Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α₃β₁ and α_vβ₃, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α₃β₁ integrin was more variously glycosylated than α_vβ₃; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α₃β₁ integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of α_vβ₃ integrin glycans in melanoma or in any cancer cells.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.     

Binding of HIV-1 virions to α4β7 expressing cells and impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells

HIV-1 envelope glycoprotein is reported to interact with α4β7, an integrin mediating the homing of lymphocytes to gut-associated lymphoid tissue, but the significance of α4β7 in HIV-1 infection remains controversial. Here, using HIV-1 strain Ba L, the gp120 of which was previously shown to be capable of interacting with α4β7, we demonstrated that α4β7 can mediate the binding of whole HIV-1 virions to α4β7-expressing transfectants. We further constructed a cell line stably expressing α4β7 and confirmed the α4β7-mediated HIV-1 binding. In primary lymphocytes with activated α4β7 expression, we also observed significant virus binding which can be inhibited by an anti-α4β7 antibody. Moreover, we investigated the impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells. In α4β7-activated CD4+ T cells, both anti-α4β7 antibodies and introduction of shorthairpin RNAs specifically targeting α4β7 resulted in a decreased HIV-1 infection. Our findings indicate that α4β7 may serve as an attachment factor at least for some HIV-1 strains. The established approach provides a promising means for the investigation of other viral strains to understand the potential roles of α4β7 in HIV-1 infection.     

Mechanotransduction activates α5β1 integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1 week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α₅β₁ integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α₅β₁ integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α₅β₁ integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α₅β₁ integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.     

Substoichiometric inhibition of Aβ1-40 aggregation by a tandem Aβ40-1-Gly8-1-40 peptide

Aβ peptides aggregate to form insoluble and neurotoxic fibrils associated with Alzheimer’s disease. Inhibition of the aggregation has been the subject of numerous studies. Here we describe a novel, substoichiometric inhibitor of Aβ_1-40 fibrillization as a tandem dimeric construct consisting of Aβ_40-1 (reverse sequence) linked to Aβ_1-40 via an eight residue glycine linker. At molar ratios of the tandem peptide to Aβ_1-40 of 1:10 to 1:25 inhibition of fibrillization, as measured by ThioflavinT, was observed. We postulate that the tandem construct binds to a fibrillar intermediate but the reverse sequence delays or prevents further monomer association.     

A versatile entry into aqueous niobium chemistry: isolation and structure of the intermediate Nb4(μ2-O)2(μ2-OC2H5)4Cl4(OC2H5)4(HOC2H5)4

The reduction of ethanolic solutions of niobium pentachloride with zinc, followed by treatment with aqueous acids serves as a versatile entry into the aqueous solution chemistry of niobium. From the zinc-reduced solution, the major intermediate, Nb₄(μ₂-O)₂(μ₂-OC₂H₅)₄Cl₄(OC₂H₅)₄(HOC₂H₅)₄, was isolated and the crystal structure determined by X-ray crystallography. The complex crystallizes in the orthorhombic space group Pccn, with Z=4, a=21.0105(9), b=11.0387(5), c=19.1389(8), V=4438.9(3) ų, M_r=1090.19,R₁=0.0327 and wR₂=0.0876. The structure revealed a centrosymmetric tetrameric Nb(IV) complex, consisting of a pair of edge-sharing bi-octahedral Nb₂(μ₂-OC₂H₅)₄Cl₂(OC₂H₅)₂(HOC₂H₅)₂ units that are joined by two axial oxo ligands. The Nb-Nb distance of 2.7458(3) Å is consistent with a single metal-metal bond.     

Inhibiting effect of αs1-casein on Aβ1-40 fibrillogenesis

Background

α_s1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α_s1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α_s1-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimer's disease.

Methods

The aggregation kinetics of β-peptide in presence and absence of α_s1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α_s1-casein-Aβ_1-40 interaction.

Results and discussions

α_s1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α_s1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.

General significance

Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.     

Phosphatidic acid potentiates Gαq stimulation of phospholipase C-β1 signaling

Phosphatidic acid (PA) is interactive with Gα_q-linked agonists to stimulate GPCR signaling via phospholipase C-β₁ (PLC-β₁). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β₁ activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β₁ and Gα_q. Activated Gα_q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β₁ activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β₁ implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β₁ to both PMA and carbachol. PA alone thus has little effect on PLC-β₁ activity, but PA and PLD1 synergize with activated Gα_q to stimulate PLC-β₁ signaling. Coordinate interaction with activated Gα_q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.     

Thallium(III) complexes of the metalloligands [Pt2(μ-S)2(PPh3)4] and [Pt2(μ-Se)2(PPh3)4]

Reactions of [Pt₂(μ-S)₂(PPh₃)₄] with the diarylthallium(III) bromides Ar₂TlBr [Ar = Ph and p-ClC₆H₄] in methanol gave good yields of the thallium(III) adducts [Pt₂(μ-S)₂(PPh₃)₄TlAr₂]⁺, isolated as their salts. The corresponding selenide complex [Pt₂(μ-Se)₂(PPh₃)₄TlPh₂]BPh₄ was similarly synthesised from [Pt₂(μ-Se)₂(PPh₃)₄], Ph₂TlBr and NaBPh₄. The reaction of [Pt₂(μ-S)₂(PPh₃)₄] with PhTlBr₂ gave [Pt₂(μ-S)₂(PPh₃)₄TlBrPh]⁺, while reaction with TlBr₃ gave the dibromothallium(III) adduct [Pt₂(μ-S)₂(PPh₃)₄TlBr₂]⁺[TlBr₄]⁻. The latter complex is a rare example of a thallium(III) dihalide complex stabilised solely by sulfur donor ligands. X-ray crystal structure determinations on the complexes [Pt₂(μ-S)₂(PPh₃)₄TlPh₂]BPh₄, [Pt₂(μ-S)₂(PPh₃)₄TlBrPh]BPh₄ and [Pt₂(μ-S)₂(PPh₃)₄TlBr₂][TlBr₄] reveal a greater interaction between the thallium(III) centre and the two sulfide ligands on stepwise replacement of Ph by Br, as indicated by shorter Tl-S and Pt?Tl distances, and an increasing S-Tl-S bond angle. Investigations of the ESI MS fragmentation behaviour of the thallium(III) complexes are reported.     

TGF-β1对乳腺癌细胞系Snail表达的影响

目的通过TGF-β1诱导乳腺癌MCF-7发生上皮-间质转化(epithelial-mesenchymal transition,EMT)后检测锌指转录因子Snail表达的改变,探讨Snail在EMT及乳腺癌发生发展中的作用。方法常规培养乳腺癌细胞株MCF-7后,用TGF-β1诱导其发生EMT,用Transwell侵袭小室法进行细胞体外侵袭能力检测;用免疫组织化学方法及免疫荧光检测E-cadherin、Vi mentin、Snail的表达;用real ti me PCR检测E-cadherin、Vi mentin、Snail mRNA的表达。结果TGF-β1处理72h后的MCF-7细胞穿透能力明显增强。E-cadherin蛋白及mRNA表达减少,Vi mentin、Snail蛋白及mRNA表达增加。结论E-cadherin、Vi mentin是细胞发生EMT的重要生物学标志,Snail可能在转录水平上调控E-cadherin、Vi mentin蛋白的表达,Snail在EMT和乳腺癌的发生发展中起着重要的作用。     

A modified protocol to prepare seed-free starting solutions of amyloid-β (Aβ)1–40 and Aβ1–42 from the corresponding depsipeptides

Preparing reliable, seed-free stock solutions of the highly amyloidogenic peptides amyloid-β (Aβ) is difficult. Besides the formation of aggregates during synthesis and storage, dissolution of the peptide is a critical step because vortexing can induce aggregation. To overcome this, synthesis of the more water-soluble depsi-Aβ_1–42 peptide, from which the native sequence is easily obtained, has been suggested. We further refined this technique, including a cutoff filtration step and switching the depsipeptide in basic conditions, to stabilize the formed native peptide. The obtained solutions of native Aβ_1–40 and Aβ_1–42 peptides were homogeneous and aggregate free, as indicated by thioflavin T and circular dichroism analysis.     

In brown adipocytes,adrenergically induced β1-/β3-(Gs)-, α2-(Gi)- and α1-(Gq)-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α₁-adrenoceptor coupled via G_q), clonidine (α₂ via G_i) or CL316243 (β₃ via G_s) or via β₁-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC₅₀ 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades.     

A one-dimensional copper (II) coordination polymer [Cu3(ampym)2(μ1,1-N3)4(μ1,3-N3)2(dmf)2]n (ampym = 2-aminopyrimidine) containing both end-on and end-to-end azido bridges

A new polynuclear copper (II) complex, derived from the azido-bridging ligand and 2-aminopyrimidine, has been synthesized and its 3-D structure has been determined by X-ray diffraction methods at two different temperatures. The compound crystallizes in the triclinic system space group, with the central copper atom lying on an inversion centre. The crystal structure is built up by trinuclear units (each of them contains two double end-on azido bridges) linked through two azide ions in an end-to-end (EE) fashion, to yield the polymer chain [Cu₃(ampym)₂(μ_1,1-N₃)₄(μ_1,3-N₃)₂(dmf)₂]_n. Magnetic susceptibility measurement shows a ferromagnetic interaction above 30 K, whereas a weak anti-ferromagnetic interaction prevails in the range of 30-2 K.     

Synthesis and characterisation of adducts of [Pt2(μ-S)2(PPh3)4] with organo-palladium and platinum-hydride substrates

The reactions of [Pt₂(μ-S)₂(PPh₃)₄] towards a range of palladium(II) complexes containing organometallic ligands (cyclopalladated N-donor ligands, η³-allyl, phenyl) have been explored, leading to the formation of a series of cationic, trinuclear sulfido-bridged aggregates containing {Pt₂PdS₂} cores. [Pt₂(μ-S)₂(PPh₃)₄] also reacts with the platinum(II) hydride complex trans-[PtHCl(PPh₃)₂] giving the adduct [Pt₂(μ-S)₂(PPh₃)₄PtH(PPh₃)]⁺. X-ray crystal structure determinations on the complexes [Pt₂(μ-S)₂(PPh₃)₄PdPh(PPh₃)]PF₆ and [Pt₂(μ-S)₂(PPh₃)₄PtH(PPh₃)]PF₆ are reported, and show the expected bis μ₃-sulfido aggregates with three square-planar metal centres.     

佛波酯(TPA)促进NIH3T3细胞α5β1整合蛋白的合成

佛波酯(TPA)是潜在促肿瘤剂,也是蛋白激酶PKC激活剂.TPA能在极低浓度下替代DG激活PKC,从而导致一系列细胞功能变化.应用100 nmol/L TPA作用于NIH3T3细胞,观察NIH3T3细胞的粘附变化,发现TPA可促进NIH3T3细胞与基质纤连蛋白的粘附,进一步研究Fn的主要受体α₅β₁整合蛋白在细胞表面含量,发现TPA作用24 h使α₅及β₁含量分别增加52.3%和51.6%.应用³H-甘露糖标记N-糖链和凝集素柱层析方法分析TPA作用后细胞N-糖链总量和组分比,结果均与对照组相仿,说明是通过增加细胞合成整合蛋白α₅及β₁亚基含量实现的.在TPA作用于细胞的同时,加入PKC抑制剂Sphingosine,发现α₅、β₁含量和细胞与Fn的粘附均回复至对照组水平,提示TPA增加α₅β₁整合蛋白合成而增加的细胞与Fn粘附作用,是由PKC介导完成的.此外还发现酪氨酸蛋白激酶抑制剂也阻断TPA增加α₅β₁整合蛋白含量的作用.     

Fe-Rh and Fe-Ir clusters substituted by diphenylacetylene: Synthesis, solid state structure and electrochemical behaviour of [Fe2Ir2(CO)10(μ4:η-PhCCPh)], [FeIr2(CO)9(μ3:η-PhCCPh)], and [Fe2Rh(CO)8(μ3:η-PhCCPh)]

The reaction between [Fe₂Ir₂(CO)₁₂]²⁻ and diphenylacetylene in refluxing CH₃CN yields the substituted cluster [Fe₂Ir₂(CO)₁₀(PhC₂Ph)]²⁻ (1). In the crystals, the four metal atoms define a butterfly arrangement whose Ir-Ir hinge is parallel to the acetylenic C₂ unit. The neutral triangular cluster [FeIr₂(CO)₉(PhC₂Ph)] (2) is obtained by the treatment of 1 with acids at room temperature; in this 48 valence electrons species, the C-C and the Ir-Ir bonds are also parallel, in the coordination mode.The cluster [Fe₂Rh(CO)₁₀]⁻ reacts with diphenylacetylene in refluxing THF yielding [Fe₂Rh(CO)₈(PhC₂Ph)]⁻ (3). In this 46 C.V.E.’s cluster, the C₂ unit is perpendicular to the Fe-Fe edge, exemplifying the bonding mode. According to ¹³C NMR spectra, the structure of the three clusters is maintained in solution. Electrochemical investigations show that the one-electron oxidation of [Fe₂Ir₂(CO)₁₀(L)]²⁻ (L = 2CO, PhC₂Ph) as well as the one-electron reduction of [Fe₂Rh(CO)₈(PhC₂Ph)]⁻ only generates the respective short lived products.     

Ru2(CO)4(OOCR)2(PPh3)2 sawhorse-type complexes containing μ2-η-carboxylato ligands derived from biologically active acids

The thermal reaction of Ru₃(CO)₁₂ with the biologically active acids acetyl salicylic acid (Aspirin), α-methyl-4-(isobutyl)phenylacetic acid (Ibuprofen) and 3α,7α,12α-trihydroxy-5β-cholanic acid (cholic acid) in refluxing tetrahydrofuran, followed by addition of triphenylphosphine, gives the dinuclear complexes Ru₂(CO)₄(OOCR)₂(PPh₃)₂ (1: R = C₆H₄-2-OCOMe, 2: R = CHMe-C₆H₄-4-Buⁱ, 3: C₂₃H₃₉O₃). The single-crystal structural analysis of 1 and 2 reveals a dinuclear Ru₂(CO)₄ sawhorse structure, the diruthenium backbone being bridged by the carboxylato ligands, while the two phosphine ligands occupy the axial positions at the ruthenium atoms. However, chiral carbon atoms in the carboxylic acid undergo racemisation during the thermal reaction.