The “bone morphogenic proteins” pathways in bone and joint diseases: Translational perspectives from physiopathology to therapeutic targets

A large body of evidence supports an important role of bone morphogenic proteins (BMPs) pathways in skeletal development in the embryo. BMPs are also involved in skeletal homeostasis and diseases in the adult. They were first identified as major bone anabolic agents and recent advances indicate that they also regulate osteoclastogenesis and joint components via multiple cross-talks with other signaling pathways. This review attempts to integrate these data in the pathogenesis of bone and joints diseases, such as osteoporosis, fracture healing, osteoarthritis, inflammatory arthritis, or bone metastasis. The use of recombinant BMPs in bone tissue engineering and in the treatment of skeletal diseases, or future therapeutic strategies targeting BMPs signal and its regulators, will be discussed based on these considerations.     

Interfacial bioconjugation on emulsion droplet for biosensors

Interfacial bioconjugation methods are developed for intact liquid emulsion droplets. Complex emulsion droplets having internal hydrocarbon and fluorocarbon immiscible structured phases maintain a dynamic interface for controlled interfacial reactivity. The internal morphological change after binding to biomolecules is readily visualized and detected by light transmission, which provides a platform for the formation of inexpensive and portable bio-sensing assays for enzymes, antibodies, nucleic acids and carbohydrates.     

Multicopy suppression screen in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain lacking the Rab GTPase-activating protein Msb3p

The yeast proteins, Msb3p and Msb4p, are two Ypt/Rab-specific GTPase-activating proteins sharing redundant functions in exocytosis, organization of the actin cytoskeleton, and budding site selection. To see if Msb3p might play an additional, specific role, we first tested the sensitivities of msb3 and msb4 mutant strains to different drugs and then screened a genomic library for multicopy suppressors of msb3 sensitivity to CdCl₂ or to the calcium channel blocker diltiazem hydrochloride. Three genes (ADH1, RNT1, and SUI1) were found to suppress the CdCl₂ sensitivity of the msb3 strain and three others (YAP6, ZEO1, and SLM1) its diltiazem-HCl sensitivity. The results suggest a possible involvement of Msb3p in calcineurin-mediated signalling.     

Two promising alkaline β-glucosidases isolated by functional metagenomics from agricultural soil,including one showing high tolerance towards harsh detergents,oxidants and glucose

New β-glucosidase activities were identified by screening metagenomic libraries constructed with DNA isolated from the topsoil of a winter wheat field. Two of the corresponding proteins, displaying an unusual preference for alkaline conditions, were selected for purification by Ni-NTA chromatography. AS-Esc6, a 762-amino-acid enzyme belonging to glycoside hydrolase family 3, proved to be a mesophilic aryl-β-glucosidase with maximal activity around pH 8 and 40 °C. A similar pH optimum was found for AS-Esc10, a 475-amino-acid GH1-family enzyme, but this enzyme remained significantly active across a wider pH range and was also markedly more stable than AS-Esc6 at pH greater than 10. AS-Esc10 was found to degrade cellobiose and diverse aryl glycosides, with an optimal temperature of 60 °C and good stability up to 50 °C. Unlike AS-Esc6, which showed a classically low inhibitory constant for glucose (14 mM), AS-Esc10 showed enhanced activity in the presence of molar concentrations of glucose. AS-Esc10 was highly tolerant to hydrogen peroxide and also to sodium dodecyl sulfate, this being indicative of kinetic stability. This unique combination of properties makes AS-Esc10 a particularly promising candidate whose potential in biotechnological applications is worth exploring further.     

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.     

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.     

Intercalation of Zn(II) and Cu(II) complexes of the cyclic polyamine Neotrien into DNA: equilibria and kinetics

The equilibria and kinetics of the interaction of the Zn(II) and Cu(II) complexes of the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) with calf thymus DNA have been investigated at pH=7.0 and T=25 degrees C by spectrophotometry, spectrofluorimetry and stopped-flow method. At low dye/polymer ratios both complexes bind to DNA according to the excluded site model. At high dye/polymer ratios the binding displays cooperative features. The logarithm of the binding constant depends linearly on -log[NaCl]. The kinetic results suggest the D + S <==> D, S <==> DS mechanism where the metal complexes (D) react with the DNA sites (S) leading to fast formation of an externally bound form (D,S) which, in turn, is converted into internally bound complex (DS) by intercalation. The binding constants, evaluated as ratios of rate constants, agree with those obtained from equilibrium binding experiments, thus confirming the validity of the proposed model. Fluorescence titrations, where the metal-Neotrien complexes were added to DNA previously saturated with ethidium bromide (EB), show that both complexes displace EB from the DNA cavities. The reverse process, i.e. the addition of excess ethidium to the DNA/metal Neotrien systems, leads to fluorescence recovery for DNA/ZnNeotrien but not for DNA/CuNeotrien. This observation suggests that the binding of CuNeotrien induces deep alterations in the DNA structure. Experiments with Poly(dA-dT)*Poly(dA-dT) and Poly(dG-dC)*Poly(dG-dC) reveal that CuNeotrien mainly affects the structure of the latter polynucleotide.     

Dye-induced aggregation of single stranded RNA: a mechanistic approach

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.