Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

The antigenic binding site(s) of antibodies to factor XII associated with the antiphospholipid syndrome.

Phospholipid binding proteins, including factor XII (FXII), are known to be targeted by antiphospholipid antibodies (aPA). Factor XII antibodies (FXIIab) have been described in some patients with the antiphospholipid syndrome (APS) and have been shown to lead to reduced levels of FXII. The antigenic binding site(s) and the pathophysiological effects of FXIIab are unknown. In an attempt to elucidate the binding site of these antibodies, immobilized plasma kallikrein was used to cleave FXII into its 52-kDa heavy-chain (HCFXII) and 28-kDa light-chain (LCFXII) components. Plasma samples from 12 female patients with definite APS and FXIIab were investigated for the presence of antibodies to FXII, HCFXII and LCFXII. All but one patient's plasma reacted to FXII, HCFXII and LCFXII in a similar manner. One patient gave markedly reduced positivity to HCFXII and LCFXII, suggesting that the FXIIab in this patient had a higher affinity for the intact FXII molecule. To further investigate the antigenic binding site(s) of FXII, 150 biotinylated peptides of the known FXII sequence were synthesized using a Multipin(TM) peptide synthesis procedure. The IgG and IgM fractions of the 12 patients' plasma were purified by affinity chromatography. The synthesized peptides were captured on streptavidin plates and individual patients' purified FXIIab assayed against the peptides in a modified enzyme-linked immunosorbent assay (ELISA). Two regions were identified as possible antigenic binding site(s) for FXIIab: one in the growth factor domain and the other in the catalytic domain.

Self-reported medication use among adolescents in Kuwait.

OBJECTIVE: The objectives of this study were to describe and examine the pattern of medication use, including age and gender differences among adolescents in Kuwait, and to establish the sources of information on medicines in this age group. SUBJECTS AND METHODS: A cross-sectional survey of 1,110 male and female students (14-21 years) from 10 randomly selected public schools in Kuwait was conducted. The prevalence of self-medication was estimated. RESULTS: The prevalence of self-medication among the high school students was 92%. The prevalence increased by age from 87% among 14-year-olds to 95% among 18-year-olds. Sixty-five percent of medicines used were for pain relief, 54% for respiratory conditions, 39% for allergic conditions, and 37% for dermatological conditions. Twenty-two percent of medicines were nutritional supplements and vitamins, 21% gastrointestinal products, 17% antidandruff products, 15% hair products, 13% for migraine while 8% were for athlete's foot. Pain relief, respiratory, dermatologic and hair products were more prevalent in female adolescents than in male while antidandruff and athlete's foot preparations were used more by male adolescents. The most common sources of information on medicines were parents. CONCLUSION: The prevalence of self-medication among adolescents in Kuwait is high. Self-medication tended to increase with age and differed between male and female students. Few students consulted pharmacists for information on drugs. There is need to promote the image of the pharmacist in Kuwait as a provider of medication information.

Receptor regulatory properties evident in the molecular similarity of serotonin receptor ligands and purine nucleotides.

Previous computational studies have explored the relative molecular similarity inherent in the ligands of neurotransmitter-regulated cell receptors and purine nucleotides. This study presents the results of an investigation of the major serotonin (5-HT) receptor classes, using molecular superimposition and fitting data. Ligands for 5HT(1B/C/D) and 5HT(4/7) receptors identified pharmacophores in the adenine ring of ATP. 5-HT(2) and 5-HT(3) receptor ligands identified pharmacophores in the guanosine nucleotide and cyclic nucleotide, respectively. The described molecular similarity is consistent with the cyclic nucleotide responses observed during signal transduction events initiated by 5-HT, and the reported similarity between ligands of the 5-HT(1B) and 5-HT(1D), 5-HT(1A) and 5-HT(7), and 5-HT(4) and 5-HT(3) receptors. The results are discussed in terms of current pharmacophoric models and signal transduction events involving interaction between G-protein receptors and catalytic sites.

Post-traumatic stress after terrorist attack: psychological reactions following the US embassy bombing in Nairobi: Naturalistic study.

BACKGROUND: Most studies of post-traumatic stress disorder following terrorist attacks are of small samples in industrialised nations and take place months or years after the incident. AIMS: To describe reactions following the US embassy bombing in Nairobi and the characteristic features of and risk factors for post-traumatic stress symptoms in a large, non-Western sample soon after the attack. METHOD: A self-report questionnaire which assessed potential risk factors and identified symptoms matching DSM-IV criteria for post-traumatic stress disorder was answered by 2883 Kenyans, 1-3 months after the bombing. RESULTS: Symptoms approximating to the criteria for post-traumatic stress disorder occurred in 35%. Factors associated with post-traumatic stress included female gender, unmarried status, lack of college education, seeing the blast, injury, not recovering from injury, not confiding in a friend, bereavement and financial difficulty since the blast. Many other factors were not significant. CONCLUSIONS: Specific factors often cited to predict marked short-term post-traumatic stress were confirmed in this large, non-Western sample.

Some 1,2-diphenylethane derivatives as inhibitors of retinoic acid--metabolising enzymes.

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50 = 52.1 microM; ketoconazole, 2.8 microM) and the RA-induced enzyme (100 microM, 65.9% inhibition; ketoconazole, 20 microM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.

Some coumarins and triphenylethene derivatives as inhibitors of human testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3): further studies with tamoxifen on the rat testes microsomal enzyme.

The 7-hydroxycoumarins, umbelliferone and 4-methylumbelliferone (IC50 = 1.4 and 1.9 microM, respectively) were potent inhibitors of human testes microsomal 17beta-HSD (type 3) enzyme whereas 7-methoxycoumarin, 4-hydroxycoumarin and 7-ethoxycoumarin had little or no inhibitory activity. Analogues of the weak inhibitory triphenylethenes tamoxifen and clomiphene but lacking the basic substituent, were weak inhibitors of the human microsomal enzyme. Inhibitory activity was improved by replacement of the triphenylethene structure with a triphenylmethyl (17, 52.6% inhibition) or phenylpropyl (16, 94.8%, IC50 = 42.1 microM) skeleton. Further studies on tamoxifen using rat testes microsomal 17beta-HSD showed that the inhibition was time-dependent and irreversible but not specifically mechanism-based.

Receptor regulatory properties evident in the molecular similarity of dopamine receptor ligands and purine nucleotides.

Computational studies have revealed similarities in the relative configurations of purine nucleotides and ligands for histamine, acetylcholine and adrenergic receptors. In common with other G-protein-regulated receptors, dopamine receptors are associated with specific changes in nucleotide levels during signal transduction processes. The purpose of this study was to investigate molecular similarity in dopamine receptor ligands and purine nucleotides. Molecular superimposition and fitting data for D1-like receptor ligands identified a pharmacophore in the adenine and ribose rings of ATP. D2-like agonists and antagonists related to a pharmacophore in the guanine and ribose rings of GTP. The results are consistent with the hypothesis that the dopamine receptor family may have evolved from receptors for the ATP and GTP nucleotides.

Antibodies to factor XII are distinct from antibodies to prothrombin in patients with the anti-phospholipid syndrome.

Patients with the anti-phospholipid syndrome (APS) have antiphospholipid antibodies (aPA) which are often targeted towards phospholipid binding proteins such as beta2-glycoprotein I and prothrombin. Antibodies to factor XII (FXIIabs) have also been identified in some patients with APS. Factor XII (FXII) is a member of the kringle family of proteins which include plasminogen and prothrombin. Antibodies to prothrombin have been associated with myocardial infarction and have been shown to cross react with plasminogen. Sixteen patients with APS and FXIIabs were investigated for the presence of antibodies to prothrombin, by enzyme linked immunosorbent assay in a calcium (Ca++) independent assay. All sixteen showed different antibody binding patterns than those observed for antibodies to FXII. Eight patients were further investigated using surface plasmon resonance (SPR) for antibody binding to covalently bound FXII and to covalently bound prothrombin in both Ca++ dependent and independent systems. Of three patients demonstrating antibody binding to FXII by SPR, none demonstrated antibody binding to prothrombin in a Ca++ independent system with one demonstrating antibody binding to prothrombin that was Ca++ dependent. Of five patients who did not bind FXII by SPR, one demonstrated antibody binding to prothrombin in a Ca++ independent system while two demonstrated antibody binding to prothrombin in a Ca++ dependent system. Antibodies to FXII in patients with APS appear to be distinct from antibodies to prothrombin.

Effect of lamotrigine on the pharmacokinetics of carbamazepine and its active metabolite in dogs.

The effect of lamotrigine (LTG) on the pharmacokinetics of carbamazepine (CBZ) and its active metabolite; carbamazepine-epoxine (CBZ-E), was investigated in dogs. Five male dogs received CBZ (2 x 200 mg tab, p.o.) daily for a period of 1 week. After the end of this period, blood samples were collected serially for up to 24 hrs. After a wash-out period of I week, LTG (100 mg tab, p.o.) was coadministered with the CBZ dose (2 x 200 mg tab, p.o.) for 7 days. Blood samples were again serially collected and plasma levels of CBZ and CBZ-E were analysed by high performance liquid chromatography (HPLC). Concurrent administration of LTG with CBZ did not have any significant effect on the pharmacokinetic parameters of CBZ. There was also no significant difference between the plasma concentration ratio (CBZ-E to CBZ) vs time profiles in the two schedules of drug administration signifying the absence of pharmacokinetic interaction between LTG and CBZ or its active metabolite in this animal model.

Inhibitors of human and rat testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as potential agents for prostatic cancer.

In a screening programme for inhibitors of human testis 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17beta-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17beta-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androstenedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 microM), phenyl-p-benzoquinone (5.7 microM), 7-hydroxyflavone (9.0 microM), baicalein (9.3 microM) and biochanin A (10.8 microM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17beta-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.

Enantioselectivity of some 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as aromatase (P450AROM) inhibitors.

The enantioselectivity ratio ((+)-:(-)-forms) of three substituted 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as inhibitors of aromatase (P450AROM) was 2.16, 12.3 and 1.0 for the 4-methyl-, 4-fluoro- and 4-chloro-substituted compounds, respectively. The (+/-)-compounds were all >1000 times more potent than (+/-)-aminoglutethimide (IC50 = 12 x 10(3) nM). High potency (5.3-65.0 nM) for all the enantiomers studied is unusual since activity usually resides in one form for chiral inhibitors of P450AROM. The 4-methyl derivative was fitted into the model [Furet, P., Batzl, C., Bhatnager, A.S., Francotte, E., Rihs, G. and Lang, M. (1993) J. Med. Chem. 36, pp. 1393-1400] for binding of S-(-)-fadrazole to the active site and the (R)- and (S)- forms both gave a good fitting pattern with (S)-(-)-fadrazole so accounting for their close activity. Docking of both forms into the active site model for P450AROM [Laughton, C.A., Zvelebil, M.J.J.M. and Neidel, S. (1993) J. Steroid Biochem. Mol. Biol. 44, pp. 399-407], using the orientation of (S)-(-)-fadrazole, gave similar strong binding along the position of the C and D rings of the steroid substrate and in the hydrophobic cavity below the A/B rings. The site was probed for group size accommodation using the less potent 4-phenyl analogue (IC50(+/-) = 242 nM): the (S)-form showed restricted access to the region under the A ring due to the extended bulk of the biphenyl group.

Evaluation of 7-hydroxy-flavones as inhibitors of oestrone and oestradiol biosynthesis.

A series of 4-aryl substituted 7-hydroxy-flavones were prepared using the three-step Baker-Venkataraman synthesis in good overall yields. The flavones were all evaluated in vitro for inhibitory activity against aromatase (P450AROM, CYP19), using human placental microsomes, and for inhibitory activity against 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD-1) using human placental cytosol. The phenyl, 4-fluoro-phenyl and 4-bromo-phenyl derivatives displayed moderate inhibitory activity against P450AROM (IC50 17.2, 13.5 and 10.1 microM, respectively), none of the flavones, including the standard genistein, displayed any inhibitory activity against 17beta-HSD type 1 at 100 microM concentration.

1-[(Benzofuran-2-yl)phenylmethyl]triazoles as steroidogenic inhibitors: synthesis and in vitro inhibition of human placental CYP19 aromatase.

Hormone-dependent breast cancer is stimulated by the female hormones oestrone and oestradiol, therefore compounds which inhibit the specific enzymes involved in the formation of the nitogenic hormones, namely CYP19 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1, are targets of therapeutic interest for the treatment of breast cancer. A series of novel 1-[(benzofuran-2-yl)phenylmethyl]1,2,4-triazoles were prepared using a three-step synthesis and evaluated for their inhibitory activity against human placental aromatase in vitro, using [1,2,6,7-3H]androstenedione as the substrate for the aromatase enzyme. Inhibitory activity was dependent on both substituent and position of substitution, with introduction of small electron-withdrawing groups in the phenyl ring showing optimum activity (IC50 ranging from 0.065 to 2.02 microm). Substitution in the benzofuran ring resulted in a loss of activity when substituted at C-5 (IC50 > 20 microm). The compounds were all shown to exhibit weak inhibitory activity against rat testes P450 17 (17,20-lyase), indicating good selectivity towards P450arom.

Effect of vigabatrin and gabapentin on phenytoin pharmacokinetics in the dog.

This study was aimed at investigating whether or not the kinetics of intravenously administered phenytoin (PT) was altered by oral administration of vigabatrin (VGB) or gabapentin (GBP). A daily dose of PT (12 mgkg(-1)i.v.) was given to a group of five beagle dogs for a period of 1 week. On day eight, plasma samples were serially collected over 24 h, after administration of the PT dose. PT administration was continued, along with supplementary oral VGB (60 mgkg(-1)) for another week and then plasma samples were collected for analysis of PT levels. The same protocol was followed for the PT (12 mgkg(-1), i.v.)-GBP (300 mg caps., p.o.) study on a separate group (n= 5) of dogs. Orally administered GBP did not significantly alter the pharmacokinetic parameters of parenteral PT. However VGB markedly changed the drug's kinetics, as evidenced by a 31% (P= 0.015) reduction in total body clearance (CL) and an increase of over 45% in half-life (t(1/2)), (P= 0.013) and area under the plasma PT concentration-time curve (AUC), (P= 0.044). GBP does not appear to have any pharmacokinetic interaction with PT, while coadministration of VGB and PT results in a marked reduction in systemic clearance of the latter in the dog.

[Synthesis and biological evaluation of indole derivatives acting as anti-inflammatory or antitumoral drugs]. / Dérivés indoliques à activités anti-inflammatoire ou antitumorale.

Two axes of research have been explored, one about promising non-acidic non-steroidal anti-inflammatory derivatives, with indolin-2-one as structural core and another one about aromatase inhibitors, characterized by azolylmethyl or alpha-azolylbenzyl chain on indole nucleus. Knoevenagel reaction led to indolin-2-ones substituted by either 2,6-di-tert-butylphenol chain or 1, 4-dihydropyridine chain, revealing antioxydant or anti-inflammatory activities. Aromatase is a logical target in the treatment of hormono-dependent breast cancer in postmenopausal women. Among non steroidal inhibitors of this enzyme, diverse compounds with anilino or azaheterocyclic moiety are currently used or undergoing clinical trials. Our pharmacomodulation in azolylmethylindole or alpha-azolylbenzylindole series led to compounds with high level aromatase inhibitory activity. Work to determine their selectivity by measuring their inhibitory effect on P450 17alpha enzyme was also carried out. A first molecular modeling approach with Discover software was performed to evaluate interactions between our molecules and the catalytic site of P450cam.

HPLC enantiomeric resolution of novel aromatase inhibitors on cellulose- and amylose-based chiral stationary phases under reversed phase mode.

The chiral resolution of seven aromatase inhibitors (four triazole derivatives (Ia, Ib, Ic, and Id) and three tetrazole derivatives (IIa, IIb, and IIc)) was achieved on Chiralcel OJ-R [cellulose tris (4-methyl benzoate)], Chiralcel OD-RH [cellulose tris (3,5-dimethylphenyl carbamate)], and Chiralpak AD-RH [amylose tris (3,5-dimethylphenyl carbamate)] chiral stationary phases. The mobile phases used were A: 2-PrOH-MeCN (90:10, v/v); B: 2-PrOH-MeCN (50:50, v/v); C: MeCN-H(2)O (50:50, v/v); D: MeCN-H(2)O (80:20, v/v); and E: MeCN-H(2)O (95:05, v/v). The flow rate was 0.5 mL/min for all the mobile phases. The resolution capability of these chiral stationary phases were in the order Chiralpak AD-RH > Chiralcel OD-RH > Chiralcel OJ-R. The values of alpha and Rs of the resolved enantiomers of the aromatase inhibitors varied from 1.02-5.63 and 1. 12-6.72, respectively.

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases.

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (beta(c)) and common gamma chain (gamma(c)) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with beta(c) and gamma(c) respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56(lck), generating anti-apoptotic signals, can be observed and requires the PROX domain of gamma(c). p56(lck) can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56(lck) alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both gamma(c) domains are vital for signal transduction.