New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.     

Compartmental analyses of plasma n-3 essential fatty acids among male and female smokers and nonsmokers

The effects of cigarette smoking on n-3 essential FA metabolism were studied in male and female subjects by fitting the concentration-time curves of the d(5)-labeled plasma fatty acids (FAs) originating from a dose of d(5)-18:3n-3 to a compartmental model of n-3 FA metabolism. For 3 weeks, female (smokers, n = 5; nonsmokers, n = 5) and male (smokers, n = 5; nonsmokers, n = 5) subjects subsisted on a beef-based diet. Beginning in the third week, subjects received a dose of d(5)-18:3n-3 ethyl ester (1 g). Plasma FAs were analyzed using gas chromatography (GC) and GC-mass spectrometry, and the kinetic rate parameters were determined from the concentration-time curves for d(5)-18:3n-3, d(5)-20:5n-3, d(5)-22:5n-3, and d(5)-22:6n-3. Women smokers had a 2-fold greater percent of dose in plasma (5.8% vs. 2.9%; P < 0.01) and a higher fractional rate constant coefficient for formation of d(5)-22:6n-3 from d(5)-22:5n-3 (0.03 h(-1) vs. 0.01 h(-1); P < 0.01), compared with nonsmokers. Male smokers had elevated total plasma n-3 FAs, more-rapid turnover of 18:3n-3 (13.3 mg/day(-1) vs. 4.3 mg/day(-1); P < 0.001), a disappearance rate of d(5)-20:5n-3 that was both delayed and slower (0.001 h(-1) vs. 0.012 h(-1); P < 0.05), and a percentage of d(5)-20:5n-3 directed into formation of d(5)-22:5n-3 (99% vs. 61%; P < 0.03) that was greater compared with nonsmokers. Smoking increased the bioavailability of n-3 FAs from plasma, accelerated the fractional synthetic rates, and heightened the percent formation of some long-chain n-3 PUFAs in men and women.     

Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation

Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.     

The role of dopamine and serotonin in regulating bone mass and strength: studies on dopamine and serotonin transporter null mice

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. Dopamine (DA) has been reported to have effects on calcium and phosphorus metabolism. The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released DA by rapid uptake of the neurotransmitter into presynaptic terminals. We have evaluated the histologic and biomechanical properties of the skeleton in mice homozygous for deletion of the DA transporter gene (DAT (-/-)) to help delineate the role of DA in bone biology. We have demonstrated that DAT (-/-) mice have reduced bone mass and strength. DAT (-/-) animals have shorter femur length and dry weight, and lower ash calcium content. Cancellous bone volume in the DAT (-/-) proximal tibial metaphysis is significantly decreased with reduced trabecular thickness. DAT (-/-) vertebrae have lower cancellous bone volume as a consequence of increased trabecular spacing and reduced trabecular number, and cortical thickness and bone area in the femoral diaphysis are reduced. The ultimate bending load (femoral strength) for the DAT (-/-) mice is 30% lower than the wild-type mice. Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity. Since serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we explored the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines including ROS 17/2.8, UMR 106-H5 and Py1a show mRNA expression for the 5-HTT, and the 5-HT(1A), 5-HT(1D), 5-HT(2A) and 5-HT(2B) receptors by RT-PCR analysis and immunoblot. A relatively high density of nanomolar affinity 5-HTT binding sites is present in ROS 17/2.8 and UMR 106-H5 cells. The maximal [(3)H]5-HT uptake rate in ROS cells was 110 pmol/10 min/well, with a K(m) value of 1.13 microM. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased by almost eight-fold at day 31. In mature rOB cultures, the estimated density of [(125)I]RTI-55 binding sites was 600 fmol/mg protein. PMA treatment caused a significant 40% reduction in the maximal uptake rate of [(3)H]5-HT, an effect prevented by pretreatment with staurosporine. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR 106-H5 cells. In 5-HTT (-/-) animals, cancellous bone volume (BV/TV) in the lumbar vertebrae is reduced, with a trend toward decreased trabecular thickness and trabecular number. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT, and disruption of the 5-HTT gene may cause osteopenia.     

VCD configuration of enantiopure/-enriched tetrasubstituted alpha-fluoro cyclohexanones and their use for epoxidation of trans-olefins

The configurations of three enantiopure tetrasubstituted alpha-fluoro cyclohexanones (-)-5Ia, (-)-5IIa and (-)-6a were determined by VCD and proved to be (-)-(2S,5R)-5Ia, (-)-(2R,5R)-5IIa, and (-)-(2R,5R)-6a. The VCD study also identified the conformers populated in CDCl3 solution, including higher-energy gas-phase conformers with equatorial fluorine for 5Ia and 5IIa that are stabilized in CDCl3 solution. Used as catalysts for epoxidation of trans olefins (beta-methylstyrene, stilbene, methyl p-methoxy cinnamate) by oxone, it was found that (-)-5Ia is the most efficient for all trans olefins (providing, respectively, 62%, 90% and 66% ee) but that all three ketones provide high ee% with stilbene (78-90% ee). Moreover, the configurations predicted from the stereo outcome of the epoxidation reaction are identical to those determined by VCD.     

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.     

Synthesis of enantiopure Delta2-isoxazoline derivatives and evaluation of their affinity and efficacy profiles at human beta-adrenergic receptor subtypes

The new enantiomerically pure 3-substituted-Delta(2)-isoxazolin-5-yl-ethanolamines (+)-6a/(-)-6b, (-)-6a/(+)-6b, and (+)-7a/(-)-7b, prepared via a 1,3-dipolar cycloaddition-based approach, were tested for their affinity at human beta(1)-, beta(2)-, and beta(3)-adrenergic receptor (beta-AR) subtypes stably expressed in CHO cells. The corresponding 3-isopropenyl derivatives (+)-5a/(-)-5b, (-)-5a/(+)-5b, and some isoxazole analogs were also tested. The binding affinities at the beta-ARs of the isoxazolinyl amino alcohols were significantly lower than those of the corresponding isoxazole derivatives. A stereochemical effect was observed, since the process of molecular recognition is predominantly controlled by the (S)-configuration of the stereogenic center located at the 5 position of the heterocycle rather than by that of the stereocenter carrying the secondary alcohol group. On the contrary, the stereochemical features marginally affected the efficacy response; as a matter of fact, functional tests carried out on Delta(2)-isoxazoline derivatives provided with a detectable binding affinity showed the overall profile of neutral antagonists at all three beta-AR subtypes.     

Synthesis and antibacterial activity of egonol derivatives

Synthesis of egonol derivatives, 5-(3'-chloropropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 1, 5-(3'-bromopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 2, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propanal 3, 5-(3'-iodopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 4, 5-[3-(3'-bromopropyloxy) propyl]-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 5, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylmethanoate 6, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propyloleate 7, 5-[3'-hydroxypropyl]-6-bromo-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 8, 4-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]butanenitrile 9, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylbenzoate 10, 5-[3'-hydroxypropyl]-7-methoxy-3-nitro-2-(3',4'-methylenedioxyphenyl)benzofuran 11 and their antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli are reported. The starting material egonol 5-[3'-(hydroxy)propyl]-7-methoxy-2-(3', 4'methylenedioxyphenyl)benzofuran was isolated from seeds of Styrax officinalis L. The structural elucidication of these compounds (1-11) was established using 1D ((1)H, (13)C), 2D NMR (HMBC, HMQC, COSY) and LCMS spectroscopic data. While egonol and some synthesised new compounds show similar antibacterial activity and MIC values against S. aureus, B. subtilis, C. albicans and E. coli, other new derivatives show different activity against S. aureus, B. subtilis, C. albicans and E. coli.     

The effect of alkylresorcinol on the respiration and synthesis of nucleic acids and proteins in isolated thymocytes

The effect of the membranotropic agent alkylresorcinol 5C10 on the respiration, nucleic acid and protein synthesis in isolated thymocytes was studied. Within the 5C10 concentration range of 10(-7)-10(-5) M, the inhibition of respiration and incorporation of labelled precursors into thymocyte proteins and DNA was observed. In case of respiration and protein synthesis, a 50% inhibition was observed at alkylresorcinol concentrations of 10(-8) and 0.5.10(-5) M, respectively. The rate of 3H-thymidine incorporation into DNA progressively decreased already at 5C10 concentration of 10(-7) M. At 10(-6) M alkylresorcinol its inhibiting effect on DNA synthesis was about 30% and it did not change with a further rise in the inhibitor concentration up to 10(-5) M. In contrast, the rate of RNA synthesis significantly increased (ca. by 20%) within the alkylresorcinol concentration range of 10(-6)-10(-5) M. At 5C10 concentrations above 10(-5) M, the state of thymocytes in the preagglutination period appeared to be critical and was characterized by a dramatic inhibition of all the parameters under study. The experimental results suggest that alkylresorcinol 5C10 causes the inhibition of processes that are functionally coupled with biological membranes.     

Preparation of a conjugate of 2',5'-triadenylate 5'-triphosphate and biotinylated firefly luciferase and its use for sensitive bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) for 2',5'-oligoadenylate 5'-triphosphate (pppA2'p5'A2'Ap5'A, 2-5A) was developed using a conjugate of 2-5A and firefly luciferase as a probe. The conjugate was prepared from a 2-5A derivative bearing a thiol group at the 2'(or 3') end, and streptavidin (SA) bearing maleimide via a linker-arm and biotinylated luciferase. The thiol group of the 2-5A derivative was relatively stable under aerobic conditions and remained 100% and 56% intact at 25 degrees C after 1 and 96 h, respectively, without dimerization by aerobic oxidation. The thiol-modified 2-5A was coupled with the SA-maleimide conjugate, followed by complex formation with biotinylated firefly luciferase. BLEIA using this conjugate allowed for the direct analysis of 2-5A in a range of 5-500 fmol (0.1-10 nM in a 50 microL sample) and in a reaction time of 15 min. The measurement of microquantities of 2-5 A from various sources is easy to perform by this BLEIA, because no radioisotope labeled 2-5A was required as a probe.     

Purification and analysis of murine 2-5A-dependent RNase

2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.     

Some benzoxazoles and benzimidazoles as DNA topoisomerase I and II inhibitors

Some novel fused heterocyclic compounds of 2, 5-disubstituted-benzoxazole and benzimidazole derivatives, which were previously synthesized by our group, were investigated for their inhibitory activity on both eukaryotic DNA topoisomerase I and II in a cell free system. 2-Phenoxymethylbenzimidazole (17), 5-amino-2-(p-fluorophenyl)benzoxazole (3), 5-amino-2-(p-bromophenyl)benzoxazole (5), 5-nitro-2-phenoxymethyl-benzimidazole (18), 2-(p-chlorobenzyl)benzoxazole (10) and 5-amino-2-phenylbenzoxazole (2) were found to be more potent as eukaryotic DNA topoisomerase I poisons than the reference drug camptothecin having IC(50) values of 14.1, 132.3, 134.1, 248, 443.5, and 495 microM, respectively. 5-Chloro-2-(p-methylphenyl)benzoxazole (4), 2-(p-nitrobenzyl)benzoxazole (6) and 5-nitro-2-(p-nitrobenzyl)benzoxazole (8) exhibited significant activity as eukaryotic DNA topoisomerase II inhibitors, having IC(50) values of 22.3, 17.4, 91.41 microM, respectively, showing higher potency than the reference drug etoposide.     

Synthesis and biological evaluation of some 5-nitro- and 5-amino derivatives of 2'-deoxycytidine, 2',3'-dideoxyuridine, and 2',3'-dideoxycytidine

In this article, we describe the synthesis of 5-nitro-1-(2-deoxy-alpha-D-erythro-pentofuranosyl)cytosine (4alpha), 5-nitro-1-(2-deoxy-beta-D-erythro-pentofuranosyl)cytosine (4beta), 5-amino-1-(2-deoxy-alpha-D-erythro-pentofuranosyl)cytosine (5alpha), 5-nitro-1-(2-deoxy-beta-D-erythro-pentofuranosyl)cytosine (5beta), 5-nitro-1-(2,3-dideoxy-beta-D-ribofuranosyl)uracil (6beta), 5-amino-1-(2,3-dideoxy-alpha,beta-D-ribofuranosyl)uracil (7), 5-nitro-1-(2,3-dideoxy-alpha,beta-D-ribofuranosyl)cytosine (8) and 5-amino-1-(2,3-dideoxy-beta-D-ribofuranosyl)cytosine (9beta). The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.     

Benzofurans and another constituent from seeds of Styrax officinalis

The benzofuran constituents of the seeds of Styrax officinalis were investigated. From the hexane extract, two new constituents named 5-(3"benzoyloxypropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)-benzofuran (5) and 4-[3"-(1c-methylbutanoyloxy)propyl]-2-methoxy-(3',4'-methylenedioxyphenyl)-1a, 5b-dihydrobenzo-[3,4]-cyclobutaoxirene (6) were isolated together with four known compounds, 5-[3"-(1c-methylbutanoyloxy)propyl]-7-methoxy-2-(3',4'-dimethoxyphenyl)-benzofuran (4), 5-[3"-(1c-methylbutanoyloxy)propyl]-7- methoxy-2-(3',4'-methylenedioxyphenyl)-benzofuran (3), 5-(3"-acetoxypropyl)-7-methoxy2-(3',4'-methylenedioxphenyl)-benzofuran (2) and 5-(3"-hydroxypropyl)-7-methoxy-2-(3',4'-met hylenedioxyphenyl)-benzofuran (1). Although the compounds 1, 2, and 3 have been isolated previously from the seeds of Styrax obassia, this is the first record of their isolation from seeds of Styrax officinalis. The structures of the isolated compounds were established by 1D- and 2D-NMR (HMBC, HMQC, COSY), FABMS and high-resolution ESI FTMS.     

Cyclosaligenyl pronucleotides of 5-iodo and 5-trifluoromethyl-1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzene mimics of thymidine: synthesis and evaluation of this pronucleotide monophosphate delivery system for compounds with potential anticancer activity

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes possessing a 5-I or 5-CF3 substituent, that were originally designed as thymidine mimics, were coupled via their 5'-OH group to a cyclosaligenyl (cycloSal) ring system having a variety of C-3 substituents (Me, OMe, H). The 5'-O-cycloSal-pronucleotide concept was designed to effect a thymidine kinase-bypass, thereby providing a method for the intracellular delivery and generation of the 5'-O-monophosphate for nucleosides that are poorly phosphorylated. The 5'-O-cycloSal pronucleotide phosphotriesters synthesized in this study were obtained as a 1:1 mixture of two diastereomers that differ in configuration (S(P) or R(P)) at the asymmetric phosphorous center. The (S(P))- and (R(P))-diastereomers for the 5'-O-3-methylcycloSal- and 5'-O-3-methoxycycloSal derivatives of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene were separated by silica gel flash column chromatography. This class of cycloSal pronucleotide compounds generally exhibited weak cytotoxic activities in a MTT assay (CC50 values in the 10(-3) to 10(-4) M range), against a number of cancer cell lines (143B, 143B-LTK, EMT-6, Hela, 293), except for cyclosaligenyl-5'-O-[1'-(2,4-difluoro-5-iodophenyl)-2'-deoxy-beta-D-ribofuranosyl]phosphate that was more potent (CC50 values in the 10(-5) to 10(-6) M range), than the reference drug 5-iodo-2'-deoxyuridine (IUDR) which showed CC50 values in the 10(-3) to 10(-5) M range.     

Synthesis of E- and Z-pyrazolylacrylonitriles and their evaluation as novel antioxidants.

A facile synthesis of (Z)- and (E)-2-(5-arylpyrazol-3-yl)-3-(pyrrol-2-yl)acrylonitriles and (Z)-2-(1,3-diarylpyrazol-5-yl)-3-pyrrol-2-yl)acrylonitriles, and isomerisation of (Z)-2-(5-arylpyrazolyl)acrylonitriles to (E)-2-(5-arylpyrazolyl)acrylonitriles under basic conditions have been reported. (Z)-2-(1,3-Diarylpyrazolyl)acrylonitriles did not undergo isomerisation under the similar conditions. New compounds were identified on the basis of their spectral data (1H-, 13C-, 1H-1H COSY, NOESY, NOE, HMQC NMR, IR, UV and EI mass). The structures of one acrylonitrile and five of their precursor 6-arylpyran-2-ones and cyanomnethylpyrazoles were confirmed by X-ray crystallographic studies. Effects of pyrazolylacrylonitriles and their precursors on rat liver-microsomal lipid peroxidation were evaluated in vitro with a view to establish structure activity relationship and to identify a lead compound.     

Indispensable role of Stat5a in Stat6-independent Th2 cell differentiation and allergic airway inflammation

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a(-/-)) mice, Stat6(-/-) mice, and Stat5a- and Stat6 double-deficient (Stat5a(-/-) Stat6(-/-)) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6(-/-)CD4+ T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6(-/-)CD4+ T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-gamma mAb), no Th2 cells developed in Stat5a(-/-)Stat6(-/-) CD4+ T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a(-/-)Stat6(-/-) mice compared with that in Stat6(-/-) mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.