Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

苄基异喹啉类化合物 D20抑制钙调素激活磷酸二酯酶的研究

苄基异喹啉化合物是一类钙调素拮抗剂.对新合成的双苄基异喹啉化合物 D₂₀对钙调素依赖的磷酸二酯酶的抑制作用进行了研究,IC₅₀=5μmol/L,表明其拮抗作用大于三氟啦嗪,是强的拮抗剂.荧光分析表明,钙调素与化合物 D₂₀的结合常数为2.64(μmol/L)^-1,一个化合物 D₂₀分子与两个钙调素分子结合,并显示了结合方向性及空间位阻影响.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Synthesis,docking study and relaxant effect of 2-alkyl and 2-naphthylchromones on rat aorta and guinea-pig trachea through phosphodiesterase inhibition

Chromone (4), which form the base structure of various flavonoids isolated as natural products, is capable of relaxing smooth muscle. This is relevant to the treatment of high blood pressure, asthma and chronic obstructive pulmonary disease. The former disorder involves the contraction of vascular smooth muscle (VSM), and the latter two bronchoconstriction of airway smooth muscle (ASM). One of the principal mechanisms by which flavonoids relax muscle tissue is the inhibition of phosphodiesterases (PDEs), present in both VSM and ASM. Therefore, a study was designed to analyze the structure–activity relationship of chromone derivatives in vaso- and bronchorelaxation through the inhibition of PDE. Docking studies showed that these chromones bind at the catalytic site of PDEs. Consequently, we synthesized analogs of chromones substituted at position C-2 with alkyl and naphthyl groups. These compounds were synthesized from 2-hydroxyacetophenone and acyl chlorides in the presence of DBU and pyridine, modifying the methodology reported for the synthesis of 3-acylchromones by changing the reaction temperature from 80 to 30 °C and using methylene chloride as solvent, yielding the corresponding phenolic esters 10a–10h. These compounds were cyclized with an equivalent of DBU, pyridine as solvent, and heated at reflux temperature, yielding the chromones 11a–11h. Evaluation of the vasorelaxant effect of 4, 11a–11h on rat aorta demonstrated that potency decreases with branched alkyl groups. Whereas the EC₅₀ of compound 11d (substituted by an n-hexyl group) was 8.64 ± 0.39 μM, that of 11f (substituted by an isobutyl group) was 14.58 ± 0.64 μM. Contrarily, the effectiveness of the compound is directly proportional to the length of the alkyl chain, as evidenced by the increase in maximal effect of compound 11c versus 11d (66% versus 100%) and 11e versus 11f (60% versus 96%). With an aromatic group like naphthyl as the C-2 substituent, the effectiveness was only 43%. All compounds tested on guinea pig trachea showed less than 55% effectiveness. Compounds 4, 11a–11h were evaluated as PDE inhibitors in vitro, with 11d showing the greatest effect (73%), corroborating the importance of a long alkyl chain, which inhibits the decomposition of cGMP. Docking studies showed that the compound 11d was selective for the inhibition of PDE-5.     

Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.     

Interaction of Aryl Hydrocarbon Receptor-interacting Protein-like 1 with the Farnesyl Moiety

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.     

Concurrent modulation of extracellular levels of noradrenaline and cAMP during stress and by anxiogenic- or anxiolytic-like neuropeptides in the prefrontal cortex of awake rats

The purpose of this study was to examine the effects of stress and the role of locally infused anxiogenic-like neuropeptides galanin, CCK-8, vasopressin, substance P and neurokinin A, and anxiolytic-like peptides NPY, nociceptin/orphanin FQ, somatostatin and neurotensin, on modulation of noradrenaline (NA) and cAMP efflux monitored simultaneously by microdialysis in the medial prefronatal cortex of awake rats. Concentrations of cAMP were determined by a newly developed method based on derivatization of cAMP with 2-chloroacetaldehyde followed by HPLC with fluorescence detection. Local infusion of forskolin (10 and 30 μM) dose-dependently increased the cAMP levels to 417% and 1050% of the control group, respectively. Similarly, local infusion of NA (10 μM) increased the cAMP to the peak level of 168%. A 5-min tail pinch and a 10-min swim stress rapidly increased the NA and cAMP levels to 167% and 203% (NA) and 141% and 161% (cAMP), respectively. Infusion of galanin and CCK-8 (0.5 nmol, and 1.5 nmol/0.5 μl) dose-dependently increased NA to the peak levels of 191% and 179% and cAMP levels to 174% and 166%, respectively. The peak levels following infusions of vasopressin, substance P and neurokinin A were 91%, 135% and 86% for NA and 131%, 83% and 76% for cAMP, respectively. Infusions of anxiolytic-like peptides at highest concentrations significantly increased (NPY, 136%) or decreased (nociceptin, 71%; somatostatin, 86%) the NA levels, whereas neurotensin had no effect. The cAMP levels decreased to 86% (NPY, neurotensin), 78% (nociceptin), somatostatin infusion was without effect. The present findings confirmed a close correlation between the stress-induced increases in prefrontal cortical NA and cAMP levels, as well as, concurrent changes in NA and cAMP levels following infusions of galanin and CCK-8 (increased levels) and nociceptin/orphanin FQ (decreased levels). Infusions of other neuropeptides showed a more complex pattern of NA and cAMP responses.     

Discovery of triazines as potent,selective and orally active PDE4 inhibitors

Expanding on HTS hit 4 afforded a series of [1,3,5]triazine derivatives as novel PDE4 inhibitors. The SAR development and optimization process with the emphasis on ligand efficiency and physicochemical properties led to the discovery of compound 44 as a potent, selective and orally active PDE4 inhibitor.     

Phosphodiesterase inhibitors. Part 6: Design,synthesis, and structure–activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity

We previously identified KCA-1490 [(?)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.