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.     

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.     

The arylation of [Pt2(μ-S)2(PPh3)4]

Routes to the synthesis of the mixed sulfide-phenylthiolate complex [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ have been explored; reaction of [Pt₂(μ-S)₂(PPh₃)₄] with excess Ph₂IBr proceeds readily to selectively produce this complex, which was structurally characterised as its PF₆⁻ salt. Reactions of [Pt₂(μ-S)₂(PPh₃)₄] with other potent arylating reagents (1-chloro-2,4-dinitrobenzene and 1,5-difluoro-2,4-dinitrobenzene) also produce the corresponding nitroaryl-thiolate complexes [Pt₂(μ-S){μ-SC₆H₂(NO₂)₂X}(PPh₃)₄]⁺ (X = H, F). The complex [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ reacts with Me₂SO₄ to produce the mixed alkyl/aryl bis-thiolate complex [Pt₂(μ-SMe)(μ-SPh)(PPh₃)₄]²⁺, but corresponding reactions with the nitroaryl-thiolate complexes are plagued by elimination of the nitroaryl group and formation of [Pt₂(μ-SMe)₂(PPh₃)₄]²⁺. [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ also reacts with Ph₃PAuCl to give [Pt₂(μ-SAuPPh₃)(μ-SPh)(PPh₃)₄]²⁺.     

Synthesis of some pentanuclear platinum clusters [Pt5(CO)6(L)4]. The X-ray structure of [Pt5(μ-CO)5(CO)(PCy3)4]

[Pt₅(μ-CO)₅(CO)L₄] (L = PPh₃1, PPh₂Bz 2, AsPh₃3, PEt₃4, PCy₃5) have been synthesized by reacting [Pt₃(μ-CO)₃(PR₃)₃] with H₂O₂ (1 and 2), by reduction of cis-[PtCl₂(CO)(PEt₃)] with Zn dust (4), and by the Zn reduction of [Pt₃(μ-CO)₃(PCy₃)₃] in the presence of [PtCl₂(CH₃CN)₂] (5). Complex 5 has not been observed previously and has been characterized by X-ray crystallography. Oxidation of the phosphine ligands with H₂O₂ is a new way to synthesize 1 and 2. The first complete NMR characterization of these complexes has also been achieved, and showed that these pentanuclear cluster complexes exhibit similar stereochemistries in solution and in the solid state. The observed ¹J_Pt-Pt values do not have any correlation with the corresponding bond lengths, again pointing out the irregular behaviour of such parameter in Pt complexes.     

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.     

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.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

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.     

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.     

Influence of CH3 group of μ-N-C-S ligand on the properties of [Fe2(C4H5N2S)2(NO)4] complex

Bi-nuclear neutral sulfur-nitrosyl iron complex [Fe₂(SR)₂(NO)₄] (I) has been obtained by replacement of thiosulfate ligands in dianion [Fe₂(S₂O₃)₂(NO)₄]²⁻ by 1-methyl-imidazole-2-yl. From X-ray analysis data, the complex has centrosymmetrical dimeric structure, with the iron atoms being linked via μ-N-C-S bridge. From Mossbauer spectroscopy, isomeric shift δ_Fe is 0.180(1) mm/s and quadrupole splitting ΔE_Q is 0.928(2) mm/s at T = 290 K. By comparative studying the mass-spectra in the gaseous phase of solid samples decomposition and kinetics of NO release in 1% aqueous solutions of dimethylsulfoxide, using of the ligand with CH₃ substituent in position 1 of imidazole-2-thiol was shown to yield a more stable donor of nitrogen monoxide than earlier obtained analog with imidazole-2-thiol, [Fe₂(C₃H₃N₂S)₂(NO)₄].     

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.     

Synthesis and characterisation of [Pt2(μ-S)(μ-I)(PPh3)4] - A cationic iodo analogue of the metalloligand [Pt2(μ-S)2(PPh3)4]

Reaction of [Pt₂(μ-S)₂(PPh₃)₄] with a number of transition metal-iodo complexes leads to the formation of the cationic iodo analogue [Pt₂(μ-S)(μ-I)(PPh₃)₄]⁺, identified using electrospray ionisation mass spectrometry (ESI MS). Synthetic routes to this complex were developed, using the reaction of [Pt₂(μ-S)₂(PPh₃)₄] with either [PtI₂(PPh₃)₂] or elemental iodine. The complex was characterised by NMR spectroscopy, ESI MS and an X-ray structure determination, which reveals the presence of a planar, disordered {Pt₂SI}⁺ core. Monitoring the iodine reaction by ESI MS allows the identification of various iodine species, including the short-lived intermediate [Pt₂(μ-S)₂(PPh₃)₄I]⁺, which allows a mechanism for the reaction to be proposed.     

Synthesis and structural characterization of the molecular loop [cis-Mo2(N,N-di-p-anisylformamidinate)2]2(O2C-CHCCH-CO2)2

A new molecular loop composed of two quadruply bonded Mo₂(DAniF)₂ units (DAniF=N,N^′-di-p-anisylformamidinate) linked by two chiral allene-1,3-dicarboxylate anions has been prepared from the reaction of [cis-Mo₂(DAniF)₂(MeCN)₄](BF₄)₂ with the bis(tetraethylammonium) salt of allene-1,3-dicarboxylic acid. This compound, [cis-Mo₂(DAniF)₂]₂(O₂C-CHCCH-CO₂)₂ (1), has been characterized by X-ray crystallography and by ¹H NMR and UV-Vis spectroscopy. The molecule possesses a center of inversion and hence is meso. There is only weak electronic coupling between the two Mo₂ ⁴⁺ units as revealed by electrochemical measurements.     

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.     

Chalcogenide-capped triruthenium clusters: X-ray structures of [Ru3(CO)6(μ3-CO){P(C4H3S)3}(μ-dppm)(μ3-O)] and [(μ-H)2Ru3(CO)6{P(C4H3S)3}(μ-dppm)(μ3-S)]

Treatment of [Ru₃(CO)₉{P(C₄H₃S)₃}(μ-dppm)] (1) [dppm = bis(diphenylphosphino)methane] with molecular oxygen in benzene at 60 °C affords oxo-capped [Ru₃(CO)₆(μ₃-CO){P(C₄H₃S)₃}(μ-dppm)(μ₃-O)] (2), while with elemental sulfur and selenium related chalcogenide-capped clusters [Ru₃(CO)₆(μ₃-CO){P(C₄H₃S)₃}(μ-dppm)(μ₃-E)] (3, E = S; 5, E = Se) and bis(chalcogenide) clusters [Ru₃(CO)₆{P(C₄H₃S)₃}(μ-dppm)(μ₃-E)₂] (4, E = S; 6, E = Se) result. Reaction of 1 with H₂S in refluxing THF affords the previously reported [(μ-H)₂Ru₃(CO)₇(μ-dppm)(μ₃-S)] (7) together with the new sulfido-capped dihydride [(μ-H)₂Ru₃(CO)₆{P(C₄H₃S)₃}(μ-dppm)(μ₃-S)] (8). All new compounds have been characterized by spectroscopic data, and 2 and 8 by single-crystal X-ray diffraction analyses. Oxo-capped 2 consists of a triangular ruthenium framework capped on opposite sides by oxo and carbonyl groups, while 8 consists of a ruthenium triangle by a capping sulfido ligand and two inequivalent bridging hydride ligands.     

内生真菌研究进展*

１内生真菌研究历史 内生菌（Ｅｎｄｏｐｈｙｔｅ）一词由 Ｄｅ Ｂａｒｙ（１８６６）首先提出,是指生活在植物组织内的微生物用以区分完那些生活在植物表面的表生菌（Ｅｐｉｐｈｙｔｅ）。按此定义植物的致病菌、菌根菌也归属于内生菌的概念范畴。Ｃａｒｒｏｌｌ（１９８６）将内生菌定义为生活在地上部分、活的植物组织内并不引起明显病害症状的真菌,突出强调内生菌与植物的互惠共生关系,因此,在这个内生菌概念的范畴内不包含植物致病菌和菌根菌。Ｐｅｔｒｉｎｉ（１９９１）把Ｃａｒｒｏｌｌ的概念范畴进一步扩展,将内生菌定义为那…