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Biomedical subjects

E J Wachtel

Publications and source records attributed to E J Wachtel.

At least 19 recordsLinked to original sources
PubMed2001

Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Bacterial Proteins↗
PubMed1997

Ligand-specific oligomerization of T-cell receptor molecules.

T cells initiate many immune responses through the interaction of their T-cell antigen receptors (TCR) with antigenic peptides bound to major histocompatibility complex (MHC) molecules. This interaction sends a biochemical signal into the T cell by a mechanism that is not clearly understood. We have used quasielastic light scattering (QELS) to show that, in the presence of MHC molecules bound to a full agonist peptide, TCR/peptide-MHC complexes oligomerize in solution to form supramolecular structures at concentrations near the dissociation constant of the binding reaction. The size of the oligomers is concentration dependent and is calculated to contain two to six ternary complexes for the concentrations tested here. This effect is specific as neither molecule forms oligomers by itself, nor were oligomers observed unless the correct peptide was bound to the MHC. These results provide direct evidence for models of T-cell signalling based on the specific assembly of multiple TCR/peptide-MHC complexes in which the degree of assembly determines the extent and qualitative nature of the transduced signal. They may also explain how T cells maintain sensitivity to antigens present in only low abundance on the antigen-presenting cell.

Amino Acid Sequence↗
PubMed1996

Flow of structural information between four DNA conformational levels.

Closed-circular supercoiled DNA molecules have been shown to form a cholesteric assembly within bacteria as well as in vitro under physiological DNA and salt concentrations. Circular dichroism and X-ray scattering studies indicate that the macroscopic structural properties of the chiral mesophase are directly and uniquely dictated by the supercoiling parameters of the constituent molecules. Specifically, we find that the pitch of the DNA cholesteric phase derived from supercoiled DNA is determined by the superhelical density, which, in turn, is modulated by secondary conformational changes. A direct interrelationship among four DNA structural levels, namely, DNA sequence, secondary structural transitions, the tertiary superhelical conformation, and the quaternary, supramolecular organization is accordingly pointed out. Since secondary conformational changes are both sequence and environment dependent, alterations of cellular conditions may effectively modulate the properties of the packed DNA organization, through their effects on secondary structural transitions and hence on the superhelical parameters. On the basis of these results we suggest that liquid crystallinity represents an effectively regulated packaging mode of plectonemic, nucleosome-free DNA molecules in living systems.

Base Sequence↗
PubMed1995

Phase behavior of mixtures of cholesterol and saturated phosphatidylglycerols.

The interaction of cholesterol with a series of saturated phosphatidylglycerols was investigated using differential scanning calorimetry and X-ray diffraction. We find that the miscibility of cholesterol in phosphatidylglycerol bilayers is lower than in the corresponding phosphatidylcholine bilayers and decreases with increasing acyl chain length of the phospholipid. The influence of the negative charge of the phosphatidylglycerol on cholesterol miscibility is discussed.

Calorimetry, Differential Scanning↗
PubMed1995

In vivo quantitative characterization of intermolecular interactions.

Extensive effort has been directed toward a quantitative evaluation of forces which operate between biomacromolecules since the characterization of such forces is essential to a thorough understanding of fundamental biological processes. However, all studies hitherto reported were conducted in vitro, using isolated species. Here we report the first quantitative characterization of forces operating between DNA molecules within living bacteria. Evaluation of x-ray scattering studies conducted on intact bacteria indicates that, at DNA-DNA surface separations characteristic of DNA assemblies, interactions are dominated by repulsive hydration forces which originate from the structuring of water molecules. The results support the notion that the mechanisms by means of which macromolecules function, fold, and interact with each other crucially depend upon their hydration properties.

Base Composition↗
PubMed1994

Liquid-crystalline mesophases of plasmid DNA in bacteria.

Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.

Birefringence↗
PubMed1992

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.

Circular Dichroism↗
PubMed1991

The effect of protons or calcium ions on the phase behavior of phosphatidylserine-cholesterol mixtures.

The influence of protons or calcium ions on the miscibility of cholesterol in phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. At pH 2.6, where the carboxyl group of the serine moiety is protonated, two endothermic transitions are observed in cholesterol-phosphatidylserine mixtures. The midpoint of the first is at 35 degrees C in the absence of cholesterol and decreases to approx. 15 degrees C for molar fraction of cholesterol 0.5. The second transition is centered at approx. 44 degrees C, almost independent of cholesterol content. The two lower temperature phases are lamellar and the high temperature phase has hexagonal symmetry. Cholesterol is more miscible in protonated phosphatidylserine than in the sodium form: cholesterol crystals are detected at a molar ratio of phosphatidylserine to cholesterol of about 1.7:1 as compared to about 2.3:1 at neutral pH. In the presence of calcium ions (1.3 Ca2+ per phosphatidylserine), a lamellar phase is observed with layer spacing 53 A which is independent of temperature (25 degrees C-65 degrees C) and of cholesterol content. Calcium ions cause reduced cholesterol solubility: crystallites are detected already at a molar ratio of 4:1.

Animals↗
PubMed1990

States of aggregation and phase transformations in mixtures of phosphatidylcholine and octyl glucoside.

The result of mixing varying concentrations of the nonionic detergent octyl glucoside (OG) with small unilamellar vesicles (SUV) of egg phosphatidylcholine (PC) made by sonication depends on the ratio between OG and PC in the mixed aggregates. When this molar ratio (Re) is lower than 1.4, the detergent partitions between the PC vesicles and the aqueous medium with a partition coefficient of K = 0.033 mM-1. As a consequence of introduction of OG into the bilayers, the vesicles grow in size. The resultant vesicles have a mean diameter that is an increasing function of Re and is independent of the total PC concentration. Experiments in which the vesicles were loaded with high molecular weight dextran prior to being exposed to OG suggest that the mechanism responsible for the size growth involves lipid transfer rather than fusion. Mixtures with Re values within the range of 1.4-3.2 separate into two macroscopic phases: The lower phase is clear but very viscous. It contains constant OG and PC concentrations and is characterized by an Re value of 3.2, independent of the composition of the whole dispersion. The upper phase contains vesicles of varying concentrations of OG and PC, but a constant Re of 1.4. When the saturating level of 1.4 OG molecules per PC molecule is approached, the concentration of OG monomers in the aqueous medium reaches the value of 16.6 +/- 0.3 mM, which is the apparent cmc of OG in the lipid-containing medium. OG-PC mixed micelles contain at least 3.2 OG molecules per PC molecule. The mixed micelles present at Re = 3.2 apparently have the shape of oblate ellipsoids with a minor axis of about 2 nm and two major axes of about 25 nm. The surface area of the mixed micelles at this point is just sufficient for them to undergo conversion into the smallest possible spherical vesicles of a radius of 12 nm. At Re values above 3.2, the major axis of the mixed micelles becomes smaller as Re increases, while at values of Re below 3.2 the micelles would have been expected to grow very rapidly with decreasing Re. This may explain the partial vesicle closure occurring below Re = 3.2.

Detergents↗
PubMed1989

X-ray-scattering of turkey skeletal-muscle troponin C in solution at low pH.

The solution structure of troponin C from turkey skeletal muscle was studied at low pH by small-angle X-ray-scattering. We find that troponin C at pH 5.3 in the presence of Mg2+ has a triaxial radius of gyration and maximum dimension comparable with those of the crystallized protein. However, the relative disposition of domains is more similar to that found for the highly homologous rabbit protein in solution at pH 7.4.

Animals↗
PubMed1987

X-ray diffraction study of cholesterol-phosphatidylserine mixtures.

Phosphatidylserine-cholesterol mixtures at a molar ratio of 2:1 were investigated by X-ray diffraction. Phase separation of cholesterol independent of temperature was detected, indicating limited solubility of cholesterol in phosphatidylserine bilayers. The second phase present, the mixed phospholipid-cholesterol phase, continued to undergo melting as determined by changes with temperature in both the small angle scattering profile and in the acyl chain packing.

Cholesterol↗
PubMed1982

Ionic strength dependent conformational changes of transfer ribonucleic acid studied by circular polarization of phosphorescence.

The circularly polarized phosphorescence emitted by the 4-thiouridine base was measured for bulk Escherichia coli tRNA, E. coli tRNA1Val, E. coli tRNAfMet, and E. coli tRNAPhe as a function of ionic strength in the presence of 1 mM Mg2+. The emission anisotropy factor, gem, was found to be dependent on the degree of local stacking in the vicinity of the chromophore. For bulk tRNA and tRNAfMet a marked similarity was observed between the behavior of gem and the translational diffusion coefficient D020,w [Pots, R. O., Wang, C. C., Fritzinger, D. C., Ford, N. C. & Fournier, M. J. (1979) in Cold Spring Harbor Monograph Series (Schimmel, P. R; Söll, D., & Abelson, J. N., Eds.) Part A, p 207, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. This was interpreted to mean that a relationship exists between the local stacking around the 4-thiouridine chromophore and the overall general similarity of behavior was observed, each species of tRNA examined exhibited a distinctive dependence of conformation on salt concentration. tRNAPhe was found to be a particular exception in the low-salt region (less than 50 mM). The value of the phosphorescence anisotropy factor was found to be remarkably larger (about 50-fold). Thus minor conformational changes in the tRNA molecule are more readily detected by circular polarization of phosphorescence than by circular dichroism

Escherichia coli↗