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Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor β1, driving expansion of CD44v6 + epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial–mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

The operation of Active Clamp circuit is presented for an Isolated DC/AC Converter based on the principle of Pulsating DC link (PDL). This innovative two stage converter, made of a Phase Shifted Full Bridge and a Three Phase Inverter, is featured by the pulsating evolution of the voltage on the intermediate DC link resulting from the absence of the output filter of the first stage. The zero voltage phases of the PDL are used to achieve the Zero Voltage Transition (ZVT) for all the switches of the second stage. The Active Clamp circuit is used to clamp the voltage overshoots on the Pulsating DC link due to the parasitic inductance which resonates with stray capacitance of the converter. Moreover, the circuit permits the storage of the energy associated with the overvoltage and its restitution to the DC-link during the subsequent energizing phases. The role of the Active Clamp capacitor is discussed on the basis of a comprehensive simulation analysis and experimental data obtained from a 5kW prototype.

Synthetic hydrogels formed from poly(ethylene glycol) (PEG) are widely used to study how cells interact with their extracellular matrix. These in vivo-like 3D environments provide a basis for tissue engineering and cell therapies but also for research into fundamental biological questions and disease modeling. The physical properties of PEG hydrogels can be modulated to provide mechanical cues to encapsulated cells; however, the impact of changing hydrogel stiffness on the diffusivity of solutes to and from encapsulated cells has received only limited attention. This is particularly true in selectively cross-linked "tetra-PEG" hydrogels, whose design limits network inhomogeneities. Here, we used a combination of theoretical calculations, predictive modeling, and experimental measurements of hydrogel swelling, rheological behavior, and diffusion kinetics to characterize tetra-PEG hydrogels' permissiveness to the diffusion of molecules of biologically relevant size as we changed polymer concentration, and thus hydrogel mechanical strength. Our models predict that hydrogel mesh size has little effect on the diffusivity of model molecules and instead predicts that diffusion rates are more highly dependent on solute size. Indeed, our model predicts that changes in hydrogel mesh size only begin to have a non-negligible impact on the concentration of a solute that diffuses out of hydrogels for the smallest mesh sizes and largest diffusing solutes. Experimental measurements characterizing the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran molecules of known size aligned well with modeling predictions and suggest that doubling the polymer concentration from 2.5% (w/v) to 5% produces stiffer gels with faster gelling kinetics without affecting the diffusivity of solutes of biologically relevant size but that 10% hydrogels can slow their diffusion. Our findings provide confidence that the stiffness of tetra-PEG hydrogels can be modulated over a physiological range without significantly impacting the transport rates of solutes to and from encapsulated cells.

The effects of the CF2H moiety on H-bond acidity and lipophilicity of various compounds, when attached directly to an aromatic ring or to other functions like alkyls, ethers/thioethers or electron withdrawing groups, are discussed. It was found that the CF2H group acts as a HB-donor with a strong dependence on the attached functional group (A = 0.035-0.165). Regarding the lipophilicity, the CF2H group may acts as a more lipophilic bioisostere of OH but as a similar or less lipophilic bioisostere of SH and CH3, respectively, when attached to Ar or alkyl. In addition, the lipophilicity of ethers, sulfoxides and sulfones is dramatically increased upon CH3/CF2H exchange at the  position. Interestingly, this exchange significantly affects not only the polarity and the volume of the solutes but also their HB accepting ability, the main factors influencing log Poct. Accordingly, this study may be helpful in the rational design of drugs containing this moiety.

A novel modulation technique is presented for an Isolated DC/AC multistage Converter, based on Pulsating DC Link principle. In this converter, the absence of a filter at the output of the first stage provides high reliability features, fast dynamic response and Zero Voltage Transitions (ZVT) of the secondary stage switches thanks to the zero voltage time intervals of the pulsating DC link. The proposed modulation techniques allows the use of two different switching frequencies for the two converter stages in such a way that each stage can be modulated with the best switching frequency to improve the efficiency, dynamic performances and converter size. Moreover, the modulation technique permits the optimization of the ratio between the switching frequencies of the two power stages which is fundamental to avoid that the pulsating DC link can compromise the Total Harmonic Distortion THD of the output voltage. An exhaustive description of the presented modulation logic is provided, highlighting the benefits due to the increase of the switching frequency of the first stage. The synchronization between the power stages is also used to guarantee the ZV commutations of the second stage, during the freewheeling phases of the first stage with consequent reduction of losses. Furthermore the effects of the predistortion of modulating signals on the distortion of output voltages are discussed and the simulations results are presented. The experimental results, obtained by the characterization of 20kW prototype, are presented and discussed to validate the features of the proposed modulation logic.

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the &ldquo, drug-like&rdquo, Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1&ndash, 4 &mu, M. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased &beta, catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases.

Small-molecule detection is important for many applications including clinical diagnostics, drug discovery, environmental screening, and food technology. Current techniques suffer from various limitations including cost, complex sample processing, massive instrumentation, and need for expertise. To overcome these limitations, a new optical immunosensing assay for the detection of small molecules was developed and assessed with the targets estrone (E1) and estradiol (E2). For this purpose, phosphorescent indicators were designed based on the tetrabenzoporphyrin skeleton directly linked to E1 or E2, or attached through a linker, with phosphorescence lifetimes in the range of ~100–~300 μs. The assay is an indicator displacement assay (IDA). The best performances of our optical immunosensor were obtained with the indicators E1-L-Por and E2-L-Por. As they bound to specific polyclonal antibodies, their phosphorescence (τ ~200 μs) was quenched. When an endogenous competitor was added, the indicator was displaced, and the phosphorescence was immediately recovered. These effects were measured with a new optical device, described here, and able to detect picograms of luminescent molecules emitting in the NIR range, simply by measuring phosphorescence decay. This radical switch-off/switch on process demonstrates that E1-L-Por and E2-L-Por are good candidates for in vivo and in vitro immunosensing of E1 and E2. Importantly, the present immunosensing assay can be easily adapted to other small molecules such as other hormones and drugs.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we developed gut organoid co-cultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate for the first time that murine and human ILC1 secrete TGFβ1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

An isolated DC/AC converter based on Pulsating DC Link principle is presented. It includes a H-bridge stage, operated in Phase Shift mode (PS), feeding the primary winding of the insulating transformer, followed by a full-wave rectifier, fed by the secondary winding of the transformer. The rectifier is connected to a pulsating DC link which feeds a three phase inverter stage. The switches of the inverter are turned on and off during the free-wheeling phase of the PS-H-bridge when the voltage on the dc-link is zero allowing the switches of the inverter stage to commutate in fully ZV conditions. Due to the absence of a large dc-link capacitor, an active-clamp network provides a clamp of the voltage across the inverter stage. An FPGA based modulation logic is also presented. It is conceived to perform the change of the pattern during the free-wheeling phase of the first stage. The proposed topology is typically aimed to convert a DC power to a line-frequency AC power, as required, for example, in photovoltaic applications. This solution allows us to obtain at the same time: galvanic isolation, reduction in weight and cost of the transformer in comparison with standard inverter solutions which adopt a line-frequency transformer, and high overall efficiency due to soft-switching operations of each switch of the circuit.

In this paper we present the operation principle of a Metal Oxide Vacuum Field Effect Transistor (MOVFET). The idea is to exploit a technology proposed in the literature to realize nano devices with low threshold voltage for power applications. The principle of operation of the MOVFET is based on the emission of electrons from a cold cathode in a vacuum in such a way to obtain a pentode-like characteristics with a very low on resistance. This last feature is particularly interesting for the application as switches in power electronics applications. Moreover, the absence of a gate oxide which is always present in traditional MOSFETs suggests that this device can have better reliability features even in rad hard applications. A numerical simulator is used to evaluate the impact of the technological parameters on the static characteristics of the device and the perspective performances in power electronics applications.

An exhaustive analysis of the evolution of current in the input power stage of insulated multistage DC/AC power converter based on the Pulsating DC link principle, characterized by Zero Voltage Transition (ZVT) of the output stage, is provided in this work. This topology is featured by an input power stage which provides on the second stage a pulsed voltage characterized by zero phases (pulsating DC link) which are used to achieve the ZVT conditions for the commutations of this stage. The specific and innovative topology of this converter influences the evolution of the main electrical characteristics and requires more attention on the design. The parameters that affects the behavior of the current in this particular topology, especially in the crucial working phases, are identified and their influence are analyzed to define the constraints of this system. A complete study of the current has been accomplished by LTSPICE simulator for different combination of leakage parameters of the architecture and switching frequency of the two power stages. The obtained results are presented and discussed to provide the limits of the design of the main components that compose this particular power converter. For the experimental validation of the results of the simulation, a 3.5kW prototype of the power converter was assembled and tested at different load conditions.

The objective of the paper is to present a trap assisted conduction mechanism able to explain the creation of a conductive path in the gate oxide of SiC power MOSFET during the impact of heavy ions. The consequent large current flow through the oxide can induce damages to the gate structure. The proposed mechanism combines a Fowler-Nordheim tunneling at the SiC/SiO2 interface with a trap-assisted valence band conduction mechanisms based on Poole-Frenkel effect. The model is based on the results of 2D finite element simulation and is supported by previous works dealing with trap assisted tunneling hole injection in silicon dioxide.

The effective difluoromethylations of various S- and O- based- nucleophiles, presenting a wide range of pKa values, using diethyl(bromodifluoromethyl) phosphonate (1) under basic conditions is described. These reactions, which rely on the quantitative generation of difluorocarbene formed through the hydrolysis of 1, were found to be effective only once the starting materials had pKa values of less than ca. 11. Importantly, in cases in which the substrates held two or three nucleophilic centers this feature was successfully implemented to achieve a high chemo- or regioselective difluoromethylation of the center exhibiting the lowest pKa value and the highest polarizability.

The effects of the CF

Phosphonate and/or bisphosphonate-functionalized mesoporous silica (MS) sorbents were developed and studied using 31P-MAS-NMR, SEM-EDS, TEM, BET, and batch sorption experiments before and/or after metal ion scavenging (Bi+3, Ba+2, Cd+2, U+6, Ag+, Pd+2 and Zn+2). The amount of phosphorus inside the sorbents was ascertained by SEM-EDS, and the phosphonate vs bisphosphonate content derived from NMR. The organic matter loading was between 15 and 38 %w/w and inversely correlated with BET. Pore size (4.9–6.6 nm) and surface areas (219–473 m2/g) were derived from nitrogen sorption isotherms. The NMR signals of the phosphonates are shifted downfield by the metal cations while the bisphosphonate signals are shifted upfield for urea-derived sorbents and downfield for the amino sorbent. The sorbents are composed of prolate particles of micrometric dimensions. The prolate particles of the urea-derived sorbents are stable following the adsorption of metal cations, while they are disrupted in the case of the amino sorbent, due to the repulsion between negative charges at basic pH. Only metal cations with relatively large ionic diameters were adsorbed revealing the need for multi-point interactions inside the sorbent, which is not possible for smaller cations. For all the sorbents, the sorption data of uranyl fit the Freundlich isotherm model, which predicts a heterogeneous surface. The K and Kd values derived from this model show good sorption capabilities. Kds for metal cation solutions (20 μg/mg) on the amino sorbent were between 500 and 35000 mL/g. Both selectivity and sorption capabilities of the sorbents allow their use in environmental analysis and remediation.

The effects solvents have on fluoride-promoted heterogeneous hydrolysis and alcoholysis of various organo-phosphorus (OP) compounds on the surface of KF/Al2O3 are described. Solid-state magic angle spinning NMR analyses and SEM microscopy have shown that not only is the identity of the solvent important in these reactions but also its quantity. That is, minimal solvent amounts are favored and much more effective in such solid-supported reactions (and maybe generally) than those featuring solvent-free or excess solvent (50 wt %) conditions. The addition of a minute quantity of the correct solvent (3-10 wt %, molar equivalent scale) avoids reagents leaching from the matrix, permits mobility (mass transport) of the reaction components and ensures their very high local concentration in close proximity to the solid-support large porous surface area. Accordingly, significant acceleration of reactions rates by orders of magnitude is obtained. Fascinatingly, even challenging phosphoesters with poor leaving groups, which were found to be very stable in the presence of solvent-free KF/Al2O3 or wetted with excess water, were efficiently hydrolyzed with a minute amount of this solvent.

There is a growing interest in organic compounds containing the difluoromethyl group, as it is considered a lipophilic hydrogen bond donor that may act as a bioisostere of hydroxyl, thiol, or amine groups. A series of difluoromethyl anisoles and thioanisoles was prepared and their druglike properties, hydrogen bonding, and lipophilicity were studied. The hydrogen bond acidity parameters A (0.085–0.126) were determined using Abraham’s solute 1H NMR analysis. It was found that the difluoromethyl group acts as a hydrogen bond donor on a scale similar to that of thiophenol, aniline, and amine groups but not as that of hydroxyl. Although difluoromethyl is considered a lipophilicity enhancing group, the range of the experimental Δlog P(water–octanol) values (log P(XCF2H) – log P(XCH3)) spanned from −0.1 to +0.4. For both parameters, a linear correlation was found between the measured values and Hammett σ constants. These results may aid in the rational design of drugs containing the difluoromethyl moiety.

Following our ongoing studies on the reactivity of the fluoride ion toward organophosphorus compounds, we established that the extremely toxic and environmentally persistent chemical warfare agent VX (O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothioate) is exclusively and rapidly degraded to the nontoxic product EMPA (ethyl methylphosphonic acid) even in dilute aqueous solutions of fluoride. The unique role of the P-F bond formation in the reaction mechanism was explored using both experimental and computational mechanistic studies. In most cases, the "G-analogue" (O-ethyl methylphosphonofluoridate, Et-G) was observed as an intermediate. Noteworthy and of practical importance is the fact that the toxic side product desethyl-VX, which is formed in substantial quantities during the slow degradation of VX in unbuffered water, is completely avoided in the presence of fluoride. A computational study on a VX-model, O,S-diethyl methylphosphonothioate (1), clarifies the distinctive tendency of aqueous fluoride ions to react with such organophosphorus compounds. The facility of the degradation process even in dilute fluoride solutions is due to the increased reactivity of fluoride, which is caused by the significant low activation barrier for the P-F bond formation. In addition, the unique nucleophilicity of fluoride versus hydroxide toward VX, in contrast to their relative basicity, is discussed. Although the reaction outcomes were similar, much slower reaction rates were observed experimentally for the VX-model (1) in comparison to VX.

The catalytic degradation of the nerve agent VX (O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothioate) by water-swelled polymer-supported ammonium fluorides is described. VX (0.06-0.53 mol/mol F(-)) is rapidly degraded (t(1/2) ∼ 10-30 min) to form the "G-analogue" (O-ethyl methylphosphonofluoridate), which hydrolyzes (t(1/2) ∼ 1-1.5 h) to the nontoxic EMPA (ethyl methylphosphonic acid). The toxic desethyl-VX is not formed. The catalytic effect of fluoride is maintained even when 6 equiv of VX are loaded. GB (O-isopropyl methylphosphonofluoridate) and desethyl-VX agents are also degraded under these conditions.

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG-q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 10(3) bacteria per ml in the sample.

Ras proteins play a role in receptor-mediated signaling pathways and are activated after traumatic brain injury. S-trans-trans-farnesylthiosalicylic acid (FTS), a synthetic Ras inhibitor, acts primarily on the active, GTP-bound form of Ras and was shown to improve neurobehavioral outcome after closed head injury (CHI) in mice. To gain a better understanding of the neuroprotective mechanism of FTS, we used diffusion-weighted imaging (DWI) in a rat model of CHI. Apparent diffusion coefficients (ADC) and transverse relaxation times (T2) were measured in injured rat brains after treatment with vehicle or FTS (5 mg/kg). Neuroprotection by FTS was also assessed in terms of the neurological severity score. One week after injury, significantly better recovery was observed in the FTS-treated rats than in the controls (p = 0.0191). T2 analysis of the magnetic resonance images revealed no differences between the two groups. In contrast, they differed significantly in ADC, particularly at 24 h post-CHI (p0.05): in the vehicle-treated rats ADC had decreased to approximately 26% below baseline, whereas it had increased to about 10% above baseline in the FTS-treated rats. As the magnitude of ADC reduction is strongly linked to blood perfusion deficit, these results suggest that the neuroprotective mechanism of FTS might be related to an improvement in cerebral perfusion. We propose that FTS, which is currently being tested in humans for anti-cancer indications, should also be considered as a new strategy for the management of head injury.

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C15 and C20 S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras–membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimer's disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimer's disease.

The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming . activities. We showed previously that S-farnesylthiosalicylic acid FTS affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated . protein kinase ERK , of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue 20 was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C S-geranyl derivative of thiosalicylic acid, farnesol, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne- 10 sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C farnesyl group linked to a rigid backbone seems to be necessary and 15 sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substituents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane. q 1998 Elsevier Science B.V. Abbreviations: AFC, N-acetyl-S-farnesyl-L-cysteine; 5-Cl-FTS, S-farnesyl-5-Cl-thiosalicylic acid; 4-Cl-FTS, S-farnesyl-4-Cl-thiosali- cylic acid; 3-Cl-FTS, S-farnesyl-3-Cl-thiosalicylic acid; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulfoxide; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; 5-F-FTS, S-farnesyl-5-fluoro-thiosalicylic acid; 4-F-FTS, S-farnesyl-4-fluoro- thiosalicylic acid; 3-F-FTS, S-farnesyl-3-fluoro-thiosalicylic acid; FTP, farnesylthiopropionic acid; FTS, S-farnesyl thiosalicylic acid; FTS-Me, S-farnesyl thiosalicylic acid carboxyl methyl ester; GTS, S-geranyl thiosalicylic acid; GGTS, S-geranylgeranyl thiosalicylic acid; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PPMTase, prenylated protein methyltransferase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

Inhibitors of a prenylated protein methyltransferase were synthesized and evaluated. S-farnesyl-5-fluorothiosalicylic acid and the 5-chloro analog (but not the 4-fluoro, 4-chloro or 3-chloro analogs) were potent inhibitors, as was the parent compound S-farnesyl thiosalicylic acid (FTS), whose methyl ester was far less active. S-geranyl and S-geranylgeranyl thiosalicylic acids were more than ten times less potent than FTS.

The processing or ras and of other GTP-binding proteins includes a final reversible step in which the carboxy terminal prenylated cysteine is methylated by the enzyme prenylated protein methyltransferase (PPMTase). The significance of this modification and of the role of PPMTase in human tumors has yet to be fully elucidated. Here we characterize the PPMTase of human endometrical carcinomas (tumors in which the frequency of ras gene mutations is relatively high) and compare it to the PPMTase of the normal endometrium. Our results show that in both types of tissues the enzyme is bound to the membranes. It can utilize synthetic substrate such as N- acetyl-S-farnesyl- l -cysteine (K m = 18–20 μ M ) and is blocked by the PPMTase inhibitor S-farnesylthioacetic acid (Ki = 2 μM). In vitro methylation assays and [α-32P]GTP blot-overlay assays showed that the major endogenous PPMTase substrates are small GTP-binding proteins. Methylation of these proteins in vitro is blocked by farnesylthioacetic acid. The kinetic properties of PPMTase from the carcinomas and the normal tissues are very similar. however, levels of PPMTase activity (but not of its endogenous substrates) are higher in the carcinomatous endometrium than in the normal one. The elevated enzyme activity is restricted to the crude mitochondrial fraction (8.0 ± 0.4 vs. 5.4 ± 0.1 pmol N-acetyl farnesylcysteine methyl ester formed/min/mg protein by the carcinoma and by the normal endometrial preparations, respectively). As this fraction is enriched in plasma membranes, it appears that the elevated enzyme activity could be related to ras protein methylation; if so, selective methylation blockers might inhibit the growth of endometrial carcinomas.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. Notably, the selectivity of the new m1 agonists is reflected also by activation of select second messenger systems via distinct G-proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand-selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Sulfur extraction from the tripeptide thiol, glutathione (α-glu-cys-gly) (1) via reaction with syn-(1-bromo-ethyl, methyl)bimane (2) yields glutathione slfide and the thiabridged bimane, μ(S)-syn-(methylmethylene, methyl)bimane (3) [1]. The reaction with 2 has been extended to dithiols as models for important biological thiols such as reduced trypanothione. The fluorescent dithiols were derived through reductive cleavage with triphenylphosphine (tetrahydrofuan, Hcl−Kbr solution, pH 1.5) of the dithiatriclic bimane esters, μ(O2C(CH2)SS(CH2)nCO2)-syn-(CH2,CH3)B, n = 1, 2, 3, prepared from syn-(bromomethyl, methyl)bimane (4) and the corresponding dithiadicarboxylic acids. Sulfur extraction led to 3 and the cyclic sulfide derived from the dithiol in moderate yields. The dithiols, dithiothreitol and dithioerythritol, also yielded moderate amounts of 3. Sterically hindered thiols (e.g., those in hemoglobin) gave 3 in lower yields. Treatment of human red blood cells and red cells membranes (hemoglobin-free ghosts) with 2 gave rise to some 3. A side product in some reactions was the oxabridged bimane, μ(O)-syn-(methylmethylene, methyl)bimane.

Inhibitors of a prenylated protein methyltransferase were synthesized and evaluated. S-farnesyl-5-fluorothiosalicylic acid and the 5-chloro analog (but not the 4-fluoro, 4-chloro or 3-chloro analogs) were potent inhibitors, as was the parent compound S-farnesyl thiosalicylic acid (FTS), whose methyl ester was far less active. S-geranyl and S-geranylgeranyl thiosalicylic acids were more than ten times less potent than FTS.

Rigid analogs of acetylcholine offer an opportunity for selective actions at muscarinic receptor subtypes, since restricted conformational mobility alters the capacity of ligands to adapt to subtle differences in receptor structure. AF102B , a highly rigid analog of acetylcholine, is a centrally active M1 agonist and is evaluated in light of some currently available therapeutic strategies in Alzheimer's disease.

Sulfur extraction from the tripeptide thiol, glutathione (α-glu-cys-gly) (1) via reaction with syn-(1-bromo-ethyl, methyl)bimane (2) yields glutathione slfide and the thiabridged bimane, μ(S)-syn-(methylmethylene, methyl)bimane (3) [1]. The reaction with 2 has been extended to dithiols as models for important biological thiols such as reduced trypanothione. The fluorescent dithiols were derived through reductive cleavage with triphenylphosphine (tetrahydrofuan, Hcl−Kbr solution, pH 1.5) of the dithiatriclic bimane esters, μ(O2C(CH2)SS(CH2)nCO2)-syn-(CH2,CH3)B, n = 1, 2, 3, prepared from syn-(bromomethyl, methyl)bimane (4) and the corresponding dithiadicarboxylic acids. Sulfur extraction led to 3 and the cyclic sulfide derived from the dithiol in moderate yields. The dithiols, dithiothreitol and dithioerythritol, also yielded moderate amounts of 3. Sterically hindered thiols (e.g., those in hemoglobin) gave 3 in lower yields. Treatment of human red blood cells and red cells membranes (hemoglobin-free ghosts) with 2 gave rise to some 3. A side product in some reactions was the oxabridged bimane, μ(O)-syn-(methylmethylene, methyl)bimane.

Conversion of the dibromo compound, 4,6-bis(bromomethyl)-3,7-dimethyl- 1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione (syn-(BrCH 2 ,CH 3 )B) or the related 3,7-dichloro derivative (syn-(BrCH 2 ,Cl)B) to a 4,6-sulfone-bridged bimane via oxidation of a 4,6-thiatrimethylene derivative is described. Elimination of SO 2 via thermolysis of the sulfones at 525 o C in a quartz reactor yields the strained tricyclic bimanes containing a 4,6-dimethylene group («zero-bridge») μ-0-syn-(CH 2 ,CH 3 )B (3e) and μ-0-syn-(CH 2 ,Cl)B (3a)

An effective synthesis of tricyclic bimanes (μ(C n )-syn-(CH 2 ,R 1 )B, R 1 =CH 3 or Cl)) (3a-g, n=1,2,3) from bis-acid chlorides via bis-3-keto esters and bis-pyrazolinones is described. The success of the synthesis is strong support for the previously proposed mechanism of bimane formation, via a diazacyclopentadienone and a diazoketene. Crystal structures for three chloro derivatives (3b-d, n=1,2,3) reveal that the bimane dihedral ring angle and crystal packing details vary with the size of the third ring

Syntheses and useful reactions of syn-vinylbimanes are reported. The precursors are syn-(ethyl,methyl)bimane (4,6-diethyl-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione) (syn-(CH 3 CH 2 ,CH 3 )B) (2) and syn-(ethyl,chloro)bimane (4,6-diethyl-3,7-dichloro-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione) (syn-(CH 3 CH 2 ,Cl)B) (4)

Merkel cell carcinoma (MCC) is a neuroendocrine malignancy showing poor response to a variety of therapeutic strategies. We evaluated the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a new inhibitor of Ras signal transduction, in a newly established SCID mouse xenotransplantation model for human MCC (seven animals per group). FTS injected intraperitoneally at 5 mg/kg per day for 2 weeks up-regulated the tumor suppressor p53 and induced tumor cell apoptosis in established MCCs growing subcutaneously in SCID mice. These effects led to a statistically significant inhibition of MCC growth (P0.002). The mean tumor weights following FTS or control treatment were 0.32+/-0.15 g and 1.08+/-0.29 g, respectively. There was no evidence of FTS related toxicity at the effective dose used. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for MCC and possibly for other tumors that rely on tyrosine kinase signaling.

Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans, trans-farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.

Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.

Inhibition of Ras-dependent signaling and of oncogenic Ras function by farnesyl transferase inhibitors that block Ras membrane anchorage is limited due to alternative prenylation of Ras. Here we demonstrate that inhibition of the Ras-dependent Raf-1-MAPK (mitogen activated protein kinase) cascade is achieved by S-farnesylthiosalicylic acid (FTS) which affects Ras membrane association but not Ras farnesylation. FTS interferes with the activation of Raf-1 and MAPK and inhibits DNA synthesis in Ras-transformed EJ cells at concentrations similar to those at which it inhibits EJ cell growth (5-25 microM). FTS also inhibits MAPK activity and DNA synthesis stimulated by serum, EGF or thrombin in serum-starved untransformed Rat-1 cells, demonstrating the generality of its effects on Ras-dependent signaling. The effects of FTS on MAPK activity developed relatively rapidly (within 2-6 h) consistent with its rapid effect on Ras membrane anchorage. FTS represents a new class of Ras antagonists that may be useful for the inhibition of various types of oncogenic Ras isoforms independently of their prenylation.

Restoration of ACh levels or replacement with an M1 (or m1) muscarinic agonist may be effective in treating at least some of the cognitive symptoms in Alzheimer’s disease (AD), (see Court and Perry, 1991; Fisher and Barak, 1994). Treatment approaches for AD have to address abnormalities occurring also along various signal transduction pathways (Harrison et al., 1991). Novel activities associated with m1 muscarinic receptors (m1 mAChR) indicate that m1 agonists may also activate hypofunctional signalling pathways in AD (Fisher and Barak, 1994; Gurwitz et al., 1994).

We report on the first secondary and tertiary complexes of the pillar[5]arene derivative 3 with xenon in water. We show that the chemical shift of the encapsulated xenon provides information on the type of the formed complex suggesting that 3 has the potential to be used as a platform for NMR biosensors.

To date five structurally different human muscarinic acetylcholine receptor (mAChR) subtypes (ml-m5) proteins have been cloned and expressed in suitable cell systems (Bonner et al., 1987). It is likely that the ml, m2, m3, and m4 AChRs fit the pharmacological definition of the M1, M2, M3 and M4 AChRs, respectively (Buckley et al., 1989; reviewed by Hulme et al., 1990). Muscarinic receptors are members of the G-protein coupled receptor superfamily. The mAChRs have two binding domains, a ligand-binding extracellular (and including membrane-spanning) domain and a G-protein binding intracellular domain. This second domain, by interaction with various G-proteins, controls and modulates second messenger systems. It was shown that the ml, m3 and m5 AChRs are closely related in sequence and apparently are functionally almost similar. When expressed in mammalian cells, these receptor subtypes couple efficiently to phosphoinositide (PI) turnover. The m2 and m4 AChRs are less related to the ml, m3 and m5 AChRs, and when expressed in mammalian cells are efficiently coupled to the inhibition of adenylate cyclase (Bonner et al., 1987; Hulme et al., 1990).

Secretion of amyloid precursor protein (APP) by cultured cells is coupled to several receptors, including m1 muscarinic (m1AChR), and is associated with decreased production of βA4 amyloid. Secreted and cell-associated APP levels were measured in m1AChR-transfected PC12 cells stimulated with the non-selective agonist carbachol or the Mi-selective agonist, AF102B. Secreted APP levels following stimulation with AF102B (5-60 min) were about half compared with carbachol. Yet, following 24 h stimulation with carbachol or AF102B, cell-associated APP levels were similarly decreased. This may be associated with a smaller reduction in APP secretion following 24 h stimulation with AF102B as compared with carbachol. AF102B may therefore have an advantage over non-selective muscarinic ligands for sustained decrease of cell-associated APP.