2-(2-Benzofuranyl)-2-Imidazoline Attenuates the Disruption of the Blood–Brain Barrier in EAE via NMDAR

Blood–brain barrier (BBB) disruption has been recognized as an early hallmark of multiple sclerosis (MS) pathology. Our previous studies have shown that 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) protected against experimental autoimmune encephalomyelitis (EAE), a classic animal model of MS. However, the potential effects of 2-BFI on BBB permeability have not yet been evaluated in the context of EAE. Herein, we aimed to investigate the effect of 2-BFI on BBB permeability in both an animal model and an in vitro BBB model using TNF-α to imitate the inflammatory damage to the BBB in MS. In the animal model, 2-BFI reduced neurological deficits and BBB permeability in EAE mice compared with saline treatment. The Western blot results indicated that 2-BFI not only alleviated the loss of the tight junction protein occludin caused by EAE but also inhibited the activation of the NR1-ERK signaling pathway. In an in vitro BBB model, 2-BFI (100 μM) alleviated the TNF-α-induced increase in permeability and reduction in expression of occludin in monolayer bEnd.3 cells. Similar protective effects were also observed after treatment with the NMDAR antagonist MK801. The Western blot results showed that the TNF-α-induced BBB breakdown and increase in NMDAR subunit 1 (NR1) levels and ERK phosphorylation could be blocked by pretreatment with 2-BFI or MK801. However, no additional effect was observed on BBB permeability or the expression of occludin and p-ERK after pretreatment with both 2-BFI and MK801. Our study indicates that 2-BFI alleviates the disruption of BBB in the context of inflammatory injury similar to that of MS by targeting NMDAR1, as well as by likely activating the subsequent ERK signaling pathway. These results provide further evidence for 2-BFI as a potential drug for the treatment of MS.

     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

Is hsp70 the cellular thermometer?

Cells respond to an increase in temperature by inducing the synthesis of the heat shock proteins, which are a small set of evolutionarily conserved proteins. We review the evidence leading us to suggest that the free pool of one of these proteins, hsp70, serves as a cellular thermometer that regulates the expression of all heat shock proteins.     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

Synthesis of the Selenium Analog of the Cytokinin 6-(3-Methyl-2-Butenylamino)-2-Methylthio-9-(β-D-Ribofuranosyl)Purine

Abstract

6-(3-methyl-2-butenylamino)-2-methylthio-9-(β-D-ribofuranosyl)purine (1) is a naturally occurring nucleoside with potent cytokinin activity. It has been isolated and identified in the t-RNA of E. coli,^1,2 the t-RNA from wheat germ^3,4 and from Staphylococcus epidermidis.⁵ In addition, compound 1 has been found in t-RNA species corresponding to each of six amino acids whose codons start with uridine, i.e., t-RNA^Cys     

Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?

The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication. There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.     

5-氮杂-2＇-脱氧胞苷对卵巢癌细胞系CP70顺铂耐药性的逆转作用

目的：探究5-氮杂-2＇-脱氧胞苷体外逆转卵巢癌铂类耐药细胞系CP70对顺铂的耐药性,并探讨与SOCS-2表达的关系。方法：免疫细胞化学和Western blot方法检测使用5-氮杂-2＇-脱氧胞苷处理细胞前后细胞内SOCS-2表达水平。MTT法检测单独使用5-氮杂-2＇-脱氧胞苷或顺铂及两药联合使用对CP70细胞的抑制作用。结果：两药联合处理CP70细胞后顺铂耐药逆转倍数为1.34、1.63、2.34,联合处理组的细胞中SOCS-2表达明显升高。结论：5-氮杂-2＇-脱氧胞苷可以部分逆转CP70细胞对顺铂的耐药性,且此作用可能与增加细胞的SOCS-2表达有关。     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

Synthesis of the Fully Protected Phosphoramidite of the Benzene-DNA Adduct,N 2-(4-Hydroxyphenyl)-2′-Deoxyguanosine and Incorporation of the Later into DNA Oligomers

N²- (4-Hydroxyphenyl)-2 ′-deoxyguanosine-5 ′-O-DMT-3 ′-phosphoramidite has been synthesized and used to incorporate the N²-(4-hydroxyphenyl)-2 ′-dG (N²-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N²-arylation (Buchwald-Hartwig reaction) of a fully protected 2 ′-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5 ′-O-DMT-3 ′-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N²-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.     

β-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and β-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, β-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gβ5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D(3)R in collaboration with Gβ5. β-Arrestin2 competed with R7BP and Gβ5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for β-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that β-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.     

Redefining Escherichia coli σ(70) promoter elements: -15 motif as a complement of the -10 motif

Classical elements of σ(70) bacterial promoters include the -35 element ((-35)TTGACA(-30)), the -10 element ((-12)TATAAT(-7)), and the extended -10 element ((-15)TG(-14)). Although the -35 element, the extended -10 element, and the upstream-most base in the -10 element ((-12)T) interact with σ(70) in double-stranded DNA (dsDNA) form, the downstream bases in the -10 motif ((-11)ATAAT(-7)) are responsible for σ(70)-single-stranded DNA (ssDNA) interactions. In order to directly reflect this correspondence, an extension of the extended -10 element to a so-called -15 element ((-15)TGnT(-12)) has been recently proposed. I investigated here the sequence specificity of the proposed -15 element and its relationship to other promoter elements. I found a previously undetected significant conservation of (-13)G and a high degeneracy at (-15)T. I therefore defined the -15 element as a degenerate motif, which, together with the conserved stretch of sequence between -15 and -12, allows treating this element analogously to -35 and -10 elements. Furthermore, the strength of the -15 element inversely correlates with the strengths of the -35 element and -10 element, whereas no such complementation between other promoter elements was found. Despite the direct involvement of -15 element in σ(70)-dsDNA interactions, I found a significantly stronger tendency of this element to complement weak -10 elements that are involved in σ(70)-ssDNA interactions. This finding is in contrast to the established view, according to which the -15 element provides a sufficient number of σ(70)-dsDNA interactions, and suggests that the main parameter determining a functional promoter is the overall promoter strength.     

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10^?4 and P = 7.1 × 10^?4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.     

Age-related lung tissue remodeling due to the local distribution of MMP-2, TIMP-2, TGF-β and Hsp70

Lung tissue remodeling requires complex interactions of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), transforming growth factor (TGF) family and heat shock protein 70 (Hsp70). We evaluated the appearance and distribution of MMP-2, TIMP-2, TGF-β1 and Hsp70 in lung tissue using immunohistochemistry. Stained structures were graded semiquantitatively. Overall, more MMP-2, TIMP-2, TGF-β1 and Hsp70 were observed in bronchial cartilage, bronchial and alveola repithelium, and among alveolar macrophages. We evaluated mostly alveolar macrophages, bronchial epithelial cells and mucosal fibroblasts stained for TGF-β1, MMP-2 and TIMP-2. We also assessed strong or moderate correlations between numbers of cells containing TGF-β1, MMP-2, TIMP-2 in patients ≥ 60 years old. The presence of less TGF-β1 and more MMP-2, TIMP-2 and Hsp70 containing cells in all tissue groups indicated that local regulation was more dependent on MMP-2, TIMP-2 and Hsp70 distribution. Fewer TIMP-2, Hsp70 and TGF-β1 immunoreactive cells in younger individuals and increased expression of Hsp70 in elderly individuals demonstrated the influence of aging in lung remodeling. Findings of MMP-2, TIMP-2 and TGF-β1 immunoreactive cells in elderly individuals indicate lung remodeling due to aging.     

Synthesis of Oligodeoxynucleotides Containing the C-Nucleoside and 2′- Deoxy-2′-Fluoro-ara-Nucleoside Moieties by the H-Phosphonate Method.1,2

Abstract

A module type, computer-controlled, multipurpose synthesizer displaying a novel device for the transport of liquids, was constructed and used in the synthesis of oligomers containing some C-nucleosides and 2′-deoxy-2′-fluoro-ara-nucleoside moieties. H-Phosphonate method was applied in terms of a further adjustment of construction features of the synthesizer versus chemistry of the process. Results of preliminary studies on the effects of the modified nucleosides on the stability of duplexes showed a clear tendency of destabilization of duplexes in the case of C-nucleosides while fluorinated nucleosides in most cases stabilize the formed duplexes.     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

5- 氮杂-2'－脱氧胞苷对卵巢癌细胞系CP70 顺铂耐药性的逆转作用

目的:探究5-氮杂-2’-脱氧胞苷体外逆转卵巢癌铂类耐药细胞系CP70对顺铂的耐药性,并探讨与SOCS-2表达的关系。方法:免疫细胞化学和Western blot方法检测使用5-氮杂-2’-脱氧胞苷处理细胞前后细胞内SOCS-2表达水平。MTT法检测单独使用5-氮杂-2’-脱氧胞苷或顺铂及两药联合使用对CP70细胞的抑制作用。结果:两药联合处理CP70细胞后顺铂耐药逆转倍数为1.34、1.63、2.34,联合处理组的细胞中SOCS-2表达明显升高。结论:5-氮杂-2’-脱氧胞苷可以部分逆转CP70细胞对顺铂的耐药性,且此作用可能与增加细胞的SOCS-2表达有关。     

Chimeric evolution of the 2-μm genome in Saccharomyces cerevisiae

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 m) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-m variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-m plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection. Correspondence to: G.H. Rank