Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 3 1, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3 beta. We also present evidence that PV signaling is activated via the alpha 6 beta 4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection. (c) 2006 Elsevier Inc. All rights reserved

Mechanism of attenuation of muscle protein degradation induced by tumor necrosis factor-alpha and angiotensin II by beta-hydroxy-beta-methylbutyrate

Both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with beta-hydroxy-beta-methylbutyrate (HMB; 50 microM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-alpha/IFN-gamma and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRDelta6, in response to TNF-alpha/IFN-gamma, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and -8. HMB also attenuated activation of PKR, suggesting that it was important in protein degradation. Formation of ROS was attenuated by rotenone, an inhibitor of the mitochondrial electron transport chain, nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, and SB 203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), which also attenuated total protein degradation. Activation of p38 MAPK by PKR provides the link to ROS formation. These results suggest that TNF-alpha/IFN-gamma and ANG II induce muscle protein degradation by a common signaling pathway, which is attenuated by HMB, and that this involves the initial activation of caspase-3 and -8, followed by autophosphorylation and activation of PKR, which then leads to increased ROS formation via activation of p38 MAPK. Increased ROS formation is known to induce protein degradation through the ubiquitin-proteasome pathway.

Highly efficient palladium-catalyzed Suzuki-Miyaura reactions of potassium aryltrifluoroborates with 5-iodo-1,3-dioxin-4-ones in water: an approach to alpha-aryl-beta-ketoesters

The high efficient palladium-catalyzed Suzuki-Miyaura reactions of potassium aryltrifluoroborates 3 with 5-iodo-1,3-dioxin-4-ones 2a-b in water as only solvent in the presence of n-Bu(4)NOH as base is reported. The respective 5-aryl-1,3-dioxin-4-ones 4a-n were obtained in good to excellent yields. The catalyst system provides high efficiency at low load using electronically diverse coupling partners. The obtained 2,2,6-trimethyl-5-aryl-1,3-dioxin-4-ones were transformed into corresponding alpha-aryl-beta-ketoesters 6 by reaction with an alcohol in the absence of solvent. (C) 2009 Elsevier Ltd. All rights reserved.

Functionalization of (2S)-Isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones by a Suzuki-Miyaura Cross-Coupling Reaction Using Aryltrifluoroborate Salts: Convenient Enantioselective Preparation of alpha-Substituted beta-Amino Acids

A simple protocol for the Pd(OAc)(2)-catalyzed cross-coupling reaction of 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones with potassium aryltrifluoroborates was developed. The reaction is performed at 110 degrees C with a ligand-free catalyst. In all cases, complete conversion of the 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones and aryltrifluoroborates into the C-C coupling products was observed within 30-360 min. It is noteworthy that a large variety of groups present in the potassium aryltrifluoroborates (-CF(3), -OMe, -SEt, -CN, -CHO, -Cl, -Cbz, -NCbz, -OH, -CO(2)H) could be tolerated. Hydrogenation of the endocyclic double bonds in the Suzuki-Miyaura products followed by acid hydrolysis afforded highly enantioenriched alpha-aryl-substituted beta-amino acids.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta(4)-adrenoceptors in both wild-type and beta(3)-adrenoceptor knockout mice

Some blockers of beta(1)- and beta(2)-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta(4)-adrenoceptor) that resembles the beta(3)-adrenoceptor. It is likely but not proven that the putative beta(4)-adrenoceptor is genetically distinct from the beta(3)-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta(3)-adrenoceptor gene (beta(3)KO). (-)-CGP 12177, a beta(1)- and beta(2)-adrenoceptor blocker that causes agonist effects through both beta(3)-adrenoceptors and cardiac putative beta(4)-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta(3)KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 mu M) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta(3)KO. (-)-[H-3]CGP 12177 labeled a similar density of the putative beta(4)-adrenoceptor in ventricular membranes from the hearts of both WT (B-max = 52 fmol/mg protein) and beta(3)KO (B-max = 53 fmol/mg protein) mice. The affinity of (-)-[H-3]CGP 12177 for the cardiac putative beta(4)-adrenoceptor was not different between WT (K-d = 46 nM) and beta(3)KO (K-d = 40 nM). These results provide definitive evidence that the cardiac putative beta(4)-adrenoceptor is distinct from the beta(3)-adrenoceptor.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

The contribution of classical (beta(1/2)-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta(3)-adrenoceptor agonists of differing selectivities

The role of beta(3)- and other putative atypical beta-adrenaceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta(3)-adrenoceptor (beta(3)AR) agonists with varying intrinsic activities and selectivities for human cloned PAR subtypes. The ability to demonstrate beta(1/2)AR antagonist-insensitive (beta(3) or other atypical beta AR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta(3)AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta(1/2)AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta(3)AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta(1/2)AR antagonism, despite it having very low efficacies at cloned beta(1)- and beta(2)ARs. A component of the response to another phenylethanolamine selective beta(3)AR agonist (SB-215691) was insensitive to beta(1/2)AR antagonism in some experiments. Because novel aryloxypropanolamine had a beta(1/2)AR antagonist-insensitive inotropic effect, these results establish more firmly that beta(3)ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta(4)ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned beta ARs which beta ARs will mediate responses to agonists in tissues that have a high number of beta(1)- and beta(2)ARs or a low number of beta(3)ARs.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.