Ellen Wachtel

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 quasi-elastic 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.

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.

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites.

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.

The effect of osmotic and mechanical pressures on water partitioning in articular cartilage.

X-ray diffraction measurements on native and proteoglycan-free articular cartilage have been made in order to test the dependence of the lateral packing of the collagen molecules on the osmotic pressure gradient, either naturally occurring or externally applied, between the intra- and extrafibrillar compartments. From the information on collagen packing we have been able to calculate, albeit with several assumptions, the amount of intrafibrillar water as a function of pressure. In parallel with the above measurements, we have quantitated, using serum albumin partitioning, the intrafibrillar water in proteoglycan-free cartilage, as a function of mechanically applied pressure. The results of both sets of experiments lead to the conclusion that the molecular packing density, and hence the intrafibrillar water content, are a function of the osmotic pressure difference between the extrafibrillar and intrafibrillar spaces or the equivalent mechanically applied pressure. The determination of intrafibrillar water has enabled us to calculate, from measured values of fixed charge density, the internal osmotic pressure of cartilage specimens, both in compressed and uncompressed states.

Collagen Turnover in Normal and Degenerate Human Intervertebral Discs as Determined by the Racemization of Aspartic Acid

Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We measured the ratio of the d- and l-isomers in collagen extracted from these tissues as a function of age between 16 and 77 years. For collagen taken from healthy discs, the fractional increase of d-Asp was found to be 6.74 × 10–4/year; for degenerate discs, the corresponding rate was 5.18 × 10–4/year. Using the racemization rate found previously for the stable population of collagen molecules in dentin, we found that the rate of collagen turnover (kT) in discs is not constant but rather a decreasing function of age. The average turnover rate in normal disc between the ages of 20 and 40 is 0.00728 ± 0.00275/year, and that between the ages of 50 and 80 is 0.00323 ± 0.000947/year, which correspond to average half-lives of 95 and 215 years, respectively. Turnover of collagen from degenerate discs may be more rapid than that found for normal discs; however, statistical analysis leaves this point uncertain. The finding of a similar correlation between the accumulation of d-Asp and that of pentosidine for three normal collagenous tissues further supports the idea that the accumulation of pentosidine in a particular tissue can, along with the racemization of aspartic acid, be used as a reliable measure of protein turnover.

Conversion of CO2 to CO by electrolysis of molten lithium carbonate

The conversion of CO 2 to CO by electrolysis of molten Li 2 CO 3 was investigated. Using a cell comprising a Ti cathode, a graphite anode and a source of CO 2 allows the continuous electrolysis of the melt at 900°C with current densities at the electrodes higher than 100 mA/cm 2 . The faradaic efficiency of the process is close to 100%, and the thermodynamic efficiency at 100 mA/cm 2 is > 85%. The proposed method has several advantages: (i) No precious metal is required, (ii) no hazardous or toxic by-products are produced, and (iii) the method may operate continuously, producing pure CO rather than a mixture of CO and CO 2 . Therefore, the process described here has a potential application for converting electrical energy into fuel.

Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid

We have used the racemization of aspartic acid as a marker for the “molecular age” of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the d/lAsp ratio of the various aggrecan species as a function of age and using the values of the racemization constant, ki, found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 ± 0.01 and 0.063 ± 0.01/year correspond to half-life values of 12 ± 2.0 and 11.23 ± 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 ± 0.022 and 0.092 ± 0.034/year for degenerate tissue correspond to half-life values of 8.77 ± 2.2 and 8.41 ± 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 ± 0.04/year, corresponding to a half-life of 5.56 ± 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 ± 0.0012/year, corresponding to a half-life of 21.53 ± 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. d/lAsp values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan.

Extrafibrillar proteoglycans osmotically regulate the molecular packing of collagen in cartilage

The molecular packing density of collagen and hence the intrafibrillar water content appears to be regulated in cartilage by the osmotic pressure gradient existing between the extrafibrillar and the intrafibrillar compartments.

A study of the microstructure of four-component sucrose ester microemulsions by SAXS and NMR

Sucrose esters form a class of surfactants with the important properties of being biodegradable, non-toxic and capable of forming temperature-insensitive microemulsions. Such microemulsions would be expected to suit a variety of food-based and pharmaceutical applications; however to date little is known about their structure and stability. In this study, the Winsor IV microemulsion systems composed of sucrose esters (SE)/1-butanol/water and oils such as n-dodecane, n-hexadecane and medium chain triglyceride (MCT), have been investigated using small angle X-ray scattering (SAXS), pulsed gradient spin echo (PGSE) NMR and viscosity measurements. The SAXS results for the sucrose monostearate (S1570) system at SE/MCT/1-butanol=1.5:1.1 clearly indicate that the periodicity d increases with increase in water content and is not sensitive to the nature of the oil. From the amphiphilicity factor, fa, and the correlation length, ξ, one can conclude that the n-dodecane-based microemulsion system is the most ordered. Microstructure investigation by PGSE NMR gives evidence of structural changes as the water content in the system increases. The oil self-diffusion remains unchanged when MCT serves as the oil phase. However, when the oil is paraffinic in nature (n-dodecane and n-hexadecane) the self-diffusion coefficient indicates participation of the oil molecules at the interface. Surfactant self-diffusion is only weakly affected by the water content. The shorter chain oils (n-dodecane and MCT) solubilize a maximum of 40 and 47 wt.% of water and cannot invert, while the long chain paraffinic (n-hexadecane-based system) inverts into an O/W microemulsion. The viscosity of these microemulsions decreases with increasing water content. The absence of a yield stress in any of the samples studied, together with the linearity of the flow curves, is evidence that there are no relaxation processes in these microemulsions which show a non-Newtonian flow behavior.

Age-related Accumulation of Pentosidine in Aggrecan and Collagen from Normal and Degenerate Human Intervertebral Discs

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.