Extent Of Oligomerization Research Articles

From dilute to concentrated solutions of intrinsically disordered proteins: Interpretation and analysis of collected data.

Intrinsically disordered proteins (IDPs) have a broad energy landscape and consequently sample many different conformations in solution. The innate flexibility of IDPs is exploited in their biological function, and in many instances allows a single IDP to regulate a range of processes in vivo. Due to their highly flexible nature, characterizing the structural properties of IDPs is not straightforward. Often solution-based methods such as Nuclear Magnetic Resonance (NMR), Förster Resonance Energy Transfer (FRET), and Small-Angle X-ray Scattering (SAXS) are used. SAXS is indeed a powerful technique to study the structural and conformational properties of IDPs in solution, and from the obtained SAXS spectra, information about the average size, shape, and extent of oligomerization can be determined. In this chapter, we will introduce model-free methods that can be used to interpret SAXS data and introduce methods that can be used to interpret SAXS data beyond analytical models, for example, by using atomistic and different levels of coarse-grained models in combination with molecular dynamics (MD) and Monte Carlo simulations.

CO2-Assisted Oxidative Dehydrogenation of Propane over VOx/In2O3 Catalysts: Interplay between Redox Property and Acid–Base Interactions

In this work, a series of VOx-loaded In2O3 catalysts were prepared, and their catalytic performance was evaluated for CO2-assisted oxidative dehydrogenation of propane (CO2-ODHP) and compared with In2O3 alone. The optimal composition is obtained on 3.4V/In2O3 (surface V density of 3.4V nm–2), which exhibited not only a higher C3H6 selectivity than other V/In catalysts and In2O3 under isoconversion conditions but also an improved reaction stability. To elucidate the catalyst structure–activity relationship, the VOx/In2O3 catalysts were characterized by chemisorption [NH3-temperature-programmed desorption (TPD), NH3-diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), CO2-TPD, and CO2-DRIFTS], H2-temperature-programmed reduction (TPR), in situ Raman spectroscopy, UV–vis diffuse reflectance spectroscopy, near-ambient pressure X-ray photoelectron spectroscopy, X-ray absorption spectroscopy, and further examined using density functional theory. The In–O–V structure and the extent of oligomerization, which play a crucial role in improving selectivity and stability, were identified in the VOx/In2O3 catalysts. In particular, the presence of surface VOx (i) inhibits the deep reduction of In2O3, thereby preserving the activity, (ii) neutralizes the excess basicity on In2O3, thus suppressing propane dry reforming and achieving a higher propylene selectivity, and (iii) introduces additional redox sites that participate in the dehydrogenation reaction by utilizing CO2 as a soft oxidant. The present work provides insights into developing selective, stable, and robust metal-oxide catalysts for CO2-ODHP by controlling the conversion of reagents via desired pathways through the interplay between acid–base interactions and redox properties.

Influence of soluble oligomeric aluminum on precipitation in the Al-KOH-H2O system.

The role of oligomeric aluminate species in the precipitation of aluminum (Al) phases such as gibbsite (α-Al(OH)3) from aqueous hydroxide solutions remains unclear and difficult to probe directly, despite its importance for developing accurate predictions of Al solubility in highly alkaline systems. Precipitation in this system entails a transition from predominantly tetrahedrally coordinated aluminate (Al(OH)4-) species in solution to octahedrally coordinated Al in gibbsite. Here we report a quantitative study of dissolved Al in the Al-KOH-H2O system using a combination of molecular spectroscopies. We establish a relationship between changes in 27Al NMR chemical shifts and the relative intensity of Raman vibrational bands, indicative of variations in the ensemble speciation of Al in solution, and the formation of unique contact ion pair interactions with the aluminate dimer, Al2O(OH)62-. A strong correlation between the extent of Al oligomerization and the amount of solvated Al was demonstrated by systematically varying the KOH : Al molar ratio. The concentration of dissolved oligomeric Al in solution also directly impacted the particle size and morphology of the precipitated gibbsite. High concentrations of dimeric Al2O(OH)62- yielded smaller and more numerous anhedral to subhedral gibbsite particles, while low concentrations yielded fewer and larger euhedral gibbsite platelets. The collective observations suggest a key role for the Al2O(OH)62- dimer in promoting gibbsite precipitation from solution, with the potassium ion-paired dimer catalyzing a more rapid transformation of Al from tetrahedral coordination in solution to octahedral coordination in gibbsite.

From swollen gels to dried films: Relating the IR spectra of ferrihydrite dried as a film on an ATRIR crystal to aqueous suspensions

In aquatic systems ferrihydrite forms as highly porous aggregates that resemble swollen gels. Much insight into the surface chemistry of ferrihydrite has been gleaned using a technique in which ferrihydrite is dried on to the surface of a crystal that acts as an Attenuated Total Reflectance (ATR) medium for in situ infrared (IR) studies of ligand sorption. In this paper BET N2 adsorption, acid-base titration, H4SiO4 isotherms and the relationship between H4SiO4 sorption and H4SiO4 interfacial oligomerization are used to systematically probe the influence of this drying step on ferrihydrite's surface chemistry. This means that insights into sorption reactions revealed from in situ ATR-IR are can be increasingly related to real environmental systems. Comparing the measured acid-base titration curves to the theoretical curve provide a specific surface area (SSA) estimation of ≈600 and 290 m2 g−1 for the fresh and film-dried ferrihydrite respectively, compared to the BET N2 adsorption values of 240 and 140 m2 g−1 where the fresh ferrihydrite was freeze dried prior to BET measurement. The H4SiO4 isotherms were identical on the two ferrihydrite samples when normalized to the titration derived surface areas. The extent of interfacial H4SiO4 oligomerization provides insights into surface structures. Within experimental uncertainty, the process of drying ferrihydrite into a film did not alter either the general trend in the relationship between sorption and oligomerization, or the extent of oligomerization as a function of surface coverage normalized to the titration derived surface areas. Therefore, in comparable laboratory studies where sorption densities for dried films are compared with fresh suspensions the SSA values of 290 and 600 m2 g−1 respectively should be a reasonable estimate.

The Power and Limitations of Influenza Virus Hemagglutinin Assays.

Influenza virus hemagglutinins (HAs) are surface proteins that bind to sialic acid residues at the host cell surface and ensure further virus internalization. Development of methods for the inhibition of these processes drives progress in the design of new antiviral drugs. The state of the isolated HA (i.e. combining tertiary structure and extent of oligomerization) is defined by multiple factors, like the HA source and purification method, posttranslational modifications, pH, etc. The HA state affects HA functional activity and significantly impacts the results of numerous HA assays. In this review, we analyze the power and limitations of currently used HA assays regarding the state of HA.

ANTI-AβX-40 ANTIBODIES EXERT NEUROPROTECTIVE EFFECT AGAINST Aβ-OLIGOMER TOXICITY

Amyloid-beta (Aβ) plays a critical role in Alzheimer's disease (AD) pathology. Aβ oligomers have been reported to be more neurotoxic than monomeric forms and correlate with AD severity and synapse loss. We have observed that active vaccination of AD patients against the C-terminal end of Aβ1–40 triggers the production of anti-Aβx-40 antibodies, and that these types of antibodies inhibit Aβ1–40 assembly into oligomers in vitro. In this study, we have compared the cytotoxic damage produced by Aβ1–40 oligomers and monomers on PC12 cells in vitro, and have also analyzed the protective effect exerted by anti-Aβx-40 antibodies against that damage. Oligomers were prepared by dissolving lyophilized Aβ1–40 monomers in a solution containing DMSO, PBS and SDS, and incubating for 18 h at 37°C. Aβ monomers were prepared immediately before their use by dissolving lyophilized Aβ1–40 monomers in a solution of the same formula used for the oligomers. Polyclonal anti- Aβx-40 antibodies were produced in our lab. PC12 cells were grown overnight in 96-well plates at 3 x 105 cells/well, and then incubated with (a) Aβ1–40 oligomers, (b) Aβ1–40 monomers, (c) Aβ1–40 oligomers + anti-Aβx-40 antibodies, (d) Aβ1–40 monomers + anti-Aβx-40 antibodies, or (e) vehicle. The different treatments were assayed at different concentrations and co-cultured for an additional 24 hours. To evaluate Aβ1–40-mediated cytotoxicity, PC12 cell viability was quantified by using the MTT assay (colorimetric assay for assessing cell metabolic activity). Aβ1–40 oligomers produced a concentration-dependent cytotoxic effect on PC12 cells whereas Aβ1–40 monomers did not. Pre-incubation of Aβ1–40 monomers with anti-Aβx-40 antibodies prevented the formation of Aβ1–40 oligomers, resulting in increased cell viability in the cultures. The beneficial effect of anti-Aβx-40 antibodies was dependent on the extent of oligomerization, being more protective when present at the beginning of oligomer formation than once the oligomers have been formed.

Synthesis and physical properties of new coco-oleic estolide branched esters

Oils derived from vegetable oils tend to not meet the standards for industrial lubricants because of unacceptable low temperature properties, pour point (PP) and/or cloud point (CP). However, a catalytic amount of perchloric acid with oleic and coconut (coco) fatty acids produced a coco-oleic estolide. The resulting coco-oleic estolide was separated into two components based on the extent of oligomerization: coco-oleic dimer estolide and coco-oleic trimer plus estolide. These two estolides were then esterified with a series of different branched alcohols; the coco-oleic dimer estolide esters had the lowest PP=−45°C when with esterified 2-hexyldecanol and PP=−39°C with 2-octyldodecanol. The best CP performer from the same series was the 2-octyldodecanol ester, CP=−37°C. The coco-oleic trimer plus estolide esters had slightly higher PPs (−24 to −39°C) with the same alcohols. The viscosities and viscosity indexes were as expected in terms of trends. The coco-oleic dimer estolide esters ranged 27.5–51.7cSt @ 40°C and 3.0–9.5 cSt @ 100°C, whereas the coco-oleic trimer plus estolide esters ranged 120.8–227.7cSt @ 40°C and 17.9–29.4cSt at 100°C for the same series as the dimer esters. Outside the series tested, an iso-stearyl trimer plus ester had the highest reported viscosity of 417.3cSt @ 40°C and 38.9cSt @ 100°C. Because these new branched estolide esters have excellent viscosity and low temperature physical properties without the addition of other chemicals, they minimize the effect on the environment while replacing nonrenewable products.

SLO induced inflammasome activation is triggered by pore-formation instead of membrane repair (INM6P.337)

Abstract Macrophages activate membrane repair and the inflammasome in response to bacterial pore-forming toxins like streptolysin O (SLO). SLO mutants that are more easily resisted by repair processes produce increased amounts of IL-1β. This suggests that membrane repair may influence inflammasome activation, though it is not clear whether pore-formation or triggering repair pathways directly activates the inflammasome. In order to determine the relationship between membrane repair and the inflammasome, we first determined the mechanism of membrane repair in macrophages. Dose-dependent cytotoxicity of SLO is lower in macrophages than in either 3T3 or HeLa cell lines, indicating increased repair efficiency in macrophages. This was not due to decreased SLO binding to macrophages. Macrophages challenged with sublytic doses of SLO shed microvesicles similar to HeLa and 3T3 cells. Although pore-formation was necessary for inflammasome activation, the pore-dead SLO N402E still promoted microvesicle shedding. However, a second SLO pore-dead mutant, SLO G395V/G396V failed to promote shedding. This suggests that membrane binding and the extent of oligomerization, rather than pore-formation, is necessary to trigger membrane repair. Since pore-formation is necessary for IL-1β secretion, these data suggest that membrane repair itself does not promote inflammasome activation. Instead membrane repair preserves the cell long enough to execute its inflammatory program.

Gly25-Ser26 Amyloid β-Protein Structural Isomorphs Produce Distinct Aβ42 Conformational Dynamics and Assembly Characteristics

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used “click peptide” chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy–mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into “pure” Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.

Observing Assembly of Complex Inorganic Materials from Polyoxometalate Building Blocks

Understanding the aqueous state of discrete metal-oxo clusters, prenucleation clusters, and even simple ions is valuable for controlling the growth of metal-oxide materials from water. Niobium polyoxometalates (Nb-POMs) are unique in the aqueous metal-oxo cluster landscape in their unusual solubility behavior: specifically, their solubility in water increases with increasing ion-pairing contact with their counterions, and thus provides a rare opportunity to observe these and related solution phenomena. Here, we isolate in the solid state the monomeric and dimeric building blocks, capped Keggin ions, of the extended Keggin chain materials that are now well-known: not only in Nb-POM chemistry, but Mo and V POM chemistry as well. Rb13[GeNb13O41]·23H2O (Rb1), Cs10.6[H2.4GeNb13O41]·27H2O (Cs1) and Cs18H6[(NbOH)SiNb12O40]2·38H2O (Cs2) were characterized by single-crystal X-ray diffraction. Small angle X-ray scattering (SAXS) of solutions of Rb1 and Cs1 in varying conditions revealed oligomerization of the monomers into chain structures: the extent of oligomerization is controlled by pH, concentration, and the counterion. We distinctly observe chains of up to six Keggin ions in solution, with the large alkali cations for charge-balance. This combined solid state and solution study reveals in great detail the growth of a complex material from discrete monomeric building blocks. The fundamentals of the processes we are able to directly observe in this study, ion-association and hydrolysis leading to condensation, universally control the self-assembly and precipitation of materials from water.

Preparation and characterization of high-solid polyurethane coating systems based on vegetable oil derived polyols

A series of bio-based polyols with high functionality and low viscosity were synthesized from 5 different vegetable oils (refined or crude). Their chemical structures and the distribution of oligomers in these polyols (known as Liprol™) were characterized. Liprol structures varied due to the fatty acid profile of the starting oils and their overall degree of unsaturation, along with the extent of oligomerization during their formation. These polyols were then used as starting materials for the production of high-solid content polyurethane (PU) coatings, by reacting them with commercial petrochemical derived diisocyanate and other additives. All of the PU coatings obtained had a bio-based content of around 60% and showed good thermo-mechanical and mechanical properties. NuLin® flax PU, made from oil with the highest linolenic acid content, had the highest glass transition temperature, high contact angle with water, good abrasion resistance and Shore hardness, low degree of solvent swelling and formed highly cross-linked networks.

Synthesis of the All-l Cyclopentapeptides Versicoloritides A, B, and C

Herein we describe the first synthesis of the all-<sc>l</sc> naturally occurring cyclopentapeptides versicoloritides A, B, and C. We found that the extent of oligomerization and epimerization upon head-to-tail cyclization of the linear pentapeptide precursors could be reduced to acceptable levels by modifying the reaction conditions.

Studies on the Structure and Stability of Cyclic Peptide Based Nanotubes Using Oligomeric Approach: A Computational Chemistry Investigation

In this study, an attempt has been made to investigate the structure, dynamics, and stability of cyclic peptide nanotubes (CPNTs) formed by the self-assembly of cyclic peptides (CPs) using classical molecular dynamics (MD) simulation and semiempirical quantum chemistry calculation employing PM6 Hamiltonian with the dispersion correction and hydrogen-bonding interaction (DH2). The structure and energetics of monomer and various oligomeric CPNTs have been investigated by considering the (cyclo-[(D-Ala-L-Ala)(4)]) peptide as the model for CP. Although the formation of CPNTs has been intensively studied, the present study adds valuable information to the de novo design of CPNTs. Various geometrical parameters extracted from the MD simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. The hydrogen bonds present in the inner core regions are stronger than the terminal residues. As the degree of oligomerization increases, the stability of the tube increases due to the hydrogen-bonding and stacking interactions between the subunits. The results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond pentamer CPNT. In addition, hydrophobic and electrostatic interactions play crucial roles in the formation of CPNTs. Analysis of both structure and energetics of the formation of CPNTs unveils that the self-assembly of dimer, trimer, and tetramer CPNTs are the essential steps in the growth of CPNTs.

Oligomerization of membrane-bound Bcl-2 is involved in its pore formation induced by tBid

Both pro-apoptotic Bax and anti-apoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.

Formation of a solubilized cobalt block oligomer from a M2L-type double helicate

The multi-dentate ligand, 2,3,5,6-tetrakis(2,2'-bipyridyl)pyrazine (L) and divalent cobalt self-assemble to a block co-polymer-like oligomer in solution, which contains at least the L(7)Co(8) fragment. The extent of oligomerization is sensitive to the water content in acetonitrile solution. In the solid state, the simple monomer [LCo(2)(CH(3)CN)(4)][ClO(4)](4) is isolated. The X-ray structure of the crystallized material (containing four CH(3)CN solvate molecules) reveals a double-helical complex with two heptadentate Co(II) sites, and a helical pitch of approximately 28.1 A. Coupled Co(I/II) redox processes are observed between the two metal centres.

Constitutive Agonist-independent CCR5 Oligomerization and Antibody-mediated Clustering Occurring at Physiological Levels of Receptors

Although homo-oligomerization has been reported for several G protein-coupled receptors, this phenomenon was not studied at low concentrations of receptors. Furthermore, it is not clear whether homo-oligomerization corresponds to an intrinsic property of nascent receptors or if it is a consequence of receptor activation. Here CCR5 receptor oligomerization was studied by bioluminescence resonance energy transfer (BRET) in cells expressing physiological levels of receptors. A strong energy transfer could be observed, in the absence of ligands, in whole cells and in both endoplasmic reticulum and plasma membrane subfractions, supporting the hypothesis of a constitutive oligomerization that occurs early after biosynthesis. No change in BRET was observed upon agonist binding, indicating that the extent of oligomerization is unrelated to the activation state of the receptor. In contrast, a robust increase of BRET, induced by a monoclonal antibody known to promote receptor clustering, suggests that microaggregation of preformed receptor homo-oligomers can occur. Taken together, our data indicate that constitutive receptor homo-oligomerization has a biologically relevant significance and might be involved in the process of receptor biosynthesis.

Physical properties of estolides and their ester derivatives

Vegetable oil based lubricants have good lubricity and biodegradability but often fail to meet viscosity requirements at cold temperatures. These properties for a lubricant are often evaluated by pour point (PP), the minimum temperature at which a liquid will pour. Viscosity and viscosity index also provide information about a fluid's properties where a high viscosity index denotes a fluid that has little viscosity change over a wide temperature range. In order to develop better quality vegetable based lubricants, pour point, melting point and viscosity (40 and 100°C) were determined for a series of esters (methyl, butyl, decyl, oleyl, 2-propyl, 2-ethylhexyl, C18-Guerbet and C24-Guerbet) of meadowfoam, crambe and oleic fatty acids and estolides. Oleic estolide esters had the lowest melting points of all derivatives studied. Of the oleic estolide series the 2-ethylhexyl and C18-Guerbet esters had the lowest melting points, −34 and −43°C, respectively. Similar melting point trends within the series of esters were observed for both fatty acids and estolides. Pour points of the oleic estolides were also determined on a series of both free acid estolides and their 2-ethylhexyl esters. The series compared the effect of oligomerization on pour point. Pour points of oleic estolides were 5–10°C higher than the corresponding derivative's melting point. The extent of oligomerization (estolide number, EN) played a significant role in the pour point with higher oligomerization giving higher pour points (EN of 2.96 had a PP of 0°C; EN of 1.1 had a PP of −27°C). In a similar fashion, viscosity increased with higher oligomerization and free acid estolides were generally several hundred centistokes (cSt) more viscous than the corresponding esters. Viscosity index ranged from 151 to 238 with the fatty acid esters giving slightly higher indices than estolide esters. Hydrogenation of the estolides resulted in a dramatic increase in the pour point but only changed viscosity slightly.

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis.

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.

Improved artificial death switches based on caspases and FADD.

A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.

Optimization of the sulfuric acid‐catalyzed estolide synthesis from oleic acid

AbstractThe production of estolide from oleic acid with sulfuric acid as a catalyst was optimized for minimal acid concentration and temperature. Commercial oleic acid forms estolide optimally when reacted at 55°C with 5% vol/vol concentrated sulfuric acid for 24 h under vacuum. The extent of oligomerization was 1.2 estolide units under these reaction conditions. Temperature plays a critical role in the rate of estolide formation as well as in the overall yield, with higher temperatures providing faster rates but lower yields. The ratio of sulfuric acid to oleic acid equivalents also plays a role, where higher acid concentrations gave faster rates and higher yields of estolide. Vacuum had a minor effect on oligomerization and estolide yield.