Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

Synergistic role of HSP90α and HSP90ß to promote myofibroblast persistence in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90ß, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90ß was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-ß1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90ß stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.

Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT).

The human Na(+)/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(-)-enantiomers and the R,S-(+/-) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na(+). Under equilibrium conditions, hSMVT can simultaneously bind ~2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (Kd) of 0.9 and 7.4 µm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na(+) and exhibits an affinity of ~3 µm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (kcat) of about 1 s(-1), a value that is well in agreement with other Na(+)-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.

Cooperative involvement of zipper-interacting protein kinase (ZIPK) and the dual-specificity cell-division cycle 14A phosphatase (CDC14A) in vascular smooth muscle cell migration.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal adhesion assembly dynamics. ZIPK silencing can induce cytoskeletal remodeling with disassembly of actin stress fiber networks and coincident loss of focal adhesion kinase (FAK)-pY397 phosphorylation. The link between ZIPK inhibition and FAK phosphorylation is unknown, and critical interactor(s) and regulator(s) are not yet defined. In this study, we further analyzed the ZIPK-FAK relationship in VSMCs. The application of HS38, a selective ZIPK inhibitor, to coronary artery vascular smooth muscle cells (CASMCs) suppressed cell migration, myosin light chain phosphorylation (pT18&pS19) and FAK-pY397 phosphorylation as well. This was associated with the translocation of cytoplasmic FAK to the nucleus. ZIPK inhibition with HS38 was consistently found to suppress the activation of FAK and attenuate the phosphorylation of other focal adhesion protein components (i.e., pCas130, paxillin, ERK). In addition, our study showed a decrease in human cell-division cycle 14A phosphatase (CDC14A) levels with ZIPK-siRNA treatment and increased CDC14A with transient transfection of ZIPK. Proximity ligation assays (PLA) revealed CDC14A localized with ZIPK and FAK. Silencing CDC14A showed an increase of FAK-pY397 phosphorylation. Ultimately, the data presented herein strongly support a regulatory mechanism of FAK in CASMCs by a ZIPK-CDC14A partnership; ZIPK may act as a key signal integrator to control CDC14A and FAK during VSMC migration.

A highly selective Hsp90 affinity chromatography resin with a cleavable linker.

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.

A sulfone-based strategy for the preparation of 2,4-disubstituted furan derivatives.

2,4-Disubstituted furans are prepared by treating 2,3-dibromo-1-phenylsulfonyl-1-propene (DBP, 2) with 1,3-diketones under basic conditions. The furan-forming step involves a deacetylation, and the selectivity of this process depends upon the steric demand of the R group. The substituent in position 4 is elaborated by reaction of sulfonyl carbanions with alkyl halides, acyl halides, and aldehydes. Oxidative or reductive desulfonylation produces the 2,4-disubstituted furans in 60-92% yield. This strategy has been used to prepare rabdoketone A (12) and the naturally occurring nematotoxic furoic acid 13.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Analysis of extracted and volatile components in blackstrap molasses feed as candidate house fly attractants.

House flies are a ubiquitous insect that have the potential to spread many diseases to humans and livestock. Managing house fly populations is accomplished by having desirable baits, traps, and killing agents. Most house fly baits are designed for outdoor use or limited indoor use, and have a foul odor that is not conducive to food preparatory and dining areas. Blackstrap molasses has long been used as a house fly bait, but it is sticky and viscous, making it difficult to handle. This study sought to identify compounds present in blackstrap molasses that might be attractive to house flies, and therefore, provide the public with an indoor bait that does not have an offensive smell and is easy to handle. Indoor bioassays with house flies using 50% blackstrap molasses diluted in deionized water, a hexane extract of blackstrap molasses, and deionized water, elicited 86.2%, 70.6%, and 13.8% responses, respectively. Hexane and diethyl ether extracts of blackstrap molasses produced a large number of compounds with widely differing organic structures including substituted phenols, nitrogen and oxygen heterocycles, carboxylic acids, and many other organic compounds.

The plasma pharmacokinetics of R-(+)-lipoic acid administered as sodium R-(+)-lipoate to healthy human subjects.

BACKGROUND: The racemic mixture, RS-(+/-)-alpha-lipoic acid (rac-LA) has been utilized clinically and in a variety of disease models. Rac-LA and the natural form, R-lipoic acid (RLA), are widely available as nutritional supplements, marketed as antioxidants. Rac-LA sodium salt (NaLA) or rac-LA potassium salt (KLA) has been used to improve the aqueous solubility of LA. STUDY RATIONALE: Several in vitro and animal models of aging and age-related diseases have demonstrated efficacy for the oral solutions of LA salts in normalizing age-related changes to those of young animals. Other models and studies have demonstrated the superiority of RLA, the naturally occurring isomer over rac-LA. Despite this, RLA pharmacokinetics (PK) is not fully characterized in humans, and it is unknown whether the concentrations utilized in animal models can be achieved in vivo. Due to its tendency to polymerize, RLA is relatively unstable and suffers poor aqueous solubility, leading to poor absorption and low bioavailability. A preliminary study demonstrated the stability and bioavailability were improved by converting RLA to its sodium salt (NaRLA) and pre-dissolving it in water. The current study extends earlier findings from this laboratory and presents PK data for the 600-mg oral dosing of 12 healthy adult subjects given NaRLA. In addition, the effect of three consecutive doses was tested on a single subject relative to a one-time dosing in the same subject to determine whether plasma maximum concentration (Cmax) and the area under the plasma concentration versus time curve (AUC) values were comparable to those in animal studies and those achievable via intravenous infusions in humans. METHODS: Plasma RLA was separated from protein by a modification of a published method. Standard curves were generated from spiking known concentrations of RLA dissolved in ethanol and diluted in a phosphate-buffered saline (PBS) into each individual's baseline plasma to account for inter-individual differences in protein binding and to prevent denaturing of plasma proteins. Plasma RLA content was determined by the percent recovery using high-performance liquid chromatography (electrochemical/coulometric detection) (HPLC/ECD). RESULTS: As anticipated from the preliminary study, NaRLA is less prone to polymerization, completely soluble in water, and displays significantly higher Cmax and AUC values and decreased time to maximum concentration (Tmax) and T1/2 values than RLA or rac-LA. In order to significantly extend Cmax and AUC, it is possible to administer three 600-mg RLA doses (as NaRLA) at 15-minute intervals to achieve plasma concentrations similar to those from a slow (20-minute) infusion of LA. This is the first study to report negligible unbound RLA even at the highest achievable plasma concentrations.

Performance of painted plywood and cloth Nzi traps relative to Manitoba and greenhead traps for tabanids and stable flies.

Experiments were conducted to adapt the cloth Nzi trap to a format suitable for fixed applications in biting fly sampling or control. Catches of tabanids [Tabanus L., Chrysops (Meigen), and Hybomitra Enderlein], and stable flies [Stomoxys calcitrans (L.)] in painted plywood traps were compared with those in standard phthalogen blue cloth traps, and in similarly painted cloth traps. The Manitoba horse fly trap and the Tabanus nigrovittatus Macquart "greenhead" box trap were used as additional standards during one tabanid season. Shiny features of traps reduced catches, e.g., paint on cloth instead of wood, or use of aluminum screening instead of netting. Nevertheless, appropriately painted plywood Nzi traps caught as many biting flies as did standard cloth Nzi traps, if paint finishes were matte, and with the use of phthalogen blue colorants. Nzi traps collected about the same tabanid fauna as the Manitoba and T. nigrovittatus traps, but with improved catches of Chrysops and Tabanus. Recommendations are provided on appropriate color matching, and selection of readily-available materials for trap construction.

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease.

Tumor necrosis factor alpha (TNF-α) has both positive and negative roles in human disease. In certain cancers, TNF-α is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF-α antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNF-α-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNF-α stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNF-α-induced cell death, with general implications for cancer and autoimmune disease treatment.

Prevention of mosquitoes (Diptera: Culicidae) and house flies (Diptera: Muscidae) from entering simulated aircraft with commercial air curtain units.

Commercially available air curtain units were used to create air barriers to prevent mosquitoes and house flies from entering a simulated aircraft doorway together with passengers. Two assemblies of simulated passenger bridge and aircraft were constructed, and airflow measurements were recorded to confirm airflow characteristics for several combinations of commercial units. Three mosquito species were selected for different host-seeking characteristics, and house flies were selected to represent a large, strong-flying insect. Batches of 20 or 200 insects of four species were released into the passenger bridge just before 25 persons passed through the assembly, then insects that entered the aircraft cabin were recovered. Results showed that horizontal plus vertical or vertical-mounted air curtain units with the airflow directed at a 45 degrees angle into the passenger bridge excluded 95-99% of the mosquitoes and 95-100% of the house flies, respectively. Airflows were measured and estimated to be effective if the mean was > 4 m/s in the critical area in the center of the converging airflows. The study validates the concept that air barriers can effectively prevent the passage of flying insects into an aircraft.

Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2(+) Breast Cancer.

Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer, particularly when combined with carboplatin.

Development of granular boric acid sugar baits for house flies (Diptera: Muscidae).

Freeze-dried, granular sugar baits containing boric acid as the toxicant were evaluated against house flies, Musca domestica L., in two bioassays with toxicant dose ranges of 3, 5, 7, 8, and 12%, and 3, 5, 9, 17, and 33% boric acid, respectively, calculated and expressed on a dry weight basis. Flies in bioassay 1 were mixed sex and unstarved and flies in bioassay 2 were females only and starved 4 h before testing began. LC50s were 8.97 and 14.33%, and LT50s were 59.75 and 53.34 h in bioassays 1 and 2, respectively. Baits were tested at concentrations as high as 33%, however there was no indication of repellency as seen previously with liquid baits. Reasons for lack of repellency and potential uses for baits are discussed. Development of efficacious granular baits would allow the presentation of higher levels of boric acid in volumes much smaller than required for liquid baits.

Free-swinging attention.

Psychoanalytic listening enlists the analyst's capacity for, and relative comfort in, rapidly shifting levels of attention and organization. Such shifts are not effortless and can be characterized as part of "free-swinging attention," a term that suggests some dimensions of the analyst's work. The need to establish meaning in the individual and immediate context parallels the task of a child in learning language, and the role of the analyst as child is an important if usually overlooked one. The author compares psychoanalytic with psychotherapeutic listening, as well as some current views on free association and evenly suspended attention.

Effects of aspirin & simvastatin and aspirin, simvastatin, & lipoic acid on heme oxygenase-1 in healthy human subjects.

BACKGROUND: Heme oxygenase 1 (HO-1) degrades heme and protects against oxidative stress. In vitro and animal models suggest that HO-1 is beneficial in several diseases (e.g., postoperative ileus, gastroparesis, acute pancreatitis, and colitis). However, the only drugs (i.e., hemin and heme arginate) which pharmacologically upregulate HO-1 in humans are expensive and can only be administered intravenously. Our aims were to compare the effects of placebo, aspirin, and simvastatin alone, and with α-lipoic acid, on HO-1 protein concentration and activity in humans. METHODS: This randomized, double-blind, placebo-controlled study compared the effects of three oral regimens administered for 7 days, i.e., placebo; aspirin (325 mg twice daily) and simvastatin (40 mg twice daily); aspirin, simvastatin, and the sodium salt of R- α-lipoic acid (NaRLA, 600 mg three times daily) on markers of HO-1 activation (i.e., plasma HO-1 protein concentration and venous monocyte HO-1 protein activity) in 18 healthy subjects (14 females). Markers of HO-1 activation were evaluated at baseline, days 2, and 7. KEY RESULTS: Baseline HO-1 protein concentrations and activity were similar among the three groups. Compared to placebo, aspirin and simvastatin combined, or together with NaRLA did not affect HO-1 protein concentration or activity at 2 or 7 days. HO-1 protein concentrations and activity were correlated on day 7 (r = 0.75, p = 0.0004) but not at baseline and on day 2. CONCLUSIONS & INFERENCES: At therapeutic doses, aspirin, simvastatin, and α-lipoic acid do not increase plasma HO-1 protein concentration or venous monocyte HO-1 activity in healthy humans.