6PI, this effect is much less important owing to the weaker coupling between the amino group and the aromatic imide moiety, and in the corresponding naphthalimide, 5NI, it is absent. The latter shows the conventional solvatochromic behavior of a push-pull substituted conjugated system, that is, minor shifts in absorption and a larger change in the emission energy with solvent polarity.

Amines↗
PubMed1997

Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.

Animals↗
PubMed2000

Ethyl formate - alternative dispersed solvent useful in preparing PLGA microspheres.

In an effort to substitute methylene chloride with a less toxic solvent, this study was aimed at developing new ethyl formate-based emulsion processes to fabricate poly-D,L-lactide-co-glycolide (PLGA) microspheres. To do so, a polymeric dispersed phase was emulsified in a 1% polyvinyl alcohol aqueous solution at an ethyl formate to aqueous volume ratio of 8:20. Microsphere hardening was then achieved by solvent evaporation and quenching techniques. The average encapsulation efficiency of a model drug progesterone amounted to 95.2+/-2.7%. When the tendency of ethyl formate and methylene chloride to evaporate to air was compared, the evaporation rate of ethyl formate was 2.1 times faster than that of methylene chloride. The ease with which ethyl formate evaporated to air was beneficial in shortening the microsphere hardening step. For the solvent quenching process, only 80 ml of additional water was required to extract ethyl formate to the aqueous phase, due to its considerable water miscibility. In particular, the timing of ethyl formate quenching affected to a great extent dynamic processes of the breakup of elementary microdroplets into smaller ones. Therefore, variations in quenching time affected microsphere characteristics such as the degree of solvation, size distribution, and tendency to aggregate on drying. The results of this study showed that PLGA microspheres were successfully prepared using the new ethyl formate-based processes.

Chemistry, Pharmaceutical↗
PubMed2006

A simple quantum statistical thermodynamics interpretation of an impressive phase diagram pressure shift upon (H/D) isotopic substitution in water + 3-methylpyridine.

In a previous work (J. Phys. Chem. B 2003, 107, 9837), we reported liquid-liquid-phase splitting at negative pressures in mixtures of H2O + D2O + 3-methylpyridine (3-MP) at the limit of pure H2O as the solvent, thus extending for the first time the L-L phase diagrams to this metastable region. We showed that there is an intimate relation between pressure and solvent deuterium content. Isotopic substitution (H/D) in water provokes subtle entropic effects that, in turn, trigger a significant pressure shift, opening a pressure-wide miscibility window of as much as 1600 bar. Isotope effects are quantum in origin. Therefore, a model that is both pressure-dependent and considers quantization constitutes a necessary tool if one wishes to fully describe the p, T, x critical demixing in these systems. In the current work, the statistical-mechanical theory of isotope effects is combined with a compressible pressure-dependent model. This combination enabled us to predict successfully the overall L-L phase diagram via differences in the vibrational mode frequencies of water on its transfer from the pure state to that of dilution in 3-MP: each of the three librational modes undergo a calculated red-shift of -(250 +/- 30) cm(-1), while the overall internal frequencies contribution is estimated as a total +(400 +/- 25) cm(-1) blue-shift.

Deuterium Oxide↗
PubMed2000

A polar, solvent-exposed residue can be essential for native protein structure.

BACKGROUND: A large energy gap between the native state and the non-native folded states is required for folding into a unique three-dimensional structure. The features that define this energy gap are not well understood, but can be addressed using de novo protein design. Previously, alpha(2)D, a dimeric four-helix bundle, was designed and shown to adopt a native-like conformation. The high-resolution solution structure revealed that this protein adopted a bisecting U motif. Glu7, a solvent-exposed residue that adopts many conformations in solution, might be involved in defining the unique three-dimensional structure of alpha(2)D. RESULTS: A variety of hydrophobic and polar residues were substituted for Glu7 and the dynamic and thermodynamic properties of the resulting proteins were characterized by analytical ultracentrifugation, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. The majority of substitutions at this solvent-exposed position had little affect on the ability to fold into a dimeric four-helix bundle. The ability to adopt a unique conformation, however, was profoundly modulated by the residue at this position despite the similar free energies of folding of each variant. CONCLUSIONS: Although Glu7 is not involved directly in stabilizing the native state of alpha(2)D, it is involved indirectly in specifying the observed fold by modulating the energy gap between the native state and the non-native folded states. These results provide experimental support for hypothetical models arising from lattice simulations of protein folding, and underscore the importance of polar interfacial residues in defining the native conformations of proteins.

Dimerization↗
PubMed1999

Disentangling the perturbational effects of amino acid substitutions in the DNA-binding domain of p53.

The spectrum of somatic cancer-associated missense mutations in the human TP53 gene was studied in order to assess the potential structural and functional importance of various intra-molecular properties associated with these substitutions. Relating the observed frequency of particular amino acid substitutions in the p53 DNA-binding domain to their expected frequency, as calculated from DNA sequence-dependent mutation rates, yielded estimates of their relative clinical observation likelihood (RCOL). Several biophysical properties were found to display significant covariation with RCOL values. Thus RCOL values were observed to decrease with increasing solvent accessibility of the substituted residue and with increasing distance from the p53 DNA-binding and Zn2+ -binding sites. The number of adverse steric interactions introduced by an amino acid replacement was found to be positively correlated with its RCOL value, irrespective of the magnitude of the interactions. A gain in hydrogen bond number was found to be only half as likely to come to clinical attention as mutations involving either a reduction or no change in hydrogen bond number. When the difference in potential energy between the wild-type and mutant DNA-binding domains was considered, RCOL values exhibited a minimum around changes of zero. Finally, classification of mutated residues in terms of their protein/solvent environment yielded, for somatic p53 mutations, RCOL values that resembled those previously determined for inherited mutations of human factor IX causing haemophilia B, suggesting that similar mechanisms may be responsible for the mutation-related perturbation of biological function in different protein folds.

Amino Acid Substitution↗