MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Activation of fatty acids by non-glyoxysomal peroxisomes

Ethylene treatment (approx. 20 l ·1^-1 in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo--1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10^-5 M -naphthaleneacetic acid) and cytokinin (1.4 · 10^-6 M kinetin). Evidence is presented that cultured tobacco cells require ethylene for -1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl₂; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1^-1 norbornadiene in air) inhibited -1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of -1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.Abbreviations NAA -naphthaleneacetic acid - NDE norbornadiene     

Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings

Rengel, Z. and Kordan, H. A. 1987. Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings.
The effects of ethylene, indolyl- and naphthylacetic acids, zeatin, benzyladenine, gib-berellic acid and triiodobenzoic acid on anthocyanin production in seedlings of Zea mays L. cv. Golden Bantam were investigated. Endogenously produced and exogen-ously supplied ethylene, as well as the other growth regulators tested markedly suppressed anthocyanin formation. Except for triiodobenzoic acid, the other growth regulators stimulated ethylene production, the amounts produced in the light being larger than those in the dark. Absorption of ethylene by permanganate as well as inhibition of ethylene production or action by Co²⁺ or Ag⁺ increased anthocyanin formation in maize seedlings above the level found in the control plants. The inhibiting effect of auxins and cytokinins on anthocyanin production was reversed by Co²⁺ or Ag⁺. In contrast, decreased anthocyanin formation caused by gibberellic acid or triiodobenzoic acid seemed unrelated to ethylene and could not be alleviated by Co²⁺ or Ag+.     

Effects of ethylene oxide on soil microbial content and some soil chemical characteristics

The effects of ethylene oxide (EtO) on survival of soil microflora and on selected chemical properties of a sandy-loam soil were examined. Soil sterilization was achieved after 8 hours exposure to EtO. Ethylene oxide treatment increased soil pH and organic matter content. Extractable Mn and Fe slightly increased whereas P content decreased. Total N was not affected by the treatment.     

Effect of osmotic stress on transpiration and absorption rates in triticale and its parental species

In triticale and its parental species, the application of a root osmotic stress induced either a transient increase or an immediate decrease in transpiration rate. The response of wheat (Trticum dicoccum farrum) proved to depend on relative humidity of air. In rye (Secale cerealecv. Petkus) and triticale (T. 300) the effect of NaCl stress was less expressive, than the effect of PEG.     

Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato

Abstract. Silver ions effectively inhibited both the initiation and the continuation of tomato ( Lyeopersicon esculentum Mill) ripening. Studies of protein synthesis in vivo showed that application of 2 mol m⁻³ silver thiosulphate to mature green fruit prevented the appearance of several novel proteins associated with ripening, including the softening enzyme polygalacturonase. However, total protein synthesis, as judged by the incorporation of [³⁵S] methionine into proteins, continued unabated after silver treatment. Ripening was also arrested when silver was supplied after ripening had begun. The accumulation of several ripening-related mRNAs, including that for polygalacturonase, was studied by translation in vitro and using cDNA clones as hybridization probes. Silver was shown to prevent the appearance of polygalaturonase mRNA when supplied to mature green fruit and to cause a rapid reduction in the concentration of mRNA for polygalacturonase and other ripening-related proteins when supplied after ripening had begun. It is proposed that silver exerts its effects due to interaction with the ethylene perception mechanism. The results suggest that perception of ethylene is vital not only for the initiation of ripening but also for the continued expression of genes required for ripening.