Synthesis of 2′-Deoxyribonucleoside Derivatives of 1-Deazapurine

Abstract

5, 7-Dichloro-3H-imidazo[4, 5-b]pyridine (4) is a versatile base which can be coupled with a variety of sugar moieties and transformed in a series of 7-alkyl(aryl)amino-derivatives by reacting with the corresponding amines. In this paper synthesis, structure elucidation and ADA inhibitory activity of 2′-deoxyribonucleoside derivatives of N⁶-substituted 1-deazaapurines are described.     

一个水稻MSP1突变体的鉴定和分析

水稻(Oryza sativa)MSP1基因编码一个LRR-RK蛋白,调控花药壁和花粉细胞的分化和发育。本研究从钴-60辐射的水稻突变体库中鉴定出一个新的msp1突变体1972m,其花药明显变小、变白,花药中不含花粉粒。这一新的MSP1等位突变中,一个LRR单元的一个丝氨酸转变为脯氨酸。结构分析表明这一氨基酸残基突变可能影响了MSP1位于细胞外的LRR结构域的稳定性,使之失去结合信号分子的能力,从而阻断了花药壁细胞发育信号的转导。     

C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

2-Aminoimidazoles inhibitors of TGF-β receptor 1

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-βR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-β-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.     

Synthesis of 1-Deazaadenosine Analogues of (2′→5′) ApApA

Abstract

Synthesis of (2′ → 5′)ApApA analogues containing 1-deazaadenosine at different positions is described (32–34). The approach used the phosphotrieer methodology in solution and utilized 3′-O-benzoylated derivatives of the N⁶-protected 5′-O-monomethoxytrityl-1-deazaadenosine as starting material.

     

Action of phospholipase A2 of rabbit neuronal and glial cells on 1,2-diacyl-, 2-acyl-1-alk-1′-enyl-, and 2-acyl-1-alkyl-glycerophosphatides

Pronounced differences in the phospholipase A₂ activities were found in neurons and glia, the enzyme activity being two- to threefold higher in neurons than in glial cells. Both phospholipases A₂ hydrolyzed the 1,2-diacylglycerophosphatides more rapidly than the acylalkyl and acylalkenyl compounds. Choline plasmalogen and the corresponding alkyl derivative were cleaved at similar rates by the phospholipase A₂ from both glia and neurons. There was a tendency by the neuronal phospholipase A₂ to release arachidonic acid faster than linolenic acid from both phosphatidylcholine and ethanolamine, while arachidonic acid was removed less actively from phosphatidylethanolamine by the glial enzyme. The glial phospholipase A₂ showed a lag period of 10 or 20 min. Norepinephrine, injected into the lateral ventricle of the rabbit brain, stimulated the hydrolysis of the various 1,2-diacyl-, acylalkyl-, and acylalkenyl-glycerophosphatides by the phospholipase A₂ from both glia and neurons.     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

The dimerization of Δ1-piperidine-2-carboxylic acid

The l-amino acid oxidase of Mytilus edulis has been used to oxidize l-lysine on a large scale in the presence of catalase. The alpha-oxo acid derived from lysine cyclizes to a Schiff base, which readily dimerizes. The dimer undergoes spontaneous dehydration and decarboxylation to form 1,2,3,4,5,6,7,8-octahydropyrido[3,2-a]-indolizin-10(4bH)-one. This structure was established by a study of its molecular weight and infrared, nuclear-magnetic-resonance and mass spectra.     

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Endogenous ADP-ribosylation of elongation factor-2 by interleukin-1��

Eukaryotic elongation factor-2 (eEF-2) catalyses the motion of the growing peptide chain relative to the mRNA at the ribosomes during protein synthesis. This highly conserved G-protein is the specific target of two lethal bacterial toxins, Pseudomonas aeruginosa exotoxin A and diphtheria toxin. These toxins exert their detrimental action by ADP-ribosylating a biologically unique posttranslationally modified histidine residue (diphthamide(715)) within eEF-2, thus inactivating the enzyme. Diphthamide(715) is also the target of endogenous (mono) ADP-ribosyl transferase activity. In this article, we report the first known activator of endogenous ADP-ribosylation of eEF-2, interleukin-1β (IL-1β). Thereby, systemic inflammatory processes may link to protein synthesis regulation.     

Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser(756) of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.     

Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.     

Binding of Subdomains 1/2 of PfEMP1-DBL1α to Heparan Sulfate or Heparin Mediates Plasmodium falciparum Rosetting

The capacity of Plasmodium falciparum parasitized erythrocytes (pRBC) to adhere to the endothelial lining in the microvasculature and to red blood cells (RBC) is associated with the virulence of the parasite, the pathogenesis and development of severe malaria. Rosetting, the binding of uninfected RBC to pRBC, is frequently observed in individuals with severe malaria and is mediated by the N-terminal NTS-DBL1α domain of the adhesin Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed at the surface of the pRBC. Heparan sulfate has been suggested to be an important receptor for the NTS-DBL1α variant IT4_var60 expressed by the parasite FCR3S1.2. Here, we have determined the binding site of NTS-DBL1α (IT4_var60) to the RBC and heparin using a set of recombinant, mutated proteins expressed in and purified from E. coli. All the variants were studied for their ability to bind to RBC, their capacities to disrupt FCR3S1.2 rosettes, their affinities for heparin and their binding to rosette-disruptive mAbs. Our results suggest that NTS-DBL1α mediates binding to RBC through a limited number of basic amino acid residues localized on the surface of subdomains 1 (SD1) and 2 (SD2). The SD2-binding site is localized in close proximity to one of two previously identified binding sites in the rosetting PfEMP1 of the parasite PaloAlto-varO. The binding site in SD2 of NTS-DBL1α could represent a template for the development of anti-rosetting drugs.