A phase II clinical trial of sunitinib following hepatic transarterial embolization for metastatic neuroendocrine tumors.

BACKGROUND: The liver is the predominant site of metastases among patients with advanced neuroendocrine tumors (NETs). Prior retrospective studies have reported high response rates in patients treated with transarterial embolization (TAE). NETs are highly vascular and are known to express vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR). We hypothesized that administration of sunitinib, a VEGFR inhibitor, following TAE would extend progression-free survival (PFS). PATIENTS AND METHODS: Patients with metastatic NETs to the liver underwent a series of selective TAEs followed by sunitinib (until disease progression or maximum of 12 months). Radiographic response (by RECIST), survival, and safety parameters were monitored. RESULTS: Thirty-nine patients were enrolled. The overall response rate was 72% [95% confidence interval (CI), 0.58-0.86]. Median PFS was 15.2 months. Rates of overall survival (OS) at 1 and 4 years were 95% (95% CI, 0.88-1.00) and 59% (95% CI, 0.38-0.80), respectively. A significant 34% rise in serum VEGF was observed following the initial TAE (P = 0.03). CONCLUSIONS: Hepatic TAE is a highly active treatment option for patients with metastatic NETs to the liver. Embolization stimulates release of VEGF into the circulation. Sunitinib, an oral VEGFR inhibitor, can be safely administered following embolization. The high rates of PFS and OS associated with this sequence of therapies are encouraging.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome: a multicenter study. II. Basophil activation by nonsteroidal anti-inflammatory drugs and its impact on pathogenesis.

BACKGROUND: Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs. MATERIAL AND METHODS: We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes). RESULTS: In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index >2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique. CONCLUSIONS: The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

Vibrational autodetachment-intramolecular vibrational relaxation translated into electronic motion.

If a negative ion has vibrational energy in excess of the binding energy of its most weakly bound electron, the anion can undergo vibrational autodetachment, similar to thermionic emission. When this effect occurs after targeted infrared excitation of a specific vibrational mode in the anion, it encodes information on the intramolecular vibrational relaxation processes that take place between excitation and electron emission. We present examples on how vibrational autodetachment can be used to obtain infrared spectra of molecular anions, and we discuss how a vibrational autodetachment photoelectron spectrum can be modeled, using vibrational autodetachment after excitation of CH stretching modes of nitromethane anions as a case study.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study.

INTRODUCTION: This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result. MATERIAL AND METHODS: In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay. RESULTS: With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%). CONCLUSIONS: A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

Predictive value of the sulfidoleukotriene release assay in oral allergy syndrome to celery, hazelnut, and carrot.

BACKGROUND: Patients sensitized to birch pollen frequently suffer from a food allergy to plant foods such as celery, carrots, or hazelnut. One of the main manifestations of birch pollen-related food allergy is the oral allergy syndrome. Skin tests and allergen-specific immunoglobulin (Ig) E determinations are poor predictors of such reactions when assessed by double-blind placebo-controlled food challenge (DBPCFC). OBJECTIVE: To investigate whether a cellular test based on leukotriene release from basophils, the cellular antigen stimulation test in combination with enzyme-linked immunosorbent assay (CAST-ELISA), is predictive of pollen-related food allergy. METHODS: Birch pollen-sensitized patients with positive DBPCFC to celery (n=21), hazelnut (n=15), and carrot (n=7) underwent skin tests along with determination of specific IgE and CAST-ELISA for the respective allergens. The results were compared with those of 24 birch pollen-sensitized patients with negative open food challenge to celery, hazelnut, and carrot. RESULTS: While skin prick tests had a sensitivity of 85%, 80%, and 29% for commercial extracts of celery, hazelnut, and carrot, respectively, prick testing with self-prepared extracts yielded sensitivities of 100%, 80%, and 100%, respectively. For specific IgE determinations, sensitivities were 71%, 73%, and 57%, respectively, and the respective specificities were 67%, 73%, and 60%. For CAST-ELISA with various sources and doses of allergens, the sensitivity varied from 71% to 95% for celery, 73% to 80% for hazelnut, and 43% to 86% for carrot. The respective specificities were 67% to 92%, 75% to 88%, and 77% to 91%. Analysis of the predictive value of CAST-ELISA with receiver operating characteristic curves showed that the results of the tests were more predictive of pollen-related food allergy than quantitative allergen-specific IgE determinations. CONCLUSIONS: CAST-ELISA is more specific than routine diagnostic tests for the diagnosis of pollen-related food allergy to celery, hazelnut, and carrot.

Cellular in vitro assays in the diagnosis of Hymenoptera venom allergy.

BACKGROUND: The current diagnostic procedures of anaphylactic reactions to hymenoptera stings include intradermal tests, venom-specific IgE (sIgE) and possibly sting challenge tests. Sometimes, the culprit insect remains unidentified. The usefulness of the cellular assays CAST-ELISA and Flow-CAST in the management of hymenoptera venom allergy was investigated. METHODS: 134 patients with systemic reactions after a yellow jacket wasp and/or honey bee sting and 44 healthy controls underwent skin tests, as well as determination of sIgE (CAP-FEIA), leukocyte sulfidoleukotriene release (CAST-ELISA) and basophil CD63 expression (Flow-CAST) upon insect venom stimulation. The clinical diagnosis based on the history alone served as reference. Sensitivity, specificity, and positive and negative predictive value of all methods were compared. Concordance and correlations among methods were calculated. RESULTS: Sensitivity and specificity of all in vitro tests were consistently high. The combination of all tests (skin tests, sIgE, combined cellular assays) yielded a positive predictive value of 100% for both venoms, if all 3 were positive, and a negative predictive value of 100%, if at least 1 test was positive. Relative specificities were considerably higher for the cellular assays (honey bee: CAST 91.1%, Flow-CAST 85.7%; yellow jacket wasp: CAST 98.4%, Flow-CAST 92.1%) and allow the detection of the culprit insect in patients with reactivity to both insects. The concordance between methods was good. There is no correlation between severity of clinical reaction and cellular assays. CONCLUSION: CAST-ELISA and Flow-CAST are valuable additional diagnostic tools for establishing the true culprit insect in patients with unclear clinical history or sensitization to both insects.

Photodetachment spectroscopy of PtBr4(2-): probing the Coulomb barrier of a doubly charged anion.

We probe the repulsive Coulomb barrier of the doubly charged anion PtBr(4) (2-) by photodetachment spectroscopy. The results are discussed in terms of models for the photoemission process, the excitation spectrum of PtBr(4) (2-), and calculations of the energy-dependent tunneling probability for various model potentials.

Fuel selection in shivering humans.

Heat exchange has been thoroughly studied in cold-exposed humans, but the metabolic substrates used for thermogenesis have received less attention. This review deals with oxidative fuel selection in shivering humans. Lipids provide most of the heat during low-intensity shivering, whereas carbohydrates become dominant under more extreme cold conditions. The contribution from plasma glucose always remains minor, but muscle glycogen plays an important role during intense shivering. Whether the size of muscle glycogen stores influences endurance in the cold remains to be demonstrated. The fuel selection patterns of shivering and exercise are different, but the mechanisms underlying this difference have not been investigated. The simultaneous measurement of metabolic substrate oxidation and muscle fibre recruitment has allowed to characterize two different mechanisms of fuel selection in shivering humans: the recruitment of different pathways within the same fibres and of different fuel-specific fibres within the same muscles. This suggests that muscle fibre composition of each individual may affect survival. Future research promises to provide a combination of theoretical advances on fundamental principles of fuel selection and applied strategies to manipulate fibre composition (through training) or fuel metabolism (through diet) to prolong human survival in cold environments.

An infrared investigation of the (CO2)n- clusters: core ion switching from both the ion and solvent perspectives.

The (CO2)n- clusters are thought to accommodate the excess electron by forming a localized molecular anion, or "core ion", solvated by the remaining, largely neutral CO2 molecules. Earlier studies interpreted discontinuities in the (CO2)n- photoelectron spectra to indicate that both the CO2- and C2O4- species were present in a size-dependent fashion. Here we use vibrational predissociation spectroscopy to unambiguously establish the molecular structures of the core ions in the 2 < or = n < or = 17 size range. Spectra are reported in the 2300-3800 cm(-1) region, which allows us to independently monitor the contribution of each ion through its characteristic overtone and combination bands. These signature bands are observed to be essentially intact in the larger clusters, establishing that the CO2- and C2O4- molecular ions are indeed the only electron accommodation modes at play. The size dependence of the core ion suggested in earlier analyses of the photoelectron spectra is largely confirmed, although both species are present over a range of clusters near the expected critical cluster sizes, as opposed to the prompt changes inferred earlier. Perturbations in the bands associated with the nominally neutral CO2 "solvent" molecules are correlated with the changes in the molecular structure of the core ion. These observations are discussed in the context of a diabatic model for electron delocalization over the CO2 dimer. In this picture, the driving force leading to the transient formation of the monomer ion is traced to the solvent asymmetry inherent in an incomplete coordination shell.

Echinococcus multlocularis infections of rural, residential and urban foxes (Vulpes vulpes) in the canton of Geneva, Switzerland.

We examined 267 red foxes (Vulpes vulpes) from the canton of Geneva, Switzerland, for intestinal infections with Echinococcus multilocularis. This region is situated in the core area of the endemic range of this zoonotic cestode in Central Europe. Several factors were taken into account and urbanisation level appeared to be the most explicative to describe observed differences. The prevalence decreased significantly from rural and residential areas (prevalence of 52%, CI 43-62%, and 49%, CI 38-59 %, respectively) to the urban area (prevalence of 31%, CI 19-42%). A few juvenile foxes harboured very high burdens up to more than 120,000 worms and were significantly more heavily infected than adults. The intensity of infection decreased from rural and residential areas to the city, suggesting a lower contamination of the urban environment.

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation.

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.

Osteoblastic cells regulate the haematopoietic stem cell niche.

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Anaphylaxis to the carbohydrate carboxymethylcellulose in parenteral corticosteroid preparations.

BACKGROUND: Carboxymethylcellulose is a carbohydrate widely used as additive in tablets, cosmetics, some injectable hormone formulations, food (as E466) and as active principle in hydrocolloid dressings. Anaphylaxis to carboxymethylcellulose in parenteral corticosteroid preparations has previously been reported. Typically, skin tests were positive in such cases, occasionally specific IgE or histamine release have been demonstrated. CASE REPORT: We report on 3 patients who suffered from anaphylactic symptoms after local injection of corticosteroid preparations. Intracutaneous skin tests with carboxymethylcellulose were positive; in 2, sulfidoleukotriene release could be measured in the cellular antigen stimulation test (CAST). Specific IgE could not be identified. Oral provocation tests with typical doses of carboxymethylcellulose as found in food and tablets were negative. CONCLUSION: In patients with anaphylaxis to parenteral administration of carboxymethylcellulose, small amounts are tolerated by the oral route. Skin tests and CAST are useful diagnostic tools.

Plasma and muscle phospholipids are involved in the metabolic response to long-distance migration in a shorebird.

We studied: (1) concentrations and fatty acid compositions of plasma non-esterified fatty acids, neutral lipids, and phospholipids, and (2) fatty acid composition of flight muscle phospholipids in wintering, premigratory, and spring and fall migrating western sandpipers ( Calidris mauri). Plasma neutral lipid and phospholipid levels were elevated in migrants, reflecting high rates of fat deposition. An important role of phospholipids in fattening is suggested by the fact that the amount of fatty acids in plasma phospholipids was similar to, or in spring as much as twice, that of neutral lipids. Changes in the ratio of plasma neutral lipids to phospholipids may indicate seasonal changes in triacylglycerol stores of invertebrate prey. Monounsaturation and total unsaturation of plasma neutral lipids and phospholipids increased during migration. Muscle phospholipids were more monounsaturated in spring and fall, but total unsaturation was reduced in fall. Arachidonic acid [20:4(n-6)] was especially abundant in muscle phospholipids in winter (29%) and declined during migration (19-22%), contributing to a decline in the ratio of n-6 to n-3 fatty acids. The abundance of plasma phospholipids and variability of neutral lipid to phospholipid ratio indicates that measurement of plasma phospholipids will improve methods for assessment of fattening rates of birds. The functional significance of changes in muscle phospholipids is unclear, but may relate to depletion of essential n-6 fatty acids during exercise.

Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity.

Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (1a) as an inhibitor of Src kinase activity (IC(50) = 30 nM), several additional analogues were prepared. Optimization of the C-4 anilino group of 1a led to 1c, which contains a 2,4-dichloro-5-methoxy-substituted aniline. Replacement of the methoxy group at C-7 of 1c with a 3-(morpholin-4-yl)propoxy group provided 2c, resulting in increased inhibition of both Src kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted anilines at C-4 were also potent Src inhibitors, and the propoxy group of 2c was preferred over ethoxy, butoxy, or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiperazine group provided 31a, which had an IC(50) of 1.2 nM in the Src enzymatic assay, an IC(50) of 100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over non-Src family kinases. Compound 31a, which had higher 1 and 4 h plasma levels than 2c, effectively inhibited tumor growth in xenograft models.

High-altitude acclimation increases the triacylglycerol/fatty acid cycle at rest and during exercise.

High-altitude acclimation alters lipid metabolism during exercise, but it is unknown whether this involves changes in rates of lipolysis or reesterification, which form the triacylglycerol/fatty acid (TAG/FA) cycle. We combined indirect calorimetry with [2-(3)H]glycerol and [1-(14)C]palmitate infusions to simultaneously measure total lipid oxidation, lipolysis, and rate of appearance (R(a)) of nonesterified fatty acids (NEFA) in high-altitude-acclimated (HA) rats exercising at 60% maximal O(2) uptake (VO(2 max)). During exercise, relative total lipid oxidation (%VO(2)) equaled sea-level control (SL) values; however, acclimation greatly stimulated lipolysis (+75%) but had no effect on R(a) NEFA. As a result, TAG/FA cycling increased (+119%), due solely to an increase in recycling (+144%) within adipocytes. There was no change in either group in these variables with the transition from rest to exercise. We conclude that, in HA, 1) acclimation is a potent stimulator of lipolysis; 2) rats do not modify TAG/FA cycling with the transition to exercise; and 3) in normoxia, HA and SL derive the same fraction of their total energy from lipids and carbohydrates.

Antibiotic prophylaxis for group A streptococcal burn wound infection is not necessary.

BACKGROUND: Historically, group A beta-hemolytic streptococci (GAS) burn wound infection has been a major source of morbidity and mortality in burn patients and has prompted the prophylactic administration of antibiotics to children with burns. Wound monitoring, surveillance cultures, and early excision of deep wounds may have changed this. Our objective in this project was to determine the efficacy of routine antibiotic prophylaxis in the era of early excision and closure of deep burn wounds. METHODS: Two cohorts of burned children were compared: all children admitted during calendar years 1992 through 1994 (group 1) and during calendar years 1995 through 1997 (group 2). All group 1 children received routine GAS antibiotic prophylaxis. Only those group 2 children with documented positive admission or surveillance cultures for GAS were treated. RESULTS: There were 511 children in group 1 and 406 children in group 2. They were well matched for age (4.7 +/- 0.21 years vs. 5.3 +/- 0.26 years, p = 0.06) and burn size (11.0% +/- 0.7% vs. 12.4% +/- 0.8%, p = 0.18). GAS species were recovered at admission or during hospitalization from 11 (2.6%) of group 1 children and 18 (4.4%) of group 2 children (p = 0.05), indicating a marginally higher rate of carriage in group 2. Nevertheless, in group 1 there were three (0.6%) who developed GAS wound infection and in group 2 there were four (0.98%, p = 0.71). The incidence of GAS infection in those patients with positive admission cultures was three (27%) of group 1 and four (22%) of group 2. No child developed fulminant GAS infection. CONCLUSION: Routine antibiotic prophylaxis of burn wounds in children in not effective in further reducing a low baseline incidence of GAS wound infection if admission screening by culture is used to identify those children who carry the organism and early excision of deep burns is practiced.

Interactions of human lacrimal and salivary cystatins with adenovirus endopeptidase.

Over 100 serotypes of adenoviruses have been implicated in a variety of human and domesticated animal pathologies and some serotypes are widely used as gene transfer vectors. Aside from the limited use of vaccines for specific serotypes, little effort has been expended in the development of antivirals. The objective here was to study the effect of cystatins from human saliva (CS) and tears (CT), two points of viral entry, on adenain, the adenovirus type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both fluids at a yield of 5 mg/l. In vitro adenain activity was inhibited to 50% at a molar ratio of 5 CS:1 adenain and 3 CT:1 adenain. By comparison, papain was inhibited to 50% at a molar ratio of 2 CS:1 papain and 1.5 CT:1 papain. Adenain differed from papain in response to CS and chicken egg white (CEW) cystatin in being stimulated at low concentrations, and in being inhibited only at very high concentrations of cystatins. The presence of cleavage consensus sites specific to adenain in the human cystatins could drive the adenain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denaturation and by highly active fresh enzyme preparations. Our experiments designed to test the nature of the interaction between adenain and cystatins suggest a docking model for the adenain-human cystatin interaction, similar to that proposed for papain and CEW. At equilibrium the dissociation constant, K(d), between adenain and CT was 1.2 nM. The kinetic parameters determined here suggest a simple reversible mechanism for the inhibition of adenain by human cystatins. We conclude that the cystatins present in tears and saliva are unlikely to play a significant role in inhibiting adenovirus infections.

Synthesis and Src kinase inhibitory activity of a series of 4-phenylamino-3-quinolinecarbonitriles.

Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a) as a Src inhibitor. An enzymatic assay established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3, and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group, had an IC(50) of 3.8 nM in the Src enzymatic assay and an IC(50) of 940 nM for the inhibition of Src-dependent cell proliferation.