Abstract: The diverse physiological functions of histamine are mediated through distinct histamine receptors. Mast cells are major producers of histamine, yet effects of histamine on mast cells are currently unclear. The present study shows that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation. Mast cell chemotaxis toward histamine could be blocked by the dual H3/H4 receptor antagonist thioperamide, but not by H1 or H2 receptor antagonists. This chemotactic response is mediated by the H4 receptor, because chemotaxis toward histamine was absent in mast cells derived from H4 receptor-deficient mice but was detected in H3 receptor-deficient mast cells. In addition, Northern blot analysis showed the expression of H4 but not H3 receptors on mast cells. Activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores. Both G alpha i/o proteins and phospholipase C (PLC) are involved in histamine-induced calcium mobilization and chemotaxis in mast cells, because these responses were completely inhibited by pertussis toxin and PLC inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). In summary, histamine was shown to mediate signaling and chemotaxis of mast cells via the H4 receptor. This mechanism might be responsible for mast cell accumulation in allergic tissues.

Abstract: A shape-based Gaussian docking function is constructed which uses Gaussian functions to represent the shapes of individual atoms. A set of 20 trypsin ligand-protein complexes are drawn from the Protein Data Bank (PDB), the ligands are separated from the proteins, and then are docked back into the active sites using numerical optimization of this function. It is found that by employing this docking function, quasi-Newton optimization is capable of moving ligands great distances [on average 7 A root mean square distance (RMSD)] to locate the correctly docked structure. It is also found that a ligand drawn from one PDB file can be docked into a trypsin structure drawn from any of the trypsin PDB files. This implies that this scoring function is not limited to more accurate x-ray structures, as is the case for many of the conventional docking methods, but could be extended to homology models.

Abstract: Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.

Abstract: Purpose. We assessed the application of water-soluble polymer-based nanofibers prepared by electrostatic spinning as a means of altering the dissolution rate of the poorly water-soluble drug, itraconazole.

Abstract: Neuropathic pain is a common and often incapacitating clinical problem for which little useful therapy is presently available. Painful peripheral neuropathies can have many etiologies, among which are trauma, viral infections, exposure to radiation or chemotherapy, and metabolic or autoimmune diseases. Sufferers generally experience both pain at rest and exaggerated, painful sensitivity to light touch. Spontaneous firing of injured nerves is believed to play a critical role in the induction and maintenance of neuropathic pain syndromes. Using a well characterized nerve ligation model in the rat, we demonstrate that hyperpolarization-activated, cyclic nucleotide-modulated (HCN) “pacemaker” channels play a previously unrecognized role in both touch-related pain and spontaneous neuronal discharge originating in the damaged dorsal root ganglion. HCN channels, particularly HCN1, are abundantly expressed in rat primary afferent somata. Nerve injury markedly increases pacemaker currents in large-diameter dorsal root ganglion neurons and results in pacemaker-driven spontaneous action potentials in the ligated nerve. Pharmacological blockade of HCN activity using the specific inhibitor ZD7288 reverses abnormal hypersensitivity to light touch and decreases the firing frequency of ectopic discharges originating in Aβ and Aδ fibers by 90 and 40%, respectively, without conduction blockade. These findings suggest novel insights into the molecular basis of pain and the possibility of new, specific, effective pharmacological therapies.

Abstract: IMPLICATIONS: That IL-6 is an interesting target in the study of pain is underscored by its biomolecular properties, its localization after experimental pain, and its modulating effect on pain after administration.

Abstract: Although it is well recognized that human platelet responses to alpha-thrombin are mediated by the protease-activated receptors PAR-1 and PAR-4, their role and relative importance in platelet-dependent human disease has not yet been elucidated. Because the expression profile of PARs in platelets from nonprimates differs from humans, we used cynomolgus monkeys to evaluate the role of PAR-1 in thrombosis. Based on reverse transcription-polymerase chain reaction, PAR expression in platelets from cynomolgus monkeys consisted primarily of PAR-1 and PAR-4, thereby mirroring the profile of human platelets. We probed the role of PAR-1 in a primate model of vascular injury-induced thrombosis with the selective PAR-1 antagonist (alpha S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide (RWJ-58259). After pretreatment with RWJ-58259 or vehicle, both carotid arteries of anesthetized monkeys were electrolytically injured and blood flow was monitored for 60 min. Time to occlusion was significantly extended after RWJ-58259 administration (27 +/- 3 to 53 +/- 8 min; p < 0.048). Vessels from three of the five treated animals remained patent. Ex vivo platelet aggregation measurements indicated complete PAR-1 inhibition, as well as an operational PAR-4 response. Immunohistochemical staining of mural thrombi with antibodies to the platelet marker CD61 and fibrinogen indicated that RWJ-58259 significantly reduced thrombus platelet deposition. Drug treatment had no effect on key hematological or coagulation parameters. Our results provide direct evidence that PAR-1 is the primary receptor that mediates alpha-thrombin's prothrombotic actions in primates and suggest that PAR-1 antagonists may have potential for the treatment of thrombotic disorders in humans.

Abstract: Purpose: R115777 is a selective nonpeptidomimetic inhibitor of farnesyltransferase (FTase), one of several enzymes responsible for posttranslational modification that is required for the function of p21ras and other proteins. Given that RAS mutations are nearly universal in pancreatic cancer and R115777 demonstrated preclinical activity against pancreatic cell lines and xenografts, this phase II study was undertaken to determine its clinical activity and effect on target proteins in patients with measurable metastatic pancreatic adenocarcinoma. Patients and Methods: Twenty patients who had not received prior therapy for metastatic disease were treated with 300 mg of R115777 orally every 12 hours for 21 of 28 days. Inhibition of FTase activity in peripheral-blood mononuclear cells was measured using a lamin B C-terminus peptide as substrate. Western blot analysis was performed to monitor farnesylation status of the chaperone protein HDJ-2. Results: No objective responses were seen. Median time to progressi...

Abstract: Objective A clinical trial was conducted in healthy adult volunteers to assess the effect of levofloxacin, moxifloxacin, and ciprofloxacin on the QT and QTc interval. Methods Electrocardiograms were recorded 24 hours before and after subjects took placebo, 1000 mg levofloxacin, 800 mg moxifloxacin, and 1500 mg ciprofloxacin in a double-blind, randomized, 4-period, 4-treatment,4-sequence crossover trial. Changes in QT and QTc interval from baseline were assessed by several different methods. Results Increases in QT and QTc interval compared with placebo were consistently greater after moxifloxacin compared with either levofloxacin or ciprofloxacin. The mean postdose change from baseline QTc (Bazett) intervals for the 24-hour period after treatment with moxifloxacin ranged from 16.34 to 17.83 ms (P < .001, compared with placebo). For levofloxacin, this change ranged from 3.53 to 4.88 ms (P < .05, comparedwith placebo), and for ciprofloxacin, this change ranged from 2.27 to 4.93 ms (P < .05, compared with placebo, with the use of 3 of 5 baseline methods). Conclusions A change in QTc (Bazett) interval from baseline can be demonstrated safely in healthy volunteers after single high doses of fluoroquinolones that achieve approximately 1.5 times the maximum plasma drug concentration that occurs after recommended doses. There is substantial daily variation in both QT and QTc interval, and the magnitude and frequency of changes in QTc interval can depend on the methods used. These factors need to be considered because clinical trials measuring the effects of drugs on QT intervals are used to estimate the risk of using these drugs. Greater changes in QT and QTc intervals after treatment with moxifloxacin compared with levofloxacin or ciprofloxacin are consistent with in vitro observations related to the effect of these drugs on rapid potassium (IKr) channels. The clinical relevance of these differences is not known. Clinical Pharmacology & Therapeutics (2003) 73, 292–303; doi: 10.1016/S0009-9236(03)00009-2

Abstract: Purpose. This study was done to elucidate the physical and pharmaceutical properties of itraconazole-HPMC dispersions and the influence of water on the phase separation. Methods. Extrudates were prepared using a corotating twin-screw hot-stage extruder with fixed process parameters. Modulated-temperature differential scanning calorimetry (MTDSC) and DSC 111 were used to examine the mixing behavior of itraconazole and the carrier by evaluation of the glass transition region. High temperature diffuse reflectance infrared transform spectroscopy (HT-DRIFT) was performed to reveal interactions between itraconazole and HPMC. Dissolution was performed to investigate the pharmaceutical performance of the dispersions. Results. Although the dissolution rate of itraconazole significantly increased, we found that the solid dispersions do not form a homogeneous system. A different picture was obtained depending on the way MTDSC analysis was performed, i.e., using open or closed sample pans. Water can evaporate in open pans, which allows itraconazole to interact with HPMC and leads to a partially mixed phase. Analysis in hermetically closed pans revealed a further phase separation as water remains on the sample and impedes the interaction between drug and polymer. Conclusions. Solid dispersions of itraconazole and HPMC do not form a homogeneous phase.

Abstract: DNA microarrays provide a global view of the physiological state of the cell by parallel analysis of the expression levels of all the genes in an organism. The effects of four bactericidal agents on t

Abstract: Aging of the immune system, or "immunosenescence," is associated with both a marked reduction in responsiveness as well as functional dysregulation. These changes have been implicated in the increased morbidity and mortality of the elderly population from infectious disease, and may also play a role in autoimmunity and cancer. Though marginal alterations in B lymphocytes are apparent, the dramatic decline in humoral and cell-mediated responses is predominantly the consequence of alterations in the T-cell compartment. The effect of aging on CD4 cell function has been extensively summarized elsewhere. This review, therefore, focuses on the CD8 T-cell subset. Age-related changes in thymic function and involution, cellular homeostasis and lifespan, population shifts, T-cell activation, the process of replicative senescence, and oligoclonal expansions are discussed in terms of their effect on CD8 T cells. Age-associated alterations in CD4 T cells and antigen-presenting cells are mentioned insofar as these cells affect CD8 T-cell activation and function. Distinct patterns of immunosenescence in humans and mice are also noted.

Abstract: The reaction of molecular bromine (Br2) with arylthioureas is known to produce 2-aminobenzothiazoles (Hugerschoff reaction). We show here that benzyltrimethylammonium tribromide (1, PhCH2NMe3Br3), a stable, crystalline organic ammonium tribromide (OATB), can be readily utilized as an alternative electrophilic bromine source. It is easier to control the stoichiometry of addition with an OATB, which minimizes aromatic bromination caused by excess reagent. We have developed a direct procedure from isothiocyanates and amines using tetrabutylammonium thiocyanate (Bu4NSCN) and PhCH2NMe3Br3 to afford functionalized 2-aminobenzothiazoles.

Abstract: R214127 was shown to be a potent and noncompetitive metabotropic glutamate 1 (mGlu1) receptor-selective antagonist. The kinetics and pharmacology of [ 3 H]1-(3,4-dihydro-2H-pyrano[2,3- b ]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) binding to rat mGlu1a receptor Chinese hamster ovary (CHO)-dhfr − membranes was investigated, as well as the distribution of [ 3 H]R214127 binding in rat brain tissue and sections. Specific binding to rat mGlu1a receptor CHO-dhfr − membranes was ∼92% of total and was optimal at 4°C. Full association was reached within 5 min, and [ 3 H]R214127 bound to a single binding site with an apparent K D of 0.90 ± 0.14 nM and a B max of 6512 ± 1501 fmol/mg of protein. Inhibition experiments showed that [ 3 H]R214127 binding was completely blocked by 2-quinoxaline-carboxamide- N -adamantan-1-yl (NPS 2390), (3a S ,6a S )-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[ c ]furan-1-on (BAY 36-7620), and 7-(hydroxyimino)cyclo-propa[ b ]chromen-1a-carboxylate ethyl ester (CPCCOEt), but was not displaced by competitive mGlu1 receptor ligands such as glutamate and quisqualate, suggesting that R214127, NPS 2390, BAY 36-7620, and CPCCOEt bind to the same site or mutually exclusive sites. Experiments using rat cortex, striatum, hippocampus and cerebellum revealed that [ 3 H]R214127 labeled a single high-affinity binding site ( K D ∼ 1 nM). B max values were highest in the cerebellum (4302 ± 2042 fmol/mg of protein) and were 741 ± 48, 688 ± 125, and 471 ± 68 fmol/mg of protein in the striatum, hippocampus, and cortex, respectively. The distribution of [ 3 H]R214127 binding in rat brain was investigated in more detail by radioligand autoradiography. A high density of binding sites was detected in the molecular layer of the cerebellum. Moderate labeling was seen in the CA3 and dentate gyrus of the hippocampus, thalamus, olfactory tubercle, amygdala, and substantia nigra reticulata. The cerebral cortex, caudate putamen, ventral pallidum, and nucleus accumbens showed lower labeling. The high affinity and selectivity of [ 3 H]R214127 for mGlu1 receptors renders this compound the ligand of choice to study the native mGlu1 receptor in brain.

Abstract: Proteinase-activated receptor-2 belongs to a new subfamily of G-protein-coupled receptors. Its precise role during inflammation and the underlying mechanisms is still unclear. Our study establishes that PAR-2 plays a direct proinflammatory role during cutaneous inflammation in mice and humans in vivo. In a model of experimentally induced allergic (ACD) and toxic (ICD) contact dermatitis (CD) we show that ear swelling responses, plasma extravasation, and leucocyte adherence were significantly attenuated in PAR-2 null mutant (PAR-2−/−) mice compared with wild-type (PAR-2+/+) mice, especially at early stages. The proinflammatory effects by PAR-2 activation were significantly diminished using nitric oxide-synthase inhibitors, while NF-kappaB and neuropeptides appear to play a minor role in these mechanisms. PAR-2-mediated up-regulation of E-selectin and cell adhesion molecule ICAM-1; enhanced plasma extravasation was observed in humans and mice and of interleukin-6 in mice in vivo. Thus, PAR-2 may be a benefi...

Abstract: Of all partners involved in G-protein coupled receptor (GPCR) signalling, the regulator of G-protein signalling (RGS) proteins are the only ones showing fast gene expression changes after various stimuli. These expression changes can offer feedback regulation to GPCR signalling as RGS accelerate the return of G-proteins to their inactive form and exert regulatory functions on intracellular effectors. However, it is not yet known which RGS regulate which receptor transduction pathways in the brain. To start to answer this question, we studied the influence of specific agonists and antagonists of the dopamine D1 and D2 receptors on the gene expression of the five most abundant RGS in the striatum: RGS2, RGS4, RGS8, RGS9 and RGS10. Only changes in RGS2 and RGS4 mRNA levels were observed. The D1 agonist SKF82958 and D2 antagonist haloperidol caused an up-regulation of RGS2 (+ 38.0% and + 41.6%, respectively). The D1 antagonist SCH23390 and D2 agonist quinpirole caused a down-regulation of RGS2 (- 25.0% and - 35.0%) and an up-regulation of RGS4 (+ 57.2% and + 52.5%). D1 and D2 receptors exert opposite effects on RGS2 expression, as they do on cAMP levels, suggesting a cAMP-mediated transcription of RGS2. This was confirmed by the unique induction of RGS2 (+ 111.1%) observed in the periventricular zone of the striatum after intracerebroventricular injection of forskolin. RGS4 was up-regulated only when RGS2 was down-regulated. This suggests that both RGS exert distinct functions. Considering the coupling of D1 and D2 receptors to the intracellular effector adenylate cyclase 5 (AC5) through their respective Galpha subunits in the striatum, our data allow us to suggest that RGS2 regulates the D1/Galphaolf/AC5 pathway and RGS4 the D2/Galphao/AC5 pathway.

Abstract: Introduction: Reliable detection of drug-induced proarrhythmia, especially the potential for polymorphic ventricular tachycardia, is of great importance in the development of new compounds that are safe for the heart and was evaluated in a blinded study Methods and Results: In 142 female rabbits, the monophasic action potential was used to determine intraventricular conduction, action potential duration (APD), triangulation (APD30 to APD90), reverse use-dependence, instability and presence of chaotic behavior, early afterdepolarizations, torsades de pointes (TdP), and ventricular fibrillation In addition, 31 coded drugs were tested in a blinded fashion in another 150 hearts Prototype cardiovascular agents [quinidine (IA), lidocaine (IB), flecainide (IC), propranolol (II), sotalol (IIIB), amiodarone (IIIAB) and verapamil (IV)] were correctly characterized in terms of their effects upon conduction and APD Agents documented in clinical practice to have proarrhythmic potential (droperidol, sotalol, mibefradil, bepridil, lidoflazine, ketanserin, sertindole, terfenadine, haloperidol, astemizole, cisapride, ziprasidone, lubeluzole, dofetilide, quinidine, ibutilide) were identified as such Pimozide is reported to rarely produce TdP and was also found to elicit Class III action with few adverse effects Equally important, agents believed not to be proarrhythmic (two solvents, atenolol, propranolol, fenoximone, cetirizine, verapamil, sildenafil, lidocaine, diltiazem) were identified as having no proarrhythmic activity Conclusion: The SCREENIT method properly characterized and quantified prototype cardiovascular drugs and correctly identified proarrhythmic noncardiovascular agents of various mechanisms, but it did not produce false-positive results(J Cardiovasc Electrophysiol, Vol 14, pp 287-294, March 2003)

Abstract: Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability. We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12. Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition. In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins. Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain. Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs. Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.

Abstract: A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.

Abstract: Inflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology.